PROGRAM PLANNING COMMITTEE Jim Wells, Ph.D. (Chair) Professor and Chair, Dept of Pharmaceutical Chemistry University of California, San Francisco

Oscar Millet, Ph.D Structural Biology Unit CIC bioGUNE

Miquel Pons, Ph.D. Professor, Dept of Organic Chemistry Universitat de Barcelona

Mark A. Lemmon, Ph.D. Professor and Chair, Dept of Biochemistry and Biophysics University of Pennsylvania Perelman School of Medicine

Gary Pielak, Ph.D. Distinguished Term Professor of Chemistry, Biochemistry and Biophysics University of North Carolina, Chapel Hill

T HANK Y OU TABLE OF CONTENTS Symposium Overview .................................................................................................... 3 Executive Council & Leadership ................................................................................. 4 Committees ...................................................................................................................... 5 Protein Society Awards ................................................................................................. 6 Call for 2015 Nominations ............................................................................................... 8 Hotel Floorplan ............................................................................................................... 9 General Information.................................................................................................... 10 Travel Awards ................................................................................................................ 12 Donor Acknowledgements ......................................................................................... 13 Daily Program ............................................................................................................... 14 Day 1 ................................................................................................................................14 Day 2................................................................................................................................ 17 Day 3................................................................................................................................21 Day 4............................................................................................................................... 24 Poster Sessions............................................................................................................. 26 Exhibitors ....................................................................................................................... 47 Exhibitor Workshops ..................................................................................................54 Speaker Abstracts ......................................................................................................... 55 Poster Abstracts ............................................................................................................ 71 Author Index ............................................................................................................... 274


THE 2014 PROGRAM PLANNING COMMITTEE Todd O. Yeates, Ph.D. Professor, Department of Chemistry and Biochemistry University of California, Los Angeles

Jennifer R. Potts, Ph.D. Professor and Chair of Molecular Biophysics Department of Biology The University of York

Tarun Kapoor, Ph.D. Pels Family Professor Laboratory of Chemistry and Cell Biology The Rockefeller University

CORPORATE SUPPORT The Protein Society is grateful to the following corporate partnerns for supporting the 28th Annual Symposium & The Protein Society: 28TH ANNUAL SYMPOSIUM SPONSORS Proteins in Disease & Therapeutics Research Session Best Poster Competition

Complimentary Tote Bags


Bronze Level


DAY 2 MONDAY JULY 28TH Members Business Meeting/New Member Welcome Breakfast

7:00 AM



(75 min) - Marina Room

8:30 AM Opening Plenary Session

Protein Proteins in Protein Proteins In Closing Plenary Session & Evolution Disease & Engineering Dynamic & 2014 Stein & Moore Grand HAll C Thera- & Synthetic Driven Award Winner Talk Grand Hall C & D peutics Biology Processes Grand Hall C & D Grand HAll D Grand HAll C Grand HAll D Protein Membrane 9:40 AM Translation Bacterial & Folding Interactions DegraProteins & Grand HAll C Grand Hall D dation Receptors & 2014 Hans Neurath Award Winner Talk

Grand HAll C

11:30 -Exhibits & Poster Displays (2 hrs) - Grand Hall A & B

11:30 AM LUNCH

(2 hrs)

Grand HAll D

Exhibitor Workshop

Exhibitor Workshop Exhibitor Workshop NanoTemper GE Healthcare Technologies EW I-47 NEW Amersham™ 600 EW III-49 Multimodal Imager series (CCD-based Some like it hot: Determination chromatography: A new of Biomolecular Interactions generation of high selectivity camera systems) using MicroScale (1 hr) - Gaslamp A media for challenging Thermophoresis purifications (1 hr) - Gaslamp A (1 hr) - Gaslamp A All Lunch Workshops from Noon - 1PM

GE Healthcare

Workshop: Undergrad Educator’s Workshop: Workshop: Workshop: How to Research Luncheon How to Write WikiPedia for How to (1 hr) Improve Your Session an Effective Protein Navigate

(1 hr) Writing (1 hr) Bankers Hill Gaslamp C & D

1:30 PM

Bankers Hill

End of Program

Paper Scientists Your Career (1 hr) (1 hr) (1 hr) Gaslamp C & D Bankers Hill Gaslamp C & D

Frontier Proteins in Chemical Cellular Plenary Awards Session HighAltered Biology & Structures 1:30 PM - 5:50 PM Throughput States Enzymology Grand HAll D Grand Hall C & D Techniques Grand HAll D Grand HAll C Grand HAll C

4:30 PM Exhibits & Poster Displays (2 hrs) - Grand Hall A & B 7:00 PM Networking Dinners (3hrs) - Locations Vary 8:00 PM

Members Reception Education & Mentoring Committees’ Mixer

(3hrs) - Please View the Program Addendum for Location

(3 hrs) Kin Spa Pool Deck, 3rd Floor

Following the Symposium, please take a few moments to share your feedback when you receive the Attendee Survey





James Bowie, University of California, Los Angeles

Jacquelyn Fetrow, University of Richmond


Lynne Regan, Yale University

Lars Baltzer, Uppsala University

Paula Booth, University of Bristol

Charles Brooks, III, University of Michigan

Johannes Buchner, Technische Universität München

Zengyi Chang, Peking University

Stewart Fisher, SL Fisher Consulting

Amy Keating, Massachusetts Institute of Technology

Jacqui Matthews, University of Sydney

Haruki Nakamura, Osaka University

Andreas Plückthun, Universität Zurich

Catherine Royer, Rensselaer Polytechnic Institute

Michael Summers, University of Maryland, Baltimore County

Sarah Teichmann, EMBL-EBI/Wellcome Trust Sanger Institute

Brian Matthews (ex officio), Editor in Chief, University of Oregon

Jody McGinness (ex officio), Executive Director, The Protein Society




Mary Munson, University of Massachusetts Medical School (Chair)

John Osterhout, Angelo State University (Chair)

Judith Frydman, Stanford University

Christopher Bystroff, Rensselaer Polytechnic Institute

Matthais Mann, Max-Planck-Institut für Biochemie

Cory L Brooks, California State University - Fresno

Thomas Magliery, The Ohio State University Andrew Miranker, Yale University

Paola Di Lello, Genentech, Inc.

Mary Munson, University of Massachusetts Medical School

Francisco J. Enguita, University of Lisbon

Brenda Schulman, St. Jude Children's Research Hospital Wei Yang, LMB, NIDDK, NIH

John J. Dwyer, Ferring Research Institute Angelo M. Figueiredo, University of East Anglia Azucena G. Horta, Universidad Autonoma de Nuevo Leon Mary Konkle, Eastern Illinois Univerrsity


Suresh Kumar, University of Arkansas

Amy Keating, Massachusetts Institute of Technology (Chair)

Patricia Ann Mabrouk, Northeastern University

James Bowie, University of California - Los Angeles

Matthew Torres, Georgia Institute of Technology

Arthur Palmer, III, Columbia University Lynne Regan, Yale University

Sudha Veeraraghavan, University of Maryland School of Pharmacy

Jacquelyn Fetrow, University of Richmond

Bo Zhang, DuPont Industrial Biosciences

Brian Matthews, University of Oregon

Zhiwen Zhang, Santa Clara University

Todd Naumann, USDA-ARS-NCAUR

MENTORING COMMITTEE Andrew Vendel, Eli Lilly and Company (Chair) Blake Hill, Medical College of Wisconsin Kevin Lumb, Merck & Company Matthew Cordes, University of Arizona Patricia Jennings, University of California, San Diego Olve Peersen, Colorado State University Stephen Fuchs, Tufts University

Judith Klein-Seetharaman, University of Pittsburgh School of Medicine Giovanna Ghirlanda, Arizona State University Janel McLean, Vanderbilt University Medical Center Carly Huitema, SIAF institute Eric Sundberg, University of Maryland School of Medicine

EDUCATION COMMITTEE Ellis Bell, University of Richmond (Chair)

John Osterhout, Angelo State University

Margaret Cheung, University of Houston

Jane Richardson, Duke University

Matthew Gage, Northern Arizona University

Srebrenka Robic, Agnes Scott College

Lisa Gentile, University of Richmond Peter Kahn, Rutgers University

Michael Summers, University of Maryland Baltimore County/HHMI

Betsy Komives, University of California - San Diego

Frieda Texter, Albright College

Mary Konkle, Eastern Illinois University

Jeff Watson, Gonzaga University


2014 PROTEIN SOCIETY AWARDS The Carl Brändén Award, sponsored by Rigaku Corporation, is given to an outstanding protein scientist who has also made exceptional contributions in the areas of education and/or service to the science. The 2014 recipient of this award is Dr. Stephen H. White (University of California, Irvine) in recognition of his many contributions to the field of membrane protein folding, his long service to the protein science community, and his skills as an educator of graduate students and postdocs in particular. His research has provided much fundamental insight into the thermodynamics of folding in and on the membrane. Dr. White has made major contributions to multiple scientific societies, including The Protein Society, and --among other service activities-- maintains the widely-utilized Membrane Proteins of Known 3D Structure website. Talk Title: Membrane Protein Folding: Biology Meets Thermodynamics When: 5:20 PM Tuesday, July 29th Where: Plenary Awards Session, 1st Floor, Grand Hall C & D

The Dorothy Crowfoot Hodgkin Award, sponsored by Genentech, is granted in recognition of exceptional contributions in protein science which profoundly influence our understanding of biology. The 2014 award will be presented to Dr. Judith Frydman (Stanford University) for exceptional contributions to our understanding of protein folding in eukaryotic cells. Dr. Frydman uses creative combinations of biochemical, genetic, biophysical, and computational analysis to dissect the mechanisms by which cells manage misfolded proteins, with a particular focus on the group II cytosolic chaperonin TRiC. Talk Title: Chaperone Control of Cellular Proteostasis in Health and Disease When: 1:40 PM Tuesday, July 29th Where: Plenary Awards Session, 1st Floor, Grand Hall C & D

The Hans Neurath Award, sponsored by The Neurath Foundation, recognizes individuals who have made a recent contribution of unusual merit to basic research in the field of protein science. In 2014 the Hans Neurath Awardee is Dr. James H. Hurley (University of California, Berkeley) for his ground-breaking contributions to structural membrane biology and membrane trafficking. Throughout his career, Dr. Hurley has produced fundamental insights about proteins that function at the membrane-solvent interface, including how signaling molecules interface with and are regulated by lipids, mechanisms of vesicle formation by ESCRT machinery, and, most recently, the regulation of clathrin coated vesicle formation and the initiation of autophagy. Talk Title: The Coreography of Self-Cannibalism When: 8:30 AM Sunday, July 27th Where: Opening Plenary Session, 1st Floor, Grand Hall C & D

The Christian B. Anfinsen Award, sponsored by The Protein Society, recognizes significant technical achievements in the field of protein science. The recipient of this award in 2014 is Dr. Robert Tycko (National Institutes of Health) for his seminal contributions to the development of modern solid state nuclear magnetic resonance methods for structural investigations of proteins and influential studies on amyoloid fibrils associated with neurodegeneration and disease. Dr. Tycko’s achievements in this area are highlighted by his recent presentation of the first detailed structure of β-amyloid fibrils from Alzheimer's disease brain tissue and evidence for structural variations that underlie prion diseases.


2014 PROTEIN SOCIETY AWARDS Talk Title: The Molecular Structural Basis for Amyloid Formation, in vitro and in vivo When: 2:10 PM Tuesday, July 29th Where: Plenary Awards Session, 1st Floor, Grand Hall C & D

The Emil Thomas Kaiser Award, sponsored The Protein Society, recognizes a recent, highly significant contribution to the application of chemistry in the study of proteins. The 2014 awardee is Dr. Carol Fierke (Dept. of Chemistry, University of Michigan). Dr. Fierke is being recognized for her exceptional contributions to our understanding of the metal homeostasis, and to understanding of the structure and mechanism of ribonuclease P. Her research has combined metallochemistry, biochemistry, genetics and a wide range of spectroscopic techniques to uncover details of metal affinity, substrate specificity, and roles to catalytic activity in numerous biologically and medically important enzymes. Amongst her groundbreaking achievements include the development of protein-based metal biosensors and techniques for monitoring metal ions in vivo for probing metal transport and toxicity. Talk Title: Histone Deacetylases: Substrate Selectivity and Regulation When: 2:50 PM Tuesday, July 29th Where: Plenary Awards Session, 1st Floor, Grand Hall C & D

The Protein Science Young Investigator Award, named for the academic journal of the Society, recognizes an important contribution to the study of proteins by a scientist still in the early stages of an independent career. The 2014 awardee is Dr. M. Madan Babu (MRC Laboratory of Molecular Biology, Cambridge UK). Dr. Babu is being recognized for his contributions to our understanding of the principles of regulation in biological systems – especially in the fields of intrinsically disordered proteins and gene regulation. He was appointed in 2006 as one of the youngest independent group leaders in the Structural Studies division at the LMB. Dr. Babu employs an interdisciplinary approach, combining computational and experimental methods to investigate regulatory processes at multiple scales of complexity (ranging from the molecular to the genome level) across a wide range of model organisms. Talk Title: Please consult the Program Addendum When: 3:40 PM Tuesday, July 29th Where: Plenary Awards Session, 1st Floor, Grand Hall C & D

The Stein & Moore Award, sponsored by The Protein Society, is named for Nobel laureates Dr. William Stein and Dr. Stanford Moore. The award venerates their contribution to understanding the connection between chemical structure and catalytic activity of the active center of the ribonuclease molecule. The 2014 awardee is Dr. Nikolaus Pfanner (Institute for Biochemistry and Molecular Biology, University of Freiburg) in recognition of his contributions to understanding pathways of cellular sorting that underlie the biogenesis of mitochondria. His pioneering research includes the first comprehensive proteomic analysis of mitochondria, the discovery of three new pathways of mitochondrial import, and the reconstitution of mitochondrial import channels and unraveling mechanisms of protein translocation. Talk Title: Mitochondrial machinery for transport and assembly of proteins When: 11:00 AM Wednesday, July 30th Where: Closing Plenary Session, 1st Floor, Grand Hall C & D




2015 Awards of The Protein Society Presented annually to distinguished scientists, the Protein Society Awards recognize excellence and outstanding achievements in the multidisciplinary fields of protein science and honor distinguished contributions in the areas of leadership, education, and service. The 2015 awards will be presented at the 29th Annual Symposium of The Protein Society (July 22-25,2015, in Barcelona, Spain). The deadline for submitting complete award nomination packages for the 2015 Awards cycle will be noon (EDT) on September 22, 2014

Note: the ability to submit a nomination is a benefit of membership.






GENERAL INFORMATION Scientific Registration – Grand Hall Foyer The Registration Area will open at 4:00 PM on Saturday, July 26th (refer to hours below). Registration includes admission to all scientific and poster sessions, exhibits, one copy of the Program/Abstract book and one tote bag. Registration does not include any meals. Hours Saturday 4:00 pm – 7:00 pm Sunday 7:30 am – 5:00 pm Monday 7:00 am – 5:00 pm Tuesday 7:30 am – 5:00 pm

Registration Fees Full Member Registration $400 Emeritus Member Registration $350 Early-Career Member Registration $350 Lab Staff Member Registration $350 Graduate Student Member Registration $225 Undergrad Member Registration FREE Undergrad Non-Member Registration $25 Regular Non-Member Registration $700 Single-day Registration $400 Guest Registration (Networking Events) $95

Press Registration Complimentary Press badges will be issued on-site to members of the working press and freelance writers. Press credentials or an official letter from an editor bearing a letter of assignment must be provided to register as press. Representatives of allied fields (public relations, public information, public affairs) are not qualified for press registration and must register as nonmembers. Registration Refunds/Cancellations Refunds for registration must be made in writing and postmarked by July 1, 2014. Badge and receipt must accompany the request (except for non-U.S. xvii registrants). Refunds cannot be made if badges are not received by the Society Office, or if postmarked after this date. A $40 cancellation fee will be deducted from all registration payment refunds. Attendees that cancel AFTER July 1, 2014 will not receive a refund. Badge/Publications/Tote Bag Pickup - Grand Hall Foyer U.S. attendees who registered prior to July 2 received their badge in advance of the meeting. All other registrants including International registrants must go to the Symposium Registration Counter. All attendees are required to wear their badge at all times. In addition to being a means of identification, the name badge is required for admission to scientific sessions and exhibits. Each registrant will receive one copy of the Program/Abstract book and one tote bag. Business Center The Business Center is located on the lobby level in the Seaport Tower. It may be reached at 619232-1234 ext 4888 and is open daily, 7:00 am – 7:30 pm. Services include printing, copying, transparencies, faxing, shipping and special services such as name tags and business cards. Cameras/Video Recording The use of cameras/video recording inside session rooms or among the posters is prohibited. Mobile Devices As a courtesy to your fellow attendees, please turn off all cell phones and beepers prior to entering a session room. 10

Meal Functions Meals are not included with the Symposium registration fee. Lunchtime workshops require a $10 deposit at registration to reserve a boxed lunch. Meeting Management Office The Management Office is located in LaJolla A and will be open Saturday 8:00 am – 10:00 am, Sunday – Tuesday 8:00 am – 5:00 pm, and Wednesday 8:00 am – 11:30 am. Complimentary Wireless Internt Symposium attendees staying at the Headquarters Hotel should have received instructions for complimentary in-room WiFi internet access. If you were not informed or otherwise paid for the access, please inform the hotel at checkout and they will refund the fees to you. Complimentary WiFi access is available in the Exhibit Hall as well. Poster Set Up & Removal Posters can be mounted from 8:30 AM to 11:30 AM on Sunday, July 27th. Note that all posters must be no larger than 3 feet (36 inches / 91 centimeters) to a side. Grand Hall C & D. Posters can remain mounted until 3:30 PM on Tuesday, July 29th. Any posters remaining after that point will be discarded. Poster Viewing Times Posters are on display from Sunday morning until Tuesday afternoon. During the following shifts, exhibitors will be on hand and--during the 2 afternoon shifts--a Mix & Mingle networking reception taking place:

SUNDAY, JULY 27TH 11:30 AM – 1:30 PM 4:30 PM – 6:30 PM

MONDAY, JULY 28TH 11:30 AM – 1:30 PM 4:30 PM – 6:30 PM

TUESDAY, JULY 29TH 11:30 AM – 1:30 PM


Authors will be presenting posters on the following schedule: SUNDAY 11:30 - 1:30

SUNDAY 4:30 - 6:30

Even numbered posters P02-P08

Odd numbered posters P02-P08

MONDAY 11:30 - 1:30

MONDAY 4:30 - 6:30

Even numbered posters P09-P12

Odd numbered posters P09-P12 11

TRAVEL AWARDS The following outstanding students and early-career investigators received travel assistance to attend the 28th Annual Symopsium of The Protein Society from The Finn Wold Travel Awards fund and The Protein Science Young Investigator Travel Grants:*

Undergraduate Students Angela Harper, Wake Forest University Gabrielle Shea, Wake Forest University Lauren Sparks, Hamline University Mark Volker, Hamline University

Graduate Students Keith Ballard, University of Massachusetts, Amherst Diego Caballero, Yale University Alexandra Chadwick, Medical College of Wisconsin Genevieve Desjardins, University of British Columbia Maayan Eitan-Wexler, Faculty of Medicin, Hebrew University Keith Fraser, Rensselaer Polytechnic Institute Gilberto Garcia, Universidad Autónoma del Estado de Morelos Nathan Gardner, Purdue University Alisa Glukhova, University of Michigan Julia Hayden, Wake Forest University Kyle Heim, University of Florida Michael Jacobsen, University of Utah Agnieszka Kendrick, University of Colorado Denver Janelle Leuthaeuser, Wake Forest University Carrie Marean-Reardon, Washington State University

Tahria Najnin, University of New South Wales Robert Newberry, University of WisconsonMadison Olive Njuma, Auburn University Rachel North, University of Canterbury Satoshi Oshiro, The University Of Tokyo Rachael Parker, Virginia Tech Eamonn Reading, University of Oxford Ashley Schloss, Yale University Crystal Serano, University of Southern Mississippi Amber Smith, University of Michigan Nardos Sori, Old Dominion University Miroslava Strmiskova, University of Ottawa Danielle Williams, Yale University Daniel Woldring, University of Minnesota Meng Yang, Stony Brook University Shlomo Zarzhitsky , Ben-Gurion University of the Negev Huimei Zheng, State University of New York – Upstate Medical University

Early-Career Investigators Andisheh Abedini , New York University Medical Center Richard Besingi, University of Florida Jennifer Cash, University of Michigan Claudia Corbo, The Houston Methodist Research Institute Kendra Frederick, Whitehead Institute

Soon Goo Lee, Washington University in St. Louis Piere Rodriguez-Aliaga, University of California Berkekey Jennifer Seedorff, NIH/NIAID Kaustubh Sinha, Carnegie Mellon University Adnan Sljoka, Ryerson University/University of Colorado Boulder 12

CHARITABLE SUPPORT OUR MISSION As a not-for-profit scientific and educational organization, The Protein Society relies upon support from the public for a vital portion of its operating budget, and in particular to fund certain programs. The following recent donors have the gratitude of The Protein Society leadership and Executive Council for their generosity and their commitment to the mission of the protein society.

The Protein Society Annual Fund Ann E. Aulabaugh Len Banaszak Arpad Karsai Edyth Malin Abhinav Nath Sheena Radford Bixun Wang

The Education & Mentoring Committees’ Funds Jessica Bell Penny Beuning Norma J Greenfield Tijana Z Grove Blake Hill Peter C Kahn Jody McGinness

The Finn Wold Travel Awards Fund Joseph Alia Rodrigo Alejandro ArreolaBarroso Len Banaszak Zengyi Chang Nidhee Chaudhary Archana Chavan Supratik Dutta Anne Gershenson Jenny P Glusker Jacob M Goldberg David P Goldenberg

Elizabeth Goldsmith Mary G Hamilton John R Helliwell James C Hu Yao-Te Huang Michael N James Joel Janin Christos Karamitros Andreas Matouschek Jody McGinness C James McKnight Mary Jo Jo Ondrechen

Gavin Ray Owen Arthur Palmer Jack Preiss George Rose Madeline Shea Frank Soennichsen Melissa Starovasnik Cynthia Stauffacher Bernadine Wold Marc Wold Beulah Woodfin

*The Protein Science Young Investigator Travel Grants are sponsored by


DAILY PROGRAM Day 1 - Sunday, July 27, 2014


Welcome & Introduction Protein Society President , James Bowie

8:35- 8:40

Presentation of the Hans Neurath Award*

8:40 - 9:10

2014 Neurath Award Winner Talk The Coreography of Self-Cannibalism James H. Hurley, University of California, Berkeley Coffee & Refreshments

9:10 - 9:40



Welcome & Introduction from session Chair Policing Secretion: How Cells Enforce Protein Quality Control in the Endoplasmic Reticulum Liz Miller, Columbia University Young Investigator Talk

10:15 - 10:30 10:30 - 11:00

Continuous Tracking of Protein Folding at Microsecond Resolution by a Line Confocal Detection of Single Molecule Fluorescence Satoshi Takahashi, Tohoku University

11:00 - 11:30

Intrinsically Disordered Proteins: Kinetics and Mechanism Jane Clarke, University of Cambridge



Welcome & Introduction from session Chair The Gram-negative Cell Envelope as Seen by Protein Antibiotics Colin Kleanthous, University of Oxford

10:15 - 10:30

Young Investigator Talk

10:30 - 11:00

Bacterial Proteins that Modulate Host Membrane Transport Pathways Craig Roy, Yale School of Medicine

11:00 - 11:30

Protein Interactions Regulating Self/Non-self-recognition in Bacterial Contact-Dependent Growth Inhibition David Low, University of California – Santa Barbara

LUNCH 11:30 - 1:30


Organized by the Protein Society Mentoring Committee, this lunchtime workshop will focus on how to improve your writing skills with focus on how to prepare an impactful abstract. Participants will learn a framework to improve the clarity and crispness of their communication. NOON - 1 PM / GASLAMP C&D


3 promising undergrads will be selected by The Education Committee to present their work, gaining visibility, experience, and making valuable connections for graduate school and professional development. Support future protein science leaders and stop by! NOON - 1 PM / BANKERS HILL



2:05 - 2:20 2:20 - 2:50

2:50 - 3:15 3:15 - 3:45

3:45 - 4:00 4:00 - 4:30

Welcome & Introduction from session Chair Ribosome profiling reveals surprises in genome decoding in meiosis Gloria Brar, University of California - Berkeley Young Investigator Talk Visualizing Transcription Pausing and Backtracking Genome-wide at Nucleotide Resolution L. Stirling Churchman, Harvard Medical School Coffee & Refreshments Structural Analysis of Proteins in Native Environments, Fact or Fiction? Juri Rappsilber, University of Edinburgh Young Investigator Talk A Mass-Spectrometry-Based Map of Universally-Shared Animal Protein Complexes Ed Marcotte, University of Texas - Austin


2:05 - 2:20

Welcome & Introduction from session Chair Protein Aggregation Done Right: The Biogenesis of Functional Amyloids Matthew Chapman, University of Michigan Young Investigator Talk


DAILY PROGRAM 2:20 - 2:50

2:50 - 3:15 3:15 - 3:45

3:45 - 4:00 4:00 - 4:30

Structural Malleability of Intrinsically Disordered Proteins Underlying Alternative Functional States Peter Tompa, Vrije Universiteit Brussel Coffee & Refreshments Amyloid Assemblies and their Interactions With Cellular Components Helen Saibil, Birkbeck College Young Investigator Talk Dynamic Complexes, Folding and Phase Separation of Disordered Proteins in Biological Regulation Julie Forman-Kay, The Hospital for Sick Children

Day 2 - Monday, July 28, 2014


Welcome & Introduction from session Chair Evolution in a Test Tube Yields De Novo Enzymes with Unusual Structure and Dynamics Burckhard Seelig, University of Minnesota

9:05 - 9:20

Young Investigator Talk

9:20 - 9:50

Sequence-function-fitness Landscapes Viewed by Massively Parallel Sequencing Approaches Dan Bolon, University of Massachusetts Medical School

9:50 - 10:15

Coffee & Refreshments


DAILY PROGRAM 10:15 - 10:45

10:45 - 11:00 11:00 - 11:30

Evolution of Novel Components of the Bacterial Flagellar Motor Morgan Beeby, Imperial College London Young Investigator Talk The Remarkable Pliability and Promiscuity of Specialized Metabolism Joseph P. Noel, Salk Institute for Biological Studies / HHMI


Welcome & Introduction from session Chair

8:35 - 9:05

Structural Basis of Broad Neutralization of Viral Pathogens Ian Wilson, The Scripps Research Institute

9:05 - 9:20

Young Investigator Talk

9:20 - 9:50

9:50 - 10:15 10:15 - 10:45

10:45 - 11:00 11:00 - 11:30

The HIV-1 viral spike: Conformational Machine For Entry and Evasion Peter Kwong, Vaccine Research Center, Niaid/Nih Coffee & Refreshments Immune Sensing of Vitamin B Metabolites Jamie Rossjohn, Monash University

Young Investigator Talk Engineering Improved Antibodies Against HIV Pamela Bjorkman, California Institute of Technology / HHMI (Chair) photo: Micheline Pelletier / Gamma











Welcome & Introduction from session Chair

1:35 - 2:05

Spatially-Resolved Proteomic Mapping of Mitochondria in Living Cells Using an Engineered Peroxidase Reporter Alice Ting, Massachusetts Institute of Technology

2:05 - 2:20

2014 Protein Science Best Paper Speaker Mark Landau, Yale University

2:20 - 2:50

Structure-Based Screens for Protein De-orphanization Brian Shoichet, University of Toronto / University of California – San Francisco

2:50 - 3:15 3:15 - 3:45

Coffee & Refreshments Molecular DNA Devices in Living Systems Yamuna Krishnan, National Center for Biological Sciences


DAILY PROGRAM 3:45 - 4:00 4:00 - 4:30

Young Investigator Talk Activity-based Proteomics - Applications for Enzyme and Inhibitor Discovery Benjamin Cravatt III, The Scripps Research Institute


Welcome & Introduction from session Chair New Insights Into Microtubule Mechanics Manuel Théry, LPCV / iRTSV / DSV / CEA

2:05 - 2:20

Young Investigator Talk

2:20 - 2:50

2014 Lorne Conference on Protein Structure and Function Speaker Kaye Truscott, Latrobe University

2:50 - 3:15

Coffee & Refreshments

3:15 - 3:45

A Protein Interaction Network that Directs Human Cytoplasmic Dynein to Microtubule Ends Thomas Surrey, LRI - CRUK

3:45 - 4:00

Young Investigator Talk

4:00 - 4:30

Mechanisms of Mitosis and Intracellular Scaling in Xenopus Rebecca Heald, University of California - Berkeley


DAILY PROGRAM Day 3 - Tuesday, July 29, 2014


Welcome & Introduction from session Chair Please consult the Program Addendum Chris Voigt, Massachusetts Institute of Technology

9:05 - 9:20

Young Investigator Talk

9:20 - 9:50

Sustaining Life With Proteins Designed De Novo Michael Hecht, Princeton University

9:50 - 10:15

Coffee & Refreshments

10:15 - 10:45

10:45 - 11:00 11:00 - 11:30

Metal-Directed Protein Evolution Akif Tezcan, University of California- San Diego

Young Investigator Talk De Novo Protein Structures and Assemblies by Design Dek Woolfson, University of Bristol


Welcome & Introduction from session Chair Deciphering Protein Dynamics During Endocytic Budding by Time-resolved Electron Microscopy Maria Isabel Geli Fernandez- Penaflor, Molecular Biology Institute of Barcelona 21

DAILY PROGRAM Young Investigator Talk

9:05 - 9:20 9:20 - 9:50

The Spatial Regulation of Molecular Motors Samara Reck-Petersen, Harvard Medical School

Coffee & Refreshments

9:50 - 10:15 10:15 - 10:45

The Bacterial Magnesium Channel Cora – Dynamic Ways to Translocate Divalent Cations Emil F. Pai, University of Toronto Young Investigator Talk

10:45 - 11:00 11:00 - 11:30

Engineering Improved Antibodies Against HIV Klaus Hahn, University of North Carolina-Chapel Hill

LUNCH 11:30 - 1:30


This guided workshop, led by Wiki expert Emily Temple-Wood, will be a fun, hands-on course on wiki-editing, Wikipedia in general, and tips and tricks for improving science articles using famous protein researchers as examples. So have someone in mind, and bring your tablet or laptop! NOON - 1 PM / GASLAMP C&D


Organized by the Protein Society Mentoring Committee, this lunchtime workshop will consist of a panel of individuals with different career paths to answer your questions. The panel will cover important aspects including connecting with hiring managers/search committees, preparing resumes/CVs, interview practices, and negotiations. The panel will also be there to discuss their experiences with traversing their career paths. NOON - 1 PM / BANKERS HILL



Welcome & Introduction from session James Bowie, President

1:35 - 1:40

Presentation of The Dorothy Crowfoot Hodgkin Award*

1:40 - 2:10

2014 Hodkgin Award* Winner Talk Judith Frydman, Stanford University School of Medicine

2:10 - 2:15

Presentation of The Christian B. Anfinsen Award

2:15 - 2:45

2014 The Anfinsen Award Winner Talk Robert Tycko, NIDDK/NIH

2:45 - 2:50 2:50 - 3:20

Presentation of The Emil T. Kaiser Award 2014 Kaiser Award Winner Talk Carol Fierke, University of Michigan

3:20 - 3:40

Coffee & Refreshments

3:40 - 3:45

Presentation of The Protein Science Young Investigator Award

3:45 - 4:15

2014 Young Investigator Award Winner Talk M. Madan Babu, MRC LMB Cambridge

4:15- 4:20

Presentation of the 2013 Hans Neurath Award**


2013 Neurath Award Winner Talk Jennifer Doudna, University of California - Berkeley


Presentation of the Protein Society Service Awards


Presentation of the Best Poster Awards***


Presentation of the Carl Brändén Award**** Stephen White, University of California - Irvine



DAILY PROGRAM Day 4 - Wednesday, July 30 2014


9:05 - 9:20 9:20 - 9:50

10:05 - 10:35

Welcome & Introduction from session Chair Ubiquitin-dependent Regulation of Proliferation and Differentiation Michael Rape, University of California- Berkeley Young Investigator Talk Investigating the Mechanisms of the Proteasome by Cryo-EM Paula da Fonseca, MRC-LMB, Cambridge Chemical Tools for the Study of Proteolytic Processes Matthew Bogyo, Stanford University


9:05 - 9:20 9:20 - 9:50

9:50 - 10:05

Welcome & Introduction from session Chair Mechanism-based Tuning of Cytokine Receptor Signaling K. Christopher Garcia, The Scripps Research Institute

Young Investigator Talk From Liposomes to Fliposomes: In Vitro Reconstitution of Lipid-Dependent Dual Topology And Post-Assembly Topological Switching of a Membrane Protein William Dowhan, University of Texas Houston Medical School Young Investigator Talk 24

DAILY PROGRAM 10:05 - 10:35

Immune Sensing of Vitamin B Metabolites Douglas Rees, California Institute of Technology / HHMI


Welcome & Introduction Protein Society President , James Bowie

11:05- 11:10

Presentation of the Stein and Moore Award

11:10 - 12:30

2014 Stein and Moore Award WINNER TALK Nikolaus Pfanner, University of Freiburg





Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM Cellular Structures POST 02-224 Dissecting the repetitive C-terminal domain of RNA polymerase II Stephen Fuchs, Mohammad Mosaheb, Summer M. Morrill

POST 02-225 Bacterial Microcompartments: Substrate Transport through Shell Pores Sunny Chun, Chiranjit Chowdhury, Jiyong Park, Kendall N. Houk, Thomas A. Bobik, Todd O. Yeates POST 02-226 Sequence Signature for Recognition of Histone H3 Arginine (R2) Francisca Essel, Suvobrata Chakravarty, Tao Lin

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 03-232 Directed evolution of a highly sensitive peroxidase reporter and application to electron microscopic visualization of MICU1. Stephanie S. Lam, Jeffrey D. Martell, Kimberli J. Kamer, Vamsi K. Mootha, Alice Y. Ting POST 03-233 Characterization of the alphaproteobacteria Wolbachia pipientis protein disulphide machinery reveals a regulatory mechanism absent in gamma-proteobacteria Patricia M. Walden, Maria A. Halili, Julia K. Archbold, Fredrik Lindahl, David P. Fairlie, Kenji Inaba, Jennifer L. Martin

POST 02-227 Microfluidic control and in situ monitoring of microtubule maturation kinetics provide evidence for a new stabilising cap model of dynamic instability Christian Duellberg, Nicholas Cade, David Holmes, Thomas Surrey

POST 03-234 The metal-dependent FAD pyrophosphatase/FMN transferase activity of periplasmic flavin-trafficking protein (Ftp): a potential role in flavoprotein biogenesis Ranjit K. Deka, Chad A. Brautigam, Wei Z. Liu, Diana R. Tomchick, Michael V. Norgard

POST 02-228 Investigating the function of Suppressor of IKK-epsilon Sean W. McKinley, Kenneth F. Lawrence, Jessica K. Bell

POST 03-235 Characterization of hyperthermophilic TyrA enzymes involved in aromatic amino acid biosynthesis Irina Shlaifer, Joanne L. Turnbull

POST 02-229 Structure of BDBT reveals a role for noncanonical FK506 binding protein in regulation of the fly circadian clock. Boadi Agyekum, Jin-Yuan Fan, Anandakrishnan Venkatesan, Jeffrey Price, Samuel Bouyain

POST 03-236 Probing novel antibiotic targets within sialic acid catabolism Rachel A. North, Sarah A. Kessans, Hironori Suzuki, Michael D. Griffin, Renwick C. Dobson

POST 02-230 The PKD-related proteins ANKS6, BICC1, and ANKS3 form a SAM domain interaction network Catherine N. Leettola, Mary J. Knight, Duilio Cascio, James U. Bowie

Chemical Biology & Enzymology POST 03-231 The variations of protein splicing: regulation and mechanism of non-canonical inteins Kenneth Mills, Julie N. Reitter, Michael Nicastri, Jennie Williams, Kathryn Colelli, Michelle Marieni

POST 03-237 The Role of Protein Glycosylation in Laccases from Lentinus sp. Wei-Chun Liu, Manuel Maestre-Reyna, Wen-Yih Jeng, Cheng-Chung Lee, ChihAn Hsu, Tuan-Nan Wen, Andrew H.-J. Wang, Lie-Fen Shyur POST 03-238 FBP17 plays a role in the morphological control by regulating the activity of Rho subfamily GTPase CDC42 Jun Zhang, Lin Ming-ming, Zhang Qianying, Wang Yun-hong, Li Xin POST 03-239 Semi-Synthesis and Applications of Fluorophore/Thioamide pairs Containing Proteins Solongo Batjargal, E. James Petersson POST 03-240 Computational Design of an Unnatural Amino Acid Dependent Metalloprotein with Atomic



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM Level Accuracy Jeremy Mills, Sagar D. Khare, Jill M. Bolduc, Farhad Forouhar, Vikram K. Mulligan, Scott Lew, Jayaraman Seetharaman, Liang Tong, Barry L. Stoddard, David Baker

POST 03-241 Acrolein-modified High Density Lipoproteins Promote Atherogenesis Alexandra Chadwick, Rebecca L. Holme, Yiliang Chen, Kirkwood A. Pritchard, Daisy Sahoo POST 03-242 Control of protein production and virus replication by pharmacological blockade of degron detachment Hokyung K. Chung, Conor Jacobs, Yunwen Huo, Jin Yang, Stefanie A. Krumm, Richard K. Plemper, Rodger Y. Tsien, Michael Z. Lin POST 03-243 Understanding the metabolism of enteric pathogen Campylobacter jejuni Adnan Ayna, Peter Moody POST 03-244 Chitosan-binding modules (CBM32) of a chitosanse from Paenbacillus sp. IK-5 --- Amino acid residues responsible for chitosan binding--Shoko Shinya, Takayuki Ohnuma, Hisashi Kimoto, Hideo Kusaoke, Tamo Fukamizo POST 03-245 Crystal structure of a family GH18 (class V) chitinase from cycad, Cycas revolta ---- structural factors controling the transglycosylation activity of the enzyme Tamo Fukamizo, Naoyuki Umemoto, Takayuki Ohnuma, Toki Taira, Tomoyuki Numata POST 03-246 Split intein mediated peptide cyclization Shubhendu Palei, Henning D. Mootz POST 03-247 Exploring the Morpheein Forms of B. cenocepacia HMG-CoA Reductase Riley Peacock, Michelle Brajcich, Courtney Boyd, Jeffrey Watson POST 03-248 Dynamic Functional Switch in Poliovirus 3C Protease Yan M. Chan, David D. Boehr POST 03-249 Lighting the Cellular Fuel Gauge with Fluorescent Sensors for Imaging Single-Cell Metabolism Mathew Tantama, Juan Ramón MartínezFrançois, Rebecca Mongeon, Gary Yellen

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 03-250 Structural analysis and molecular dynamics of the self-sufficient P450 CYP102A5 and CYP102A1: A combined computational/experimental approach to increase the efficiency of biocatalyst engineering. Maximilian Ebert, Brahm Yachnin, Guillaume Lamoureux, Albert Berghuis, Joelle Pelletier POST 03-251 Isolation and characterization of proline specific dipeptidyl peptidase IV from the Tenebrio molitor larval midgut Valeriya F. Sharikova, Irina Goptar, Yulia Smirnova, Brenda Oppert, Irina Filippova, Elena Elpidina POST 03-252 Characterization of hydrolytic enzymeproducing bacteria isolated from paper mill Manel Ghribi, Fatma Meddeb-Mouelhi, Marc Beauregard POST 03-253 Isolation of NRPS and PKS Gene Clusters from Soil Microbes Danielle N. O'Hara, Connor P. Craig POST 03-254 Treatment of kraft pulp with enzymes for improving beatability and physical properties Li Cui, Fatma Meddeb, Marc Beauregard POST 03-255 Structural and biochemical investigation of the intramolecular interactions of ceramide transfer protein Jennifer Prashek, Seungkyung Kim, Xiaolan Yao POST 03-256 Evaluating interpretation of B-factors for collective motion modeling Edvin Fuglebakk, Nathalie Reuter, Konrad Hinsen POST 03-257 Structure/Function Relationships in Carboxylesterase EstGtA2 from Geobacillus thermodenitrificans Jessica K. Moisan, Fatma Meddeb-Mouelhi, Marc Beauregard POST 03-258 Up-regulation of Rich1 causes S-phase arrest and reduces cell adhesion in epithelial cells Lin Ming-ming, Zhang Qian-ying, Wang Yun-hong, Li Xin, Zhang Jun POST 03-259 Tracking wood fibers decrystallization with carbohydrate binding module Yannick Hébert-



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

Ouellet, Vinay Khatri, Fatma Meddeb-Mouelhi, Marc Beauregard

organisms Jorge D. Garcia, David G. Mendoza-Cozatl, Rafael Moreno-Sánchez

POST 03-260 Evolution of Structure and Mechanistic Divergence in Di-Domain Methyltransferases from Nematode Phosphocholine Biosynthesis Soon Goo Lee, Joseph M. Jez

POST 03-270 Nonlocal effects of metal ion binding at the catalytic site of a protein-DNA complex. Kaustubh Sinha, Michael R. Kurpiewski, Sahil Sangani, Andrew D. Kehr, Gordon S. Rule, Linda Jen-Jacobson

POST 03-261 ERK1 and 2 - Exploring Isoform Differences Jen Liddle, Natalie Ahn

POST 03-271 Recognition and Conversion of Flunitrazepam by Cyp3A4 is Altered by Caffeine Mark Volker, Lauren Sparks, Larry R. Masterson

POST 03-262 Chemical Modification of MitoNEET Megan Laffoon, Michael Menze, Mary Konkle POST 03-263 Biochemical basis for the extended spectrum Cephalosporinase activity of a clinical AMPC β-Lactamase variant Jozlyn R. Clasman, Brianna M. Jackman, Cynthia M. June, Rachel A. Powers, David A. Leonard POST 03-264 Common substitutions enhance the Carbapenemase activity of OXA-51-Like Class D βLactamases from Acinetobacter SPP. Joshua M. Mitchell, David A. Leonard POST 03-265 Exploring the Potential of Arylboronic Acids as Inhibitors of OXA-24 β-lactamase Josephine P. Werner, Rachel A. Powers POST 03-266 Structure-based Discovery of a Novel Inibitor of OXA-1 β-lactamase Leslie A. Wyman, Neil V. Klinger, Rachel A. Powers

POST 03-272 Fluorogenic Probes for Mycobacterial Esterase Profiling Katie Tallman, Kimberly Beatty POST 03-273 Visualizing cell interactions with genetically encoded bioluminescent tools Krysten A. Jones, David Li, Elliot E. Hui, Mark A. Sellmyer, Jennifer A. Prescher POST 03-274 Specific Inhibition of Enolase From Entamoeba histolytica Normande Carrillo-Ibarra, Cesar Augusto Sandino Reyes-Lopez, Jose Correa-Basurto, Elibeth Mirasol Melendez, Claudia Guadalupe Benitez Cardoza POST 03-275 Effects of Intrauterine and Extrauterine Exposure to 1800 MHz GSM-Like Radiofrequency Radiation on Liver Regulatory Enzymes Activities in one-month-old male New Zealand Rabbits Nuray N. Ulusu

POST 03-267 Kinetic and Biochemical Investigations of Thermostable Acid Phosphatase from Zea mays and Glycine max cotyledons: A therapeutically important enzyme Nidhee Chaudhary, Subhash Chand, Nameet Kaur

POST 03-276 Imaging Protein-Protein Interactions, Post-Translational Modifications, and Non-Protein Biomolecules by Correlative Light and Electron Microscopy John T. Ngo, Daniela Boassa, Stephen R. Adams, Thomas J. Deerinck, Sakina F. Palida, Varda Lev-Ram, Mark H. Ellisman, Roger Y. Tsien

POST 03-268 Investigation of the dynamic amino acid networks in a (β/α)8 barrel enzyme Jennifer M. Axe, Kathleen F. O'Rourke, Eric M. Yezdimer, Nicole E. Kerstetter, Xianrui Yuan, David D. Boehr

POST 03-277 Evaluating the role of peroxidatic reducing substrates in an unusual catalase activity of catalase-peroxidases Olive J. Njuma, Elizabeth Ndontsa, Douglas Goodwin

POST 03-269 An uncommon phytochelatin synthase gives hints on how to improve their catalytic efficiency on heavy metal hyperaccumulator

POST 03-278 Pyrrolysine-Inspired Protein Cyclization Marianne M. Lee, Tomasz Fekner, Jia Lu, Michael K. Chan



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 03-279 Functional sectors involved in thermal stability and activity in Beta-Glucosidases Fabio K. Tamaki, Larissa C. Textor, Igor Polikarpov, Sandro R. Marana POST 03-280 A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles Chu Wang, Benjamin F. Cravatt

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 03-288 Investigating Molecular Determinants that Modulate the Preference of I kBs for Specific NF kBs James D. Marion, Elizabeth A. Komives POST 03-289 The examination of the MALDI-TOF-MS analysis of the proteins and the peptides by use of the sinapic acid derivatives as the new matrix. Narumi Hirosawa, Takeshi Sakamoto, Yasushi Uemura, Yasushi Sakamoto

POST 03-281 Protein stabilization and prevention of protein aggregation by fungal sucrase of Termitomyces clypeatus and application in biotechnology and biomedical research Suman Khowala, Sudeshna Chowdhury, Sanjeeta Tamang, Sangita Majumdar, Rajib Majumder

POST 03-290 Sequential Phosphorylation of SIKE by TBK1 Hyejin Park, SoHo Kim, Jessica Bell, Ellis Bell

POST 03-282 Using Multiwavelength Collisional Quenching to Investigate the Effects of Arginine and Inhibitors on inducible Nitric Oxide Synthase Rachel Jones, Ellis Bell

POST 03-292 Multiwavelength Collisional Quenching to Study Ligand Protein Interactions in Glutamate Dehydrogenase Chun Li, Ellis Bell

POST 03-283 Enzyme Active Sites May Extend Further than We Thought Lisa Ngu, Penny J. Beuning, Mary Jo Ondrechen POST 03-284 Nucleotide pyrophosphatase/phosphonuclease possesses the zeatin cis-trans isomerase activity in vitro Tomáš Hluska, Michaela Baková, René Lenobel, Marek Šebela, Petr Galuszka POST 03-285 Exploring the Trigger for Cooperativity at the Subunit Interface of Malate Dehydrogenase Jacqunae Mays, Ellis Bell POST 03-286 Redox control of Protein Arginine Methyltransferase 1 (PRMT1) activity Yalemi Morales, Damon Nitzel, Owen Price, Shanying Gui, Joan Hevel POST 03-287 Structural and biochemical characterization of Thielavia terestris Cutinase (TtC) Abhijit N. Shirke, Danielle A. Basore, Evan Baugh, An Su, Glen Butterfoss, George I. Makhatadze, Christopher Bystroff, Richard A. Gross

POST 03-291 Effects of Phosphorylation on the Structure & Stability of SIKE Clara Kerckhove, Jessica Bell, Ellis Bell

POST 03-293 Mapping the binding sites of class D beta-lactamases for inhibitor design and discovery Joshua M. Mitchell, Rachel Powers POST 03-294 Determining the Effects of MitoNEET on Cellular Dehydrogenase Activity Sarah Banister, Matthew Woodruff, Paige Birge, Michael Menze, Mary Konkle POST 03-295 In silico and kinetic studies to verify the potency of α-glucosidase inhibitors isolated from Morus alba L. Shakeel Ahmad, Akash Chaudhary, Shadab Ahmad, Mohd. Tashfeen Ashraf POST 03-296 Molecular Dynamic Studies of the Reductase Domain of Polyketide Synthase from the Myxobacterium Stigmatella Aurantiaca Andrew J. Schaub, Jesus Barajas, Ray Luo, Shiou-Chaun (Sheryl) Tsai POST 03-297 Mechanisms regulating ribosome biogenesis by AKT and c-MYC Simone Woods, Colin House, Gretchen Poortinga, Ross Hannan, Katherine Hannan



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 03-298 Highly selective peptide substrates for the assay of cysteine peptidases from the C1 family Elena N. Elpidina, Irina Y. Filippova, Tatiana A. Semashko, Elena A. Vorotnikova, Valeriya F. Sharikova, Konstantin S. Vinokurov, Lyndsey Fallis, Brenda Oppert POST 03-299 Collagen Determination and Its Application for the Authentication of Tortoise Shell Linqiu Li, Hon-Yeung Cheung POST 03-300 The B-domain-half-deleted recombinant Coagulation factor VIII (FVIII) shows much higher coagulation activity compared with B-domain-deleted recombinant ones Zhang Jun, Zhu Chong-yang, Wen Quan, Lin Ming-ming POST 03-301 Novel Heterotetrameric Enzymes in Cholesterol Metabolism from Mycobacterium tuberculosis Meng Yang, Kip Guja, Miguel Garcia-Diaz, Suzanne Thomas, Nicole Sampson *

POST 03-302 Energetic contribution of n→π interactions to PII conformations of model peptides Liu He, Zhengshuang Shi POST 03-303 The secretion and expression of a series of B domain truncated recombinant coagulation factor VIII in hepatocyte Zhang Jun, Zhu Chong-yang, Zhang Qian-ying, Lin Ming-ming, Wen Quan, Li Xin POST 03-304 Crystal structure studies of dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24. Yasumitsu Sakamoto, Yoshiyuki Suzuki, Ippei Iizuka, Chika Tateoka, Saori Roppongi, Mayu Fujimoto, Koji Inaka, Hiroaki Tanaka, Mika Masaki, Kazunori Ohta, HIrofumi Okada, Takamasa Nonaka, Yasushi Morikawa, Kazuo T. Nakamura, Wataru Ogasawara, Nobutada Tanaka POST 03-305 Protein Recognition by Multivalent Fluorescent Molecular Sensors David Margulies POST 03-306 Assessing Student Understanding of Foundational Concepts of Protein Structure and Function Ellis Bell

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 03-307 Dietary condensed tannins and their effect on microbial protein and theoretical methane yield in ruminal fermentation on cattle in the northern Mexico Ericka Fabiola Luisillo-Quiñones, Gerardo A. Pámanes-Carrasco, Christian A. HernándezVázquez, Zaira J. Romo-Astorga, Esperanza Herrera POST 03-308 Regulation and Inter-Domain Communications in C-terminal Src Kinase (CSK) Sulyman Barkho, Levi C. Pierce, Joseph A. Adams, Patricia A. Jennings POST 03-309 Site-Specific Molecular Recognition of Proteins by Synthetic Receptors Adam Urbach POST 03-310 Structure of Dihydromethanopterin Reductase: Redox Transfer in a Cubic Protein Cage 1 Dan E. McNamar , Duilio Cascio, Julien Jorda, Cheene Bustos, Tzu-Chi Wang, Madeline E. Rasch, Thomas A. Bobik, Todd O. Yeates POST 03-311 The Linkage Between Folding Cooperativity and Allostery: Glutamate Dehydrogenase Angela Tata, Grace Kingdom, Ellis Bell POST 03-312 Hydrogen exchange of disordered proteins in living cells Austin E. Smith, Zijian Zhou, Gary J. Pielak POST 03-313 Assessment of UCH-L3 Substrate Selectivity using Engineered Ubiquitin Fusions with Variable Linker Lengths Peter Suon, John J. Love POST 03-314 Identification, Characterization, and Modification of Fatty Acid Alkyl Esterases Found in Staphylococcus aureus Benjamin D. Saylor, John J. Love Protein Degradation POST 04-315 Apoptotic protein Bax is regulated by the ubiquitin-proteasome pathway Kwang-Hyun Baek, SoRa Kim, Jin-Ok Kim POST 04-316 Discovery and characterization of small molecule fragments that bind and inhibit the



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

Ubiquitin Specific Protease 7 (USP7) Paola Di Lello, Terry Crawford, Kurt Deshayes, Joy Drobnick, Jake Drummond, James Ernst, Lorna Kategaya, Michael Kwok, Cuong Ly, Till Maurer, Jeremy Murray, Chudi Ndubaku, Rich Pastor, Lionel Rouge, Structural Biology Expression group, Vickie Tsui, Ray Zhao, Kerry Zobel, Ingrid Wertz POST 04-317 Polyglycine Hydrolases Secreted by Pathogenic Fungi Todd A. Naumann, Donald T. Wicklow, Todd J. Ward, Michael J. Naldrett, Neil P. Price POST 04-318 Higher-order assembly architecture of the AAA+ protease Lon reveals a new regulatory mechanism for substrate specificity Tejas Kalastavadi, Ellen Vieux, Breann Brown, Tania Baker POST 04-319 A robust assay for protein unfolding by AAA+ molecular machines Vladimir Baytshtok, Tania A. Baker, Robert T. Sauer POST 04-320 Mitochondrial Lace1 ATPase Lukas Stiburek, Jana Cesnekova, Josef Houstek, Jiri Zeman POST 04-321 The structure of the human hybrid proteasome Edward Morris, Fabienne Beuron, Paula da Fonseca POST 04-322 Are the Precursor Sequences of Thiol Proteases Related to Thiol Protease Inhibitors Meron Tarekegn, David Harry, Kelsey Kines, Ellis Bell POST 04-323 Thiol Proteases & Thiol Protease Inhibitors in C Elegans Kelsey Kines, David Harry, Meron Tarekegn, Ellis Bell POST 04-324 Structural Analysis of Poly-SUMO Chain Recognition by RNF4-SIMs Domain Chia-Hsiuan C. Kung, Mandar T. Naik, Hsiu-Ming Shih, Tai-huang Huang POST 04-325 Misfolded Proinsulin Retrotranslocation for Proteasome-dependent Degradation in the Cytosol Can Be Modulated by Altering the

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

Endoplasmic Reticulum Lumenal Composition PenJen Lin Frontier High-Throughput Techniques

POST 05-326 Virotrap: Abducting Protein Complexes from Mammalian Cells Sven Eyckerman, Kevin Titeca, Emmy Vanquickelberghe, Annick Verhee, Noortje Samyn, Delphine De Sutter, Evy Timmerman, Kris Gevaert, Jan Tavernier POST 05-327 Global measurement of protein localization in C. elegans with tissue and subcellular specificity Aaron Reinke, Eric Bennett, Emily Troemel POST 05-328 Broad Scope and Coverage of Functionally Relevant Groups for Sequences in the Enolase Superfamily Brian Westwood, Stacy Knutson, Janelle Leuthaeuser, Patricia Babbitt, Jacquelyn Fetrow POST 05-329 A molecular toolkit for single molecule peptide sequencing Jim Havranek, Ben Borgo POST 05-330 Novel Solubility Fusion Partners High Throughput System to Produce Soluble Proteins Saurabh Sen, Eric Steinmetz, Sally Floyd, David Mead, Mark Maffitt POST 05-331 Elucidating the global metabolic regulatory role of prokaryotic enzyme posttranslational modifications through systems biology analysis Nathan E. Lewis, Roger Chang, Chen Yang, Hooman Hefzi, Bernhard Palsson, George Church

Membrane Proteins & Receptors POST 06-332 What’s on the Menu: Identification of the Hydrocarbon Transport Systems as a first step in Marine Oil-Degradation by Alcanivorax borkumensis Swapnav Deka, Chad Brautigam POST 06-333 Keeping in touch: T-cadherin impedes dissociation of adiponectin receptor 1 dimers



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM Tobias Leimer, David Kosel, Karin Mörl, Barbara Ranscht, Annette G. Beck-Sickinger

POST 06-334 A new rigidity-based model for allosteric communication in G-protein coupled receptors. Adnan Sljoka POST 06-335 Keeping it simple: The construction of biologically active proteins with minimal chemical diversity Erin N. Heim, Jez L. Marston, Kelly M. Chacon, Lisa M. Petti, Daniel DiMaio

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 06-343 Development of a quantitative, realtime, label-free assay for ligands interacting with the HIV Envelope glycoprotein in virus-like particles Jennifer Seedorff, Edward Berger POST 06-344 Silicon transport in diatoms: the key to unlocking their full potential? Sarah Ratcliffe, Michael Knight, Laura Senior, Paul Curnow

POST 06-336 Characterization of the second immunoglobulin domain (Ig2) of the human receptor CRTAM/CD355 and its role in the formation of dimer Juan C. Barragan-Galvez, Luis Brieba-Castro, Vianney Ortiz-Navarrete

POST 06-345 Mechanistic studies of the Alternative Complex III from the photosynthetic bacterium Chloroflexus aurantiacus Erica L. Majumder, Robert E. Blankenship POST 06-346 Structure and function of the diatom silicon transporter Michael Knight, Laura Senior, Sarah Ratcliffe, Paul Curnow

POST 06-337 Expression, purification and functional refolding of human olfactory receptor expressed in Escherichia coli Heehong Yang, Sae Ryun Ahn, Tai Hyun Park

POST 06-347 Intermolecular interactions between the intracellular domains of Arabidopsis CRINKLY4 (ACR4) receptor-like kinase and homologs. Matthew R. Meyer, Shweta Shah, Gururaj A. Rao

POST 06-338 The importance of CD4 allostericity for interaction with HIV glycoprotein 120 Nichole M. Cerutti, Vinesh Jugnarain, Alexio Capovilla

POST 06-348 Expression, purification and reconstitution of the aromatic acid transporter PcaK Christian Pernstich, Laura Senior, Katherine A. MacInnes, Marc Forsaith, Paul Curnow

POST 06-339 Structural characterization of the major pilin subunit from the bacterial nanowires of Geobacter sulfurreducens Patrick N. Reardon, Karl T. Mueller POST 06-340 X-ray structures reveal bent conformation for all CNTN family members Roman M. Nikolaienko, Samuel E. Bouyain

POST 06-349 Insight into receptor-active conformation of apolipoprotein E revealed by XL-MS: new clues for a putative importance of helix 4 elongation in receptor recognition. Nicolas Henry, Stéphanie Deroo, Florian Stengel, Eva-Maria Krammer, Rouslan Efremov, Guy Vandebussche, Martine Prevost, Ruedi Aebersold, Vincent Raussens

POST 06-341 Visualization of HIV-1 envelope glycoprotein in live cells by labeling it with GFP in the gp120 subunit Shuhei Nakane, Aikichi Iwamoto, Zene Matsuda

POST 06-350 The Little Lipid That Could: Elucidating the Effects of Small Amounts of Phosphatidic Acid on the α-Synuclein Membrane Interaction Sara K. Hess, Jennifer C. Lee

POST 06-342 Hemifusion induced by the HA2 subunit of influenza virus hemagglutinin: Respective major, moderate, and minor contributions of the soluble ectodomain, fusion peptide, and transmembrane regions. Punsisi Upeka Ratnayake

POST 06-351 Structure-based Analysis of Protein Modifications in G Protein Signaling - A New Approach to Prioritize PTMs in a Protein Complex Henry Dewhurst, Shilpa Choudhury, Matthew P. Torres



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 06-352 Molecular characterization of the Arabidopsis CRINKLY4 receptor-like kinase (ACR4) intracellular domain coupled with transmembrane domain Shweta Shah, Matthew R. Meyer, Gururaj Rao POST 06-353 Improved fusion protein strategies for crystallization of G-protein coupled receptors Yi Zheng, Ling Qin, Lauren Holden, Chunxia Zhao, Tracy Handel POST 06-354 Biochemical characterization of interaction between the Arabidopsis receptor-like kinase (ACR4) and phosphatase, PP2A3. Priyanka Sandal, Matthew R. Meyer, Petra E. Gilmore, Ried Townsend, Aragula G. Rao POST 06-355 The proteomic signature of the LeukoLike Vector unveils the presence of molecules able to improve self-tolerance of the drug delivery systems Claudia Corbo, Alessandro Parodi, Roberto Molinaro, Michael Evangelopoulos, David A. Engler, Shilpa Scaria, Francesco Salvatore, Anthony C. Engler, Ennio Tasciotti POST 06-356 Crystal structure of phosphate-bound V1-ATPase of Enterococcus hirae Kano Suzuki, Kenji Mizutani, Yoshiko Ishizuka-Katsura, Takaho Terada, Mikako Shirouzu, Shigeyuki Yokoyama, Ichiro Yamato, Takeshi Murata POST 06-357 Structural analysis for the interaction of sialic T antigen glycopeptide of HSV-1 with entry receptor PILRα Takao Nomura, Jiro Sakamoto, Fumina Oosaka, Kosuke Kakita, Atsushi Furukawa, Masahiro Anada, Shunichi Hashimoto, Kimiko Kuroki, Toyoyuki Ose, Hisashi Arase, Takashi Saitoh, Katsumi Maenaka POST 06-358 Structural snapshots of the α-helical pore-forming toxin FraC reveal the molecular basis of its activation in membranes. Koji Tanaka, Jose M. Caaveiro, Kouhei Tsumoto POST 06-359 Membrane proteins bind lipids selectively to modulate their structure and function Eamonn Reading, Art Laganowsky, Timothy M. Allison, Carol V. Robinson

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 06-360 NMR Studies of G-Protein Coupled Receptors Jasmina Radoicic, Sang Ho Park, Anna De Angelis, Bibhuti Das, Sabrina Berkamp, Stanley J. Opella POST 06-361 Structure Determination of Vpu from HIV-1 by NMR Hua Zhang, Eugene Lin, Stanley Opella POST 06-362 NMR studies of a GPCR with ligand bound: CXCR1 and Interleukin-8 Sabrina Berkamp, Anna De Angelis, Bibhuti Das, Sang Ho Park, Mitchell J. Zhao, Jasmina Radoicic, Stanley J. Opella POST 06-363 Membrane Protein Folding Stability and Kinetics in Bilayers Yu-Chu Chang, James U. Bowie POST 06-364 Effects of Mercury Ion on the Structure and Function of E. coli Aquaporin Z Qingsong Lin, Hu Zhou, Lili Wang POST 06-365 Characterization of the calcium and membrane binding properties of the hearing related protein otoferlin Colin P. Johnson, Murugesh Padmanarayana, Nicole Hams, Ryan Mehl POST 06-366 TOWARDS A CRYSTAL STRUCTURE OF THE HIV-1 MEMBRANE PROTEIN, Vpu Arpan Deb, William Johnson, Dustin Srinivas, Liqing Chen, Petra Fromme, Tsafrir Leket-Mor POST 06-367 Conformational Landscape Governing the Constitutive Activity of GPCRs Ravinder Abrol, Caitlin E. Scott, William A. Goddard III, Kwang H. Ahn, Debra E. Kendall POST 06-368 Cell-free translation systems for biophysical and biochemical characterization of proteins and protein complexes Feliza A. Bourguet, Craig D. Blanchette, Nicholas O. Fischer, Paul J. Jackson, Masood Z. Hadi, Wei He, Brian K. Kay, Kit S. Lam, Ted A. Laurence, Zachary Rogers, John C. Voss, Matthew A. Coleman POST 06-369 Membrane proteins can have high kinetic stability Robert Jefferson, Tracy Blois, James Bowie POST 06-370 Advancing membrane protein crystallography using the LCLS Mark Hunter, Brent W.



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

Segelke, Nadia Zatsepin, Matt Coleman, W. Henry Benner, Stefan Hau-Riege, Ching-Ju Tsai, Xiao-dan Li, Bill Pedrini, Gebhard Schertler, Matthias Frank Translation & Folding POST 07-371 Elucidating the structure and dynamics of small heat shock protein complexes using a hybrid approach . Michelle Heirbaut, Steven Beelen, Esther Martin, Frederik Lermyte, Tim Verschueren, Frank Sobott, Sergei Strelkov, Stephen Weeks POST 07-372 Achieving folding cooperativity in a physiological environment Nathan Gardner, Chiwook Park POST 07-373 n→π* Interactions in Protein Structure and Folding Robert W. Newberry, Ronald T. Raines POST 07-374 Structural and dynamic insights about unfolding intermediates in four amyloidogenic immunoglobulin light chains. Gilberto Valdes-Garcia, Cesar Millan-Pacheco, Nina Pastor POST 07-375 Mechanistic insights into the folding of the trefoil-knotted proteins Nicole C. Lim, Sophie E. Jackson POST 07-376 Intermolecular Interactions in a Blood Clotting Mechanism Led to Protein Folding Theory Harold A. Scheraga POST 07-377 Molecular mechanism of nuclear transport mediated by flexible amphiphilic proteins Shigehiro Yoshimura, Msahiro Kumeta, Kunio Takeyasu POST 07-378 Native state dynamics of the prion protein probed by Hydrogen exchange and mass spectrometry Roumita Moulick, Jayant B. Udgaonkar POST 07-379 Mutations in the bacterial ribosomal protein S12 influence aminoglycoside antibiotic and ribosome dynamics Joanna Panecka, Cameron Mura, Joanna Trylska

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 07-380 Curious characteristics of a mutant chaperonin GroEL with multiple cysteines in the central cavity Tomohiro Mizobata, Shuhei Fusa, Masashi Ikeda, Kunihiro Hongo, Yasushi Kawata POST 07-381 Untangling ribosome biogenesis using quantitative mass spectrometry, electron microscopy and chemical probing Joseph H. Davis, Nikhil Jain, Admad Jomaa, Joaquin Ortega, Robert Britton, James R. Williamson POST 07-382 Study of E.coli GroEL using stoppedflow analysis and circular permutation Toshifumi Mizuta, Tatsuya Uemura, Kunihiro Hongo, Yasushi Kawata, Tomohiro Mizobata POST 07-383 A Global machine learning-based scoring function for protein structure prediction Andrzej Kloczkowski, Eshel Faraggi POST 07-384 Effect of Mg ‘2+ in the structure and thermal stability of enolase from Trichomonas vaginalis Elibeth Mirasol Meléndez, Jorge L. Rosas Trigueros, Luis G. Brieba de Castro, Rossana Arroyo Verástegui, Claudia G. Benítez Cardoza POST 07-385 Controlling nanostructures of insulin amyloid fibrils using metal ions Misaki Yokoyama, Yoshito Furuie, Motonari Tsubaki, Hiroshi Hori, Takamasa Nishida, Kazuo Eda, Eri Chatani POST 07-386 Structural and Thermodynamic Characterization of the X−Dimer of Human PCadherin: Implications for Homophilic Cell Adhesion Shota Kudo, Jose Caaveiro, Satoru Nagatoishi, Takao Hamakubo, Tatsuhiko Kodama, Tadashi Matsuura, Yukio Sudou, Kouhei Tsumoto POST 07-387 A Novel Protein Fold within the Nterminus of a Streptococcal Adhesin Mediates Proper Folding, Function, and Stability Kyle P. Heim, Paula Crowley, Shweta Kailasan, Robert McKenna, Jeannine Brady POST 07-388 Folding of Collagen Heterotrimeric Helices via Cation- π Interactions Jia-Cherng Horng, Chu-Harn Chiang, Tang-Chun Kao



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 07-389 Loss of Conformational Entropy in Protein Folding Calculated using Realistic Ensembles (with implications to NMR-based calculations) Tobin R. Sosnick, Michael C. Baxa, Esmael Haddadian, Karl F. Freed POST 07-390 Rare example of a protein where an isolated domain is more stable than the full-length Swati Bandi, Surinder Singh, Krishna Mallela POST 07-391 Analysis of several monomeric mutants of Triosephosphate Isomerase. MIsrain E. Gurrola Acosta, Maria E. Chánez, Edgar Vazquez Contreras POST 07-392 Probing the Denatured State of a Knotted Protein David J. Burban, Dominique Capraro, Joanna Sulkowska, Patricia Jennings POST 07-393 Transitions between different side-chain conformations in hydrophobic residues. Diego Caballero, Corey S. O'Hern, Lynne Regan POST 07-394 N-terminal domain of Luciferase controls misfolding avoidance Zackary N. Scholl, Weitao Yang, Piotr Marszalek POST 07-395 Direct measurement of the multimer stabilization in the mechanical unfolding pathway of Streptavidin Zackary N. Scholl, Piotr Marszalek POST 07-396 ATPase domain of DnaK, Escherichia coli Hsp70 molecular chaperone, experiences pHdependent ATPase activity upon linker binding due to Asp194 and Glu171 Rahmi Imamoglu, Umut Gunsel, Bulent Balta, Gizem Dinler-Doganay Protein Evolution POST 08-397 Structural Evidence for Antigen Receptor Evolution Romain Rouet, David Langley, Daniela Stock, Daniel Christ POST 08-398 Computational prediction and experimental characterization of a “size switch type

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

repacking” during the evolution of dengue envelope protein domain III (ED3) Montasir Elahi, Keiichi Noguchi, Masafumi Yohda, Hiroyuki Toh, Yutaka Kuroda POST 08-399 Evolutionary Exploitation of Promiscuous NSAR/OSBS Enzymes Andrew McMillan, Denis Odokonyero, Mariana Lopez, DaNae Woodard, Ashley Brizendine, Margaret E. Glasner POST 08-400 Active site profile-based clustering of enolase structures and sequences Janelle Leuthaeuser, Stacy Knutson, Brian Westwood, Patricia Babbitt, Jacquelyn Fetrow POST 08-401 Active site profile-based clustering of the peroxiredoxin superfamily Angela Harper, Janelle Leuthaeuser, Jacquelyn Fetrow States POST 08-402 Bioinformatics and Network Analysis of Lipocalin Superfamily Nardos Sori, Lesley H. Greene POST 08-403 A New Evolutionary Subclass of HMGCoA Reductases Jeffrey Watson, James Palmer POST 08-404 Ebola protein VP35 exploits evolutionary constraints in host defense kinase PKR to evade translational blockade Maayan Eitan-Wexler, Raymond Kaempfer POST 08-405 The substrate specificity “lock:” evolutionary epistasis in Apicomplexan lactate and malate dehydrogenases. Brian Beckett, Michelle Y. Fry, Douglas L. Theobald POST 08-406 Multi-level Iterative Functional Clustering of Glutathione Transferase Superfamily Kiran Kumar, Janelle Leuthaeuser, Brian Westwood, Jacquelyn Fetrow POST 08-407 Structural Analysis of Toxin/immunity Complexes in Contact-Dependent Growth Inhibition Systems Parker M. Johnson, Robert P. Morse, David A. Low, Christopher S. Hayes, Celia W. Goulding



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 08-408 Automated Functional Clustering of the Crotonase Superfamily based on active site motif sequences Julia Hayden, Janelle Leuthaeuser, Brian Westwood, Jacquelyn Fetrow POST 08-409 Domain exchange between membrane and soluble proteins contributes to expand intercellular communication network Hyun Jun Nam, Inhae Kim, James U. Bowie, Sanguk Kim POST 08-410 Utilization of an Iterative Clustering Method to group Radical SAMs in FunctionallyRelevant Ways Gabrielle B. Shea, Janelle B. Leuthauser, Brian Westwood, Jacquelyn S. Fetrow POST 08-411 Directed evolution of duplicated Qbeta RNA replicases in liposome Keisuke Uno, Takeshi Sunami, Yasuaki Kazuta, Norikazu Ichihashi, Tetsuya Yomo POST 08-412 Comparative Analysis of Posttranslational Modification Hot Spots in Phylogenetically Distant Heterotrimeric G proteins Shilpa Choudhury, Henry Dewhurst, Matthew P. Torres

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 08-418 Synuclein and the Coelacanth James M. Gruschus Proteins in Altered States POST 09-419 Storage of Environmental Information into Biological Structures: Temperature-dependent Fibrillar Polymorphism of α-Synuclein Ghibom Bhak, Seung Ryeoul Paik POST 09-420 Reversible Polymeric Fibers made from Low-Complexity Sequences function as a Foundation of RNA Metabolism Masato Kato, Steven McKnight POST 09-421 Flavone derivatives as inhibitors of insulin amyloid-like fibril formation Ricardas Malisauskas, Akvile Botyriute, Vytautas Smirnovas POST 09-422 Phosphorylation Releases Constraints to Domain Motion in ERK2 Yao Xiao, Thomas Lee, Michael P. Latham, Lisa R. Warner, Akiko Tanimoto, Arthur Pardi, Natalie G. Ahn

POST 08-413 The evolution of caffeine synthases in Theobroma cacao Craig D. Thulin

POST 09-423 Using mass spectrometry to define how the small HSP chaperones protect substrates from aggregation Keith Ballard, Heather O'Neill, Wenzhou Li, Vicki Wysocki, Elizabeth Vierling

POST 08-414 Evolution in a test tube yields de novo enzymes with unusual structure and dynamics Burckhard Seelig, Frank A. Chao, Aleardo Morelli, John C. Haugner, Lewis Churchfield, Gianluigi Veglia

POST 09-424 Mechanically resistant conformations in amyloid β and α-synuclein Sigurdur Æ. Jònsson, Simon Mitternacht, Anders Irbäck

POST 08-415 Quantitative Imaging with Amersham’TM Imager 600 Erik Bjerneld, Erika Svensson, Johan Johansson, Maria Winkvist, Hakan Roos POST 08-416 Solution NMR Structure of the SLED Domain from Scml2 (Sex Comb on Midleg-like 2) - a New Domain on Epigenetic Landscape. Irina Bezsonova POST 08-417 Monitoring the effect of extensive genetic drift on the evolution of novel enzyme function Linda Jäger, Peter Kast, Donald Hilvert

POST 09-425 Structural Determinants of Amyloid Fibril Formation in Triosephosphate Isomerase. Edson N. Carcamo-Noriega, Gloria Saab-Rincón POST 09-426 Developing quantitative NMR methods for predicting residue specific helicity of MetOλ unfolded state Kan Li, Roy Hughes, Terrence G. Oas POST 09-427 Towards in vivo NMR: structural studies of prion fibrils assembled in native environments at endogenous levels Kendra K. Frederick, Vladimir K. Michaelis, Bjorn Corzilius, Ta-Chung Ong, Angela C. Jacavone, Robert G. Griffin, Susan Lindquist



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 09-428 Structural and Ligand Binding Properties of Dimeric Horse Myoglobin Satoshi Nagao, Hisao Osuka, Takuya Yamada, Takeshi Uni, Yasuhito Shomura, Kiyohiro Imai, Yoshiki Higuchi, Shun Hirota POST 09-429 Study of interactions of amyloidogenic regions of Streptococcus mutans adhesin P1 by Nuclear Magnetic Resonance Wenxing Tang, L. Jeannine Brady, Joanna R. Long POST 09-430 Identification and Characterization of Functional Amyloids in Streptococcus mutans Richard N. Besingi, L. Jeannine Brady POST 09-431 Achieving selectivity in the Hippo pathway: An investigation of the inter-domain communication in TEAD transcription factors Priyanka Rauniyar, Sudha Veeraraghavan POST 09-432 Structural Projection of PTMs (SPoP): A toolkit for providing structural and functional context for sequence-specific protein features Henry Dewhurst, Matthew P. Torres POST 09-433 Investigating the Molecular Basis of Curli Amyloid Inhibition by Protein and Chemical Chaperones Neha Jain, Margery L. Evans, Matthew R. Chapman POST 09-434 The effects of mutations on the aggregation propensity of human prion-like domains Eric D. Ross, Kacy Paul, Sean Cascarina POST 09-435 Competition or forced collaboration? An unusual pattern of self-propagating polymorphism of insulin amyloid fibrils upon seeding with mixed templates. Wojciech Dzwolak, Weronika SurmaczChwedoruk POST 09-436 Nanoscale Organization of Protein Molecules within Amyloids and Prions Samrat Mukhopadhyay POST 09-437 A Temperature Sensitive Parkinsonian Mutation in DJ-1 Enhances Protein Dynamics in a Metal-dependent Fashion Nicole M. Milkovic, Steven

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

Halouska, Jonathan Catazaro, Sara Basiaga, Robert Powers, Mark A. Wilson POST 09-438 Analytical Tools in Decision Making of Suitability of Monoclonal Antibodies for Immunodiagnostic Assays Kevin R. Rupprecht, Tracey D. Rae, Svetoslava D. Gregory, Na Yang, Janet M. Bergsma, Ryan M. Bonn, Martin R. Lopez, Panfilo F. Ozaeta, Cheng Zhao, Carol S. Ramsay, Jeffrey R. Fishpaugh POST 09-439 Spectroscopic Investigation of the Structural Perturbations of Tau Microtubule Binding Domains at the Golgi Membrane as Illustrated by Membrane Mimics Lauren E. Sparks, Larry R. Masterson Proteins in Disease & Therapeutics

POST 10-50 Coupling the Antimicrobial Action of a Novel Un-natural Cationic Amphiphilic Polyproline Helix with its Cell Penetrating Ability to Target Intracellular Bacteria Manish Nepal, Jean Chmielewski POST 10-51 Rational design of the furin cleavage site of an anti-CD22 recombinant immunotoxin based on Pseudomonas exotoxin A John E. Weldon, Ira Pastan POST 10-52 Structural and functional analysis of lysosomal phospholipase A2, a close homolog of lecithin-cholesterol acyltransferase Alisa Glukhova, Robert J. Kelly, Vania Hinkovska-Galcheva, James A. Shayman, John J. Tesmer POST 10-53 Structural Basis for Antigen Recognition of a Tumor Specific Therapeutic Antibody Reza Movahedin, Teresa M. Brooks, Cory L. Brooks POST 10-54 Neutralization of Listeria monocytogenes by Single Domain Antibodies Ian Huh, Robert Gene, Jyothi Kumaran, Cory Brooks POST 10-55 Understanding regulation of P-Rex1, an enhancer of metastatic potential Jennifer Cash, Ellen Davis, John J. Tesmer



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 10-56 Expression and Purification of Functionally Active Recombinant Plasmepsin 9 from Plasmodium falciparum Folasade M. Olajuyigbe POST 10-57 Characterizing HER2 gene variation at the protein level to address racial disparities in breast cancer mortality Wei He, Matthew Saldana, Tiffany Scharadin, Steven Hoang-Phou, Denise Trans, Dennis Chang, Kermit Carraway, Paul Henderson, Matthew A. Coleman POST 10-58 The interaction between the H. pylori oncoprotein CagA and human pro-apoptotic ASPP2 is distributed throughout both proteins and involves intrinsically disordered regions Tali H. Reingewertz, Anat Iosub-Amir, Daniel A. Bonsor, Assaf Friedler, Eric J. Sundberg POST 10-59 Is there a common structural basis for amyloidosis toxicity? A new receptor-mediated mechanism of pancreatic islet amyloidosis-induced beta-cell toxicity in type 2 diabetes Andisheh Abedini, Annette Plesner, Ping Cao, Jinghua Zhang, Fanling Meng, Chris T. Middleton, Ling-Hsien Tu, Hui Wang, Fei Song, Rosa Rosario, Martin T. Zanni, Bruce Verchere, Daniel P. Raleigh, Ann Marie Schmidt POST 10-60 SUMO4 C438T polymorphism is associated with papulopustular skin lesion in Korean patients with Behçet’s disease Hyun-Sook Kim POST 10-61 Calcium-dependent proteases and their proteinaceous inhibitor in the brain regions affected by neurodegeneration in rats Liudmila A. Lysenko, Nadezda P. Kantserova, Nikolay L. Rendakov, Khristina N. Prokopenko, Nina N. Nemova POST 10-62 Monomeric IKK: Probing Activation and Specificty William E. Rogers POST 10-63 An Ultrasensitive platform for the detection of protein biomarkers in spiked human serum Stephen Vance, Marinella Sandros POST 10-64 Regulation of calpain activity in fish brain by weak low-frequency magnetic fields

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

Nadezda P. Kantserova, Liudmila A. Lysenko, Natalia V. Ushakova, Vyacheslav V. Krylov, Nina N. Nemova POST 10-65 Amyloid beta peptide Aβ40 and Aβ42 form separate fibrils in binary mixtures Xiaoting Yang, Risto Cukalevski, Georg Meisl, Birgitta Frohm, Tuomas Knowles, Sara Linse POST 10-66 Molecular engineering of L-asparaginases as therapeutic enzymes for the treatment of leukemia Manfred W. Konrad, Christos S. Karamitros POST 10-67 Tweaking the spines of Kinase Structures Lalima G. Ahuja, Jiancheng Hu, Alexandr P. Kornev, Andrey S. Shaw, Susan S. Taylor POST 10-68 Correlation between Aβ (1-40) aggregation in E.coli and in vitro amyloid fibril formation Kalyani Sanagavarapu, Irem Nasir, Celia Cabaleiro-Lago, Sara Linse POST 10-69 Structure-based design of dual smallmolecule inhibitors of Mdm2/MdmX for efficiently reactivating p53 in cancer cells Zhengding Su, David Duda, Lingyun Qin, Yao Chen, Huashan Zhang, Weiping Wang, Brenda Schulman POST 10-70 Subtle Differences Between NOS Active Sites Lends Towards the Development of a Bacterial NOS Specific Inhibitor Jeffrey K. Holden, Soosung Kang, Richard B. Silverman, Thomas L. Poulos POST 10-71 The role of extracellular EMMPRIN and its glycosylation in modulating cancerous phenotypes Agnieszka A. Kendrick, Elan Z. Eisenmesser POST 10-72 Structural insights into the role of the Smoothened cysteine rich domain in Hedgehog signalling Rajashree Rana, Candace Carroll, Ho-Jin Lee, Ju Bao, Suresh Marada, Grace Royappa, Stacey Ogden, Jie Zheng POST 10-73 CRAMP 16-33 inhibits the assembly and GTPase activity of FtsZ and perturbs the formation of the cytokinetic Z-ring in bacteria Shashikant Ray, Dulal Panda



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 10-74 Dimerized Translationally Controlled Tumor Protein (TCTP) induces Interleukin-8 secretion in human bronchial epithelial cells via MAPK and NFκB pathway. Heewon Lee, Haejun Pyun, Jeehye Maeng, Kyunglim Lee† POST 10-75 Lysolipids Modulate Aggregation of the Repeat Domain of a Human Functional Amyloid, Pmel17 Zhiping Jiang, Jennifer C. Lee POST 10-76 Extracellular clusterin suppresses the formation of cytotoxic -α-synuclein species by interacting with prefibrillar species and facilitates their lysosomal degradation: Implications in Parkinson’s disease Abdullah Sultan, Bakthisaran Raman, Ch M. Rao, Ramakrishna Tangirala POST 10-77 The adhesion regions of gingipains from P. gingivalis are composed of at least two distinct types of domains . Charles A. Collyer, Jinlong Gao, Daniele Vicari, Nan Li POST 10-78 Alzheimer’s disease and cerebral amyloid angiopathy, doppelgangers? Rabia Sarroukh, Ellen Hubin, Louise C. Serpell, Nico A. van Nuland, Kerensa Broersen, Vincent Raussens POST 10-79 Investigating of the immunomodulatory function of Plasmodium falciparum Hsp70 expressed from various LPS minus bacterial strains Ofentse J. Pooe, Gabriele Köllisch, Holger Heine, Addmore Shonhai POST 10-80 Near infrared spectral monitoring reveals water molecular system dynamics during the amyloidogenic nucleation Eri Chatani, Yutaro Tsuchisaka, Yuuki Masuda, Roumiana Tsenkova POST 10-81 Liver Internalization of Cholesterol Decoded by X-rays Hay Dvir POST 10-82 RNA structural elements and protein interactions that regulate HIV genome splicing Blanton S. Tolbert POST 10-83 Molecular mechanism by which an intrinsically disordered region in non-segmented

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

negative strand RNA virus phosphoprotein acts as a chaperone of unassembled viral nucleoprotein Cédric Leyrat, Filip Yabukarski, Malene R. Jensen, Rob Ruigrok, Martin Blackledge, Marc Jamin POST 10-84 Substrate and drug induced conformational heterogeneity in CETP Revathi Sankar, Sanjib Senapati POST 10-85 ASB9 N-terminus plays an important role in creatine kinase regulation Deepa Balasubramaniam, Jamie Schiffer, Jonathan Parnell, Stephan Mir, Elizabeth Komives POST 10-86 Limited proteolysis and dissociation of trimeric state of the BRICHOS domain increase its anti-amyloid activity Henrik Biverstål, Lisa Dolfe, Erik Hermansson, Jenny Presto, Jan Johansson POST 10-87 Thermodynamic and structural characterization of the binding of Zn(II) and other molecules to human protein DJ-1 Shinya Tashiro, Jose Caaveiro, Chun-Xiang Wu, Quyen Hoang, Kouhei Tsumoto POST 10-88 PICH and BEND3 form a complex: potential role in the processing of ultrafine anaphase DNA bridges. Ganesha p. Pitchai

POST 10-89 Computational Docking and Site Directed Mutagenesis to Identify Steroid Binding Sites on Ionotropic Glutamate Receptors Emily Bartle, Philip Varnes, Carol Parish, Ellis Bell POST 10-90 Effects of Agonist and Regulator Binding on the Structure and Conformational Flexibility of the Ligand Binding Domains of Ionotropic Glutamate Receptors Forest Barkdoll-Weil, Carol Guzman, Philip Varnes, Ellis Bell POST 10-91 A Novel CD4-based Chimeric Antigen Receptor for Functional Cure of HIV Li Liu, Bhavik Patel, Mustafa Ghanem, Zhilli Zheng, Virgilio Bundoc, Richard Morgan, Steven A. Rosenberg, Barna Dey, Edward A. Berger



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 10-92 Some Like it Hot: Determination of Biomolecular Interactions using MicroScale Thermophoresis Ana Lazic, Wyatt Strutz, Nicole Ford, Stefan Duhr POST 10-93 Effects of Regulator Binding on the Structure and Conformational Flexibility of the Amino Terminal Domains of Ionotropic Glutamate Receptors Carlos Metz, Emily Bartle, Alaina Hyde, Ellis Bell POST 10-94 The variant of CREBBP with HAT domain deletion inhibits Hela cell proliferation by downregulating the expression of TAF1 Zhang Lu-yu, Li Xin, Wang Yun-hong, Wen Quan, Zhang Jun POST 10-95 Effect of Polyethylene Glycol Conjugation on Conformational and Colloidal Stability of a Monoclonal Antibody Antigen Binding Fragment (Fab) Cristopher Roque, Anthony Sheung, Nausheen Rahman, Salvador F. Ausar POST 10-96 Biochemical and structural characterization of LiaR from Vancomycin-Resistant E. faecalis : the ‘master regulator’ of the cell-envelope stress response . Milya Davlieva, Yiwen Shi, Michael Zianni, Troy Johnson, Paul Leonard, John Ladbury, Cesar A. Arias, Yousif Shamoo POST 10-97 Potent inhibition of α-synuclein fibrillization and toxicity through an energyindependent chaperone-like activity Jan Bieschke POST 10-98 Rational search for a compound that selectively inhibits the triose phosphate isomerase from Trichomonas vaginalis José L. Vique, Luis G. Brieba, Rossana Arroyo, Jaime Ortega, Arturo Rojo, Ponciano Garcia, Claudia Benítez POST 10-99 The level of Dot1L recruitment defines the degree of MLL-AF9 hematopoietic transformation Aravinda Kuntimaddi, John Bushweller POST 10-100 Long-range Activity Regulation Mechanisms Within The IL-33/ST2/IL-1RAcp Complex Kendra Hailey, Bryan E. Jones, Patricia A. Jennings

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 10-101 Modeling and Simulation of Full-length p53 Tetramer Bound to DNA Ozlem Demir, Pek Ieong POST 10-102 Putative programmed cell death pathway of the malaria parasite and the role of cytochrome C Judith H. Prieto POST 10-103 Misfolding of the vWF A1 Domain alters the strength of platelet adhesion in Type 2 von Willebrand Disease Alexander Tischer, Pranathi Madde, Laurie Moon-Tasson, Matthew Auton POST 10-104 Predictive and experimental approaches for characterizing mutations in proteins Maria Teresa Buenavista, Rohanah Hussain, Ann-Marie Mallon, David Nutt, Liam James McGuffin POST 10-105 Structural unity in diversity in pilins of some enteric pathogens Himadri Biswas, Rajagopal Chattopadhyaya POST 10-106 Synthesis of selectively functionalized adiponectin Andreas Mattern, Annette G. BeckSickinger POST 10-107 Structural analysis of the CFA/III minor pilin subunit CofB of human enterotoxigenic Escherichia coli Hiroya Oki, Kazuki Kawahara, Shunsuke Fukakusa, Takuya Yoshida, Yuji Kobayashi, Tooru Taniguchi, Takeshi Honda, Tetsuya Iida, Shota Nakamura, Tadayasu Ohkubo POST 10-108 Analysis of the gene products related to osseointegration in the early stage of titanium implantation Masataka Horiuchi, Rumi Horiuchi, Masanori Ochiai, Atsuro Yokoyama POST 10-109 Advanced Molecular Tools for Proteomic Analyses of Microvesicles Masood KamaliMoghaddam, Liza Löf, Felipe Oliveir, Lotta Wik, Di Wu, Junhong Yan POST 10-110 Bovine Brain Ribonuclease is the Functional Homolog of Human Ribonuclease 1 Chelcie H. Eller, Jo E. Lomax, Ronald T. Raines



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM POST 10-111 Defect in the Intramolecular and Intermolecular Cross-linking of Collagen Caused by Hcy-thiolactone Marta Rusek

POST 10-112 Thermodynamic Analysis of Membrane Interactions with Tau Peptides Megan Culp, Larry Masterson POST 10-113 Molecular basis of heme capture by Isd system of Staphylococcus aureus Jose M. Caaveiro, Nhuan Vu, Koldo Morante, Yoshitaka Moriwaki, Ryota Abe, Kouhei Tsumoto POST 10-114 Biologically responsive recombinant protein anchors for macromolecular drug delivery Jason S. Buhrman, Jamie E. Rayahin, Yu Zhang, Mary Tang, Richard A. Gemeinhart POST 10-115 X-ray crystallography & Small Angle Xray Scattering studies of Interferon Regulatory Factor 4 Soumya Govinda Remesh, Carlos R. Escalante POST 10-116 Structural Characterization of Protein Aggregates of Wild-Type and Disease Associated Variants of Human γS-crystallin David M. Montelongo, Chelsea Anorma, Diana Bandak, Rachel W. Martin POST 10-117 Insight into the Catalytic Mechanism of GABA - producing Enzyme: Glutamate Decarboxylase from Sphaerobacter thermophilus Ruiying Wu, Shonda Clancy, Andrzej Joachimiak POST 10-118 Interactions of E. coli immunoglobulin binding protein D and the Fc part of IgG Kornelia M. Mikula, Robert Kolodziejczyk, Adrian Goldman POST 10-119 The Dynamic Functional Consequences of the Thrombin-Thrombomodulin Interaction Lindsey D. Handley, Elizabeth A. Komives POST 10-120 A Novel Activator of ATPase Activity of NBD1 Domain of the CFTR Jay Singh, William Balch POST 10-121 Targeting the NEET Proteins for Cancer Treatment Colin H. Lipper, Mark L. Paddock, José N. Onuchic, Ron Mittler, Rachel Nechushtai, Emmanuel A. Theodorakis, Patricia A. Jennings

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 10-122 Understanding the role of human DDX21 RNA helicase in HIV-1 Rev-RNA assembly in vitro Li Zhou, James R. Williamson

Proteins in Dynamic & Driven Processes POST 11-123 Characterization of a temperature responsive two-component regulatory system in the Antarctic methanogen, Methanococcoides burtonii POST 11-124 Crystal Structure of the Periplasmic Sensor Domain of Histidine Kinase CusS Bound to Silver Trisiani Affandi, Aaron V. Issaian, Sue A. Roberts, Megan M. McEvoy POST 11-125 Structural Investigation into the Mechanism of the Synthase Subunit of PLPS. Amber M. Smith, Janet L. Smith POST 11-126 Super Spy variants implicate flexibility in chaperone action Shu Quan, Lili Wang, Evgeniy V. Petrotchenko, Karl A. Makepeace, Scott Horowitz, Jianyi Yang, Yang Zhang, Christoph H. Borchers, James C. Bardwell POST 11-127 Elucidation of the nonspecific DNAbinding mechanism of the POU homeodomain using NMR Tsuyoshi Konuma, Erisa Harada, Takashi Oda, Mamoru Sato, Kenji Sugase POST 11-128 Time-lapsing Planaria: Studying Planarian regeneration by stop-motion imaging and by pulse-isotopic proteome labeling Wei Shen, Karsten Berning POST 11-129 Hydrogen Deuterium Exchange Mass Spectrometry and Molecular Dynamics Reveal the Interactions of Membrane Phospholipids and Inhibitors with Phospholipases A2 Varnavas Mouchlis, Denis Bucher, J. Andrew McCammon, Edward A. Dennis



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 11-130 Visualizing the inter-domain motions of a flexible protein using continuous models Yang Qi, Jeffrey W. Martin, Bruce R. Donald, Terrence G. Oas POST 11-131 Benchmarking FRET for live-cell PPI Tao Lin, Brandon Scott, Francisca Essel, Moul Dey, Adam Hoppe, Suvobrata Chakravarty POST 11-132 Dynamics and chaperone function in the small heat-shock protein αB-crystallin Georg Hochberg, Heath Ecroyd, Dezerea Cox, Michael Sawaya, Cong Liu, Duilio Cascio, Miranda Collier, James Stroud, John Carver, Andrew Baldwin, Carol Robinson, David Eisenberg, Justin Benesch, Arthur Laganowsky

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 11-140 Intrinsic GTP Hydrolysis is Observed For a Switch 1 Mutant of Cdc42 in the Presence of a Specific GTPase Inhibitor Reena Chandrashekar, Kyla Morris, Colin D. Heyes, Paul D. Adams POST 11-141 Three-dimensional Structure of the 54 kDa Subunit of the Chloroplast Signal Recognition Particle using Molecular Modeling Rory Henderson, Suresh Kumar, Colin Heyes, Ralph Henry POST 11-142 Evidence for significant changes in backbone motions between apo and Gα-bound human RGS4 Lusine Simonyan, Shayla A. Brooks, Karin A. Crowhurst

POST 11-133 Protein Flexibility and Gymnastics Drive Robust Clockwise Ticking of a three-protein KaiABC Oscillator Yonggang Chang, Roger Tseng, Andy LiWang

Protein Engineering & Synthetic Biology

POST 11-134 Characterization of Dynamic processes in substrate recognition by cytochrome P450 enzymes Nitin Jain, Nicholas Lopes, Ana Bernal

POST 12-143 Engineering Picomolar Affinity into a Rationally Identified 5 kDa Scaffold for Tumor Targeting Max Kruziki, Patrick Holec, Benjamin Hackel

POST 11-135 Mapping the Interactions between the Molecular Chaperones Hsp70, Hsp104 and Hsp110 Shankar Shastry, Shannon Doyle, Joel Hoskins, Sue Wickner

POST 12-144 Utilizing Metal-Ligand Interactions to Promote Assembly of Collagen-Based Peptides into Functional Nanostructures Jeremy Gleaton, David Przybyla, Charles M. Rubert-Perez, Jean Chmielewski

POST 11-136 The role of Phenylalanine in an intrinsically disordered protein from yeast nucleoporins. Korey M. Reid, Krish Krishnan

POST 12-145 Live Cell Imaging of Molecular Conformations and Actions Peter Yingxiao Wang

POST 11-137 The Acidic Residues of the IκBα PEST Sequence are Responsible for Actively Dissociating NFκB from DNA Holly E. Dembinski, Kevin Wismer, Elizabeth Komives POST 11-138 Nanoscale Hydrodynamic Study of Proteins under Thermal Agitation and Electric Field Yuanming Zhang, Zachary Weiner, Eric Farrell POST 11-139 Probing the clamping movement of xylanase B by NMR spectroscopy Nhung T. Nguyen, Nicolas Doucet

POST 12-146 Protein design: Preventing protein aggregation in recombinant erythropoietin Manuel A. Carballo-Amador, Jim Warwicker, Alan J. Dickson POST 12-147 Intracellular regulation of the NFE2L3 transcription factor Meenakshi B. Kannan, Volker Blank POST 12-148 Phosphorylation and Binding Partners of the NFE2L3 Transcription Factor: Insights into its Role in Oncogenesis Isadore Dodard-Friedman, Volker Blank



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 12-149 An Orthogonal Genetic System for Rapid Evolution Chang Liu POST 12-150 A recognition model of ACP-HCS interaction for programmed beta-branching in type I polyketide synthases Rohit Farmer, Anthony S. Haines, Matthew Crump, Christopher Thomas, Peter J. Winn POST 12-151 Engineering photo-control of translation initiation using photoactive yellow protein hybrids Anil Kumar, Anna S. Jaikaran, Alaji Bah, Julie FormanKay, G. Andrew Woolley POST 12-152 Gold Decorated Peptide Amphiphile Templates for Directed Silver Nanorods Growth Shlomo Zarzhitsky, Hanna Rapaport POST 12-153 Engineered oligosaccharyltransferases with greatly relaxed acceptor site specificity Anne A. Ollis, Sheng Zhang, Matthew P. DeLisa POST 12-154 The dynamic peptide recognition and stabilization mechanism of Human Leukocyte Antigen B*35:01 Saeko Yanaka, Takamasa Ueno, Kouhei Tsumoto, Kenji Sugase POST 12-155 Engineering Ordered Protein Assemblies by Helix-Fusion Strategy Yen-Ting Lai, Todd O. Yeates POST 12-156 Total synthesis and chaperonemediated folding of a 312-residue mirror-image enzyme Michael T. Jacobsen, Matthew T. Weinstock, Michael S. Kay POST 12-157 Creating self-assembling stimulusresponsive hydrogels from protein components Danielle Williams, Ashley Schloss, Lynne Regan POST 12-158 Characterization of a novel synthetic biomaterial for protein immobilization Carrie MareanReardon, Patrick Reardon, Thomas Squier, Kathleen McAteer POST 12-159 Protein Fragment Exchange: Converting an Arbitrary Binding Protein into a Robust FRET Biosensor Huimei Zheng, Jing Bi, Mira Krendel, Stewart N. Loh

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 12-160 Design of an Albumin-Binding Human Protein by Mimicking the Contact Surface of a Bacterial Albumin-Binding Domain Satoshi Oshiro, Shinya Honda POST 12-161 Developing soluble co-receptor mimetics for the study of HIV Env/receptor interactions Agnes Hajduczki, Virgilio Bundoc, Edward A. Berger POST 12-162 Constructing highly detectable fluorescence reporter protein for in vitro single molecular screening Kotaro Nishiyama, Norikazu Ichihashi, Yasuaki Kazuta, Tetsuya Yomo POST 12-163 Model building of antibody-antigen complex structures using GB/SA scores Narutoshi Kamiya, Noriko Shimba, Haruki Nakamura POST 12-164 Engineering Novel Phosphopeptide Recognition Modules that Recognize Targets In Vitro and in E. coli Nicholas Sawyer, Lynne Regan, Brandon Gassaway, Jesse Rinehart, Adrian Haimovich, Farren Isaacs POST 12-165 N-linked glycosylation of HIV-1 core gp120 is not required for native trimer formation or viral infectivity Ujjwal Rathore, Piyali Saha, Sannula Kesavardhana, Aditya A. Kumar, John R. Mascola, Raghavan Varadarajan POST 12-166 Silicon transporters: from membrane proteins to nanotechnology Laura Senior, Sarah Ratcliffe, Michael Knight, Adam Perriman, Stephen Mann, Paul Curnow POST 12-167 Characterization of Thermotoga maritima Maltotriose Binding Protein Laura Masson, Jonathan Dattelbaum POST 12-168 Zinc induces self-assembly of bacterial thermoalkalophilic lipases: a strategy for thermostability Emel Timucin, Osman U. Sezerman POST 12-169 The SasG E-G5 protein fold forms a stable contiguous rigid nanorod of tunable length Fiona Whelan, Dominika T. Gruszka, Jane Clarke, Jennifer R. Potts



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM POST 12-170 Computational design of allosteric antibody Olga Khersonsky, Sarel Fleishman

POST 12-171 Development and optimization of an E. coli -based display platform for selection of affinity proteins Filippa Fleetwood, Ken Andersson, Stefan Ståhl, John Löfblom

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 12-181 Beta-hairpin tags for increased expression of helical peptides Melissa E. Lokensgard, John J. Love POST 12-182 Gradient Diversity Enriches Combinatorial Protein Library Design Daniel R. Woldring, Benjamin J. Hackel

POST 12-173 Accurate prediction of protein stability by explicit negative multistate design James A. Davey, Christian K. Euler, Roberto A. Chica

POST 12-183 Expanding the GFP toolbox through a better understanding of its folding pathway Keith Fraser, Colleen Lamberson, Victoria Jones, Erin Gilbert, David Rosenman, Yao-ming Huang, Derek Pitman, Shounak Banerjee, Yan Xia, Angela Choi, Rachel Altshuler, Luis Garreta, John Karonicolas, Donna E. Crone, Jonathan S. Dordick, Christopher Bystroff

POST 12-174 Plastic protein design: A novel design algorithm using backbone and side-chain ensembles to model protein flexibility Christian D. Schenkelberg, Derek J. Pitman, Yao-ming Huang, Christopher Bystroff

POST 12-184 Are Peptide Nucleic Acids (PNAs) Recognized by Aminoacyl-tRNA Synthetases (aaRSs)? Crystal Serrano, Long Nguyen, Anthony Bell, Filbert Totsingan

POST 12-175 Repeat-protein directed synthesis of gold nanoparticles with tunable morphology and optical properties Tijana Grove, Xi Geng

POST 12-185 Protein Rings and Tubes as Versatile Templates for Self-Assembled Bionano Structures Ali A. Malay, Zuben Brown, Kenji Iwasaki, Jonathan G. Heddle

POST 12-172 Computational Redesign of Metagenomic Enzymes Justin B. Siegel

POST 12-176 Designed Affinity Reagents Directed to Heat Shock Protein C-terminal Motifs Robert Wells, Akiko Koide, Shohei Koide POST 12-177 Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide (MDP), a bacterial cell wall fragment Rachael Parker, Ana Mercedes-Camacho, Tijana Z. Grove POST 12-178 Solvent engineering to improve enzymatic synthesis of a sugar-based surfactant Rodrigo A. Arreola-Barroso, Gloria Saab-Rincón POST 12-179 Delivery of natural and non-natural chemical entities into cells using anthrax toxin Amy Rabideau, Xiaoli Liao, Bradley Pentelute POST 12-180 Thermostabilization of Aspergillus oryzae Cutinase Danielle A. Basore, Abhijit Shirke, Glen Butterfoss, Richard Gross, Christopher Bystroff

POST 12-186 BIO-INSPIRED TECTONS: The architecture & engineering of synthetic ring-forming proteins Francesca Manea, Bridget C. Mabbutt POST 12-187 A designed small protein for controlling site-specific mineralization of silica and calcium on DNAs Kenji Usui, Kazuma Nagai, Hiroto Nishiyama, Aoi Yamada, Makoto Ozaki, Takaaki Tsuruoka, Kin-ya Tomizaki POST 12-188 Enhancing predictions of surface entropy reduction for improved crystallizability of proteins Derek J. Pitman, Thomas Holton, Luki Goldschmidt, Zygmunt Derewenda, David Eisenberg POST 12-189 Repeat protein scaffolds for assembly of functional nanostructures Sara H. Mejias, Pierre Couleaud, Javier Lopez, Begoña Sot, Carmen Atienza, Teresa Gonzalez, Aitziber L. Cortajarena



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 12-190 Generation of Novel Amino Acid Dehydrogenase Activity through Evolution of a Highly Specific Alanine Dehydrogenase Emily Mundorff POST 12-191 Rational protein design, SPR binding analysis, and iterative optimization of studentdesigned single-chain immunoreceptors Benjamin J. McFarland, Andrew Daman

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 12-200 Protein-based stimuli-responsive hydrogels for targeted drug delivery Ashley C. Schloss, Abbie Omolu, Richard Day, Lynne J. Regan POST 12-201 β/α and α/β of TIM Barrel Proteins: Database and Structural Analysis Ramakrishna Vadrevu, Rajashekar V. Kadumoori

POST 12-192 Efficient gene disruption at hTERT promoter region by simultaneous digestion by pairs of ZFNs or guide RNAs for CRISPR/Cas system. Wataru Nomura, Akemi Masuda, Hirokazu Tamamura

POST 12-202 De Novo Design of Peptides that Assemble Lipid Nanodiscs Shao-Qing Zhang, Kazuma Yasuhara, Hyunil Jo, William F. DeGrado

POST 12-193 High-throughput sorting of the highest producing cell via a transiently protein-anchored system Kuo-Hsiang Chuang POST 12-194 Dissecting contributions to protein stability via recombination of a wild type and computationally designed protein Lucas Johnson, Lucas Gintner, Luke Minardi, Christopher Snow

POST 12-203 A Hyperstable Minimalist Protein for Molecular Recognition Michael W. Traxlmayr, Raja R. Srinivas, Elisabeth Lobner, Bruce Tidor, Karl D. Wittrup

POST 12-195 Computational Design of Protein Ligand Interfaces Using RosettaLigand Brittany Allison, Jens Meiler

POST 12-205 “PA tag”, a versatile affinity tag system that enables one-step affinity purification and high sensitive detection of recombinant proteins from dilute sample Yuki Fujii

POST 12-196 Eliminating Endotoxin at the Source - A Novel Competent Cell Line with Modified Lipopolysaccharide for Low-Endotoxin Plasmid Production Saurabh Sen, Uwe Mamat, Chad Souvignier, Eric Steinmetz, Chelsea Kovacich, David Mead, Curtis Knox POST 12-197 Computational Design of Leucine-Rich Repeat- based Protein Binding Scaffolds. Sebastian Rämisch, Ulrich Weininger, Ingemar André

POST 12-204 A Synthetic Biochemistry Molecular Purge Valve Module that Maintains Redox Balance Paul Opgenorth

POST 12-206 Computational design of protein-DNA nanowires Yun Mou POST 12-207 Reversible modification of the Nterminal cysteine residue of proteins using pyruvic acid analogs Pradeep Budhathoki, Youngha Ryu POST 12-208 AbDesign : Computational antibody design Switching species preference and humanizing an inhibitory antibody Assaf Alon, Sarel J. Fleishman

POST 12-198 Generation and Characterization of the Native-like IgG Bispecific Antibodies Steven M. Lewis, Arlene Sereno, Flora Huang, Anna Pustilnik, Heather L. Rick, Elaine M. Conner, Shane Atwell, Brian Kuhlman, Stephen J. Demarest, Xiuifeng Wu

POST 12-209 Sequence-specific cleavage of initiating methionine and RimJ-catalyzed N-terminal acetylation of the Z-domain in Escherichia coli Youngha Ryu, Lina Bernal-Perez

POST 12-199 Improving the efficiency of Concanavalin A as affinity ligand in alkaline pH range Akash Chaudhary, Shakeel Ahmad, Shadab Ahmad, Mohd. Tashfeen Ashraf

POST 12-210 In vitro selection and evolution of membrane proteins using liposome display Tomoaki Matsuura, Satoshi Fujii, Yasuaki Kazuta, Takeshi Sunami, Tetsuya Yomo



Author Presentation Schedule:

Sunday, 07/27/2014 P02 - P08 Even Numbers 11:30 AM-01:30 PM P02 - P08 Odd Numbers 4:30 PM – 6:30 PM

POST 12-211 Ubiquibodies: Engineered E3 Ubiquitin Ligases for Targeted Degradation Erin A. Stephens, Alyse D. Portnoff, Morgan R. Baltz, Jeffrey D. Varner, Matthew P. DeLisa POST 12-212 Engineering Ubiquitin to recognize noncognate proteins implicated in cancer Isabel Leung, Sachdev Sidhu, Nick Jarvik POST 12-213 Optimization of a protein labelling technique for fluorogenic, X-ray crystallography and NMR applications Miroslava Strmiskova, Natalie K. Goto, Jeffrey W. Keillor

Monday, 7/28/2014 P09-P12 Even Numbers 11:30 AM-01:30 PM P09-P12 Odd Numbers 4:30 PM – 6:30 PM

POST 12-221 Beyond Glutaraldehyde: The Search for Optimal Chemical Crosslinkers for Protein Crystal Thaddaus R. Huber, Jacob Sebesta, Christopher Snow POST 12-222 Utilization of Collagen IV NC1 Domains to Control Helical Composition: A Recombinant Strategy for the Production of Collagen IV Protomers Kyle L. Brown, Vadim Pedchenko, Selene Colon, Tim Blackwell, Ambra Pozzi, Billy Hudson POST 12-223 Secondary Antibody Cross-Reactivity Identification Using ProteOn XPR36 System Gary Ross, Perry Ripa, Mohammed Yousef

POST 12-214 Computational Design of Cystatin and Ketosteroid Isomerase Folds from Scratch: From Structure to Self-Labeling Function Enrique Marcos, Dr. David Baker POST 12-215 Development of Novel p16INK4a Peptide Mimetics as Anticancer Therapy Marian Kratzke, Yuk Sham, Mark A. Klein POST 12-216 A new protein cage architecture formed via gold cluster catalysis Ali D. Malay, Kenji Iwasaki, Zuben Brown, Jonathan G. Heddle POST 12-217 Identification of residues in TIMP-1 that are critical in interaction between MMP-9 and TIMP1. Ruiying Wang, Alexandra Hockla, Evette S. Radisky POST 12-218 Computationally Designed Green Fluorescent Protein Based Biosensors Shounak Banerjee, Yao-Ming Huang, Donna E. Crone, Diana I. Paredes, Jonathan S. Dordick, Christopher Bystroff POST 12-219 Atomic Force Microscopy Characterization of Beta-Solenoid Based Amyloid Fibrils Arpad Karsai, Maria D.R. Peralta, Alice Ngo, N. Robert Hayre, Nima Mirzaee, Alexander J. Kluber, Xi Chen, Gang-yu Liu, Michael Toney, Rajiv R.P. Singh, Daniel L. Cox POST 12-220 Reverse Protein Engineering towards a fluorescent peptide Zhiwen J. Zhang, Blake Williams


EXHIBITOR DIRECTORY Anton Paar USA Booth # 103 10215 Timber Ridge Drive Ashland, VA 23005 Phone Fax Email Web

804-550-1051 804-550-1057 [email protected]

SAXSpace is a laboratory instrument for small-angle and wide-angle X-ray scattering (SAXS and WAXS/XRD). Combined in the SAXSpace system, these two techniques form a powerful analytical solution for investigating liquid crystals, dispersions, polymers, proteins, and much more. A compact instrument that provides ultimate clarity for your materials research and quality control. AVIV Biomedical, Inc. Booth #204 750 Vassar Avenue, Suite 2 Lakewood, NJ 08701-6929 Phone Fax Email Web

732-370-1300 732-370-1303 [email protected]

Aviv Biomedical Inc. manufactures scientific and clinical instruments. Products include a fluorescence accessory (AU-FDS) for the Beckman Analytical Ultracentrifuge, model XLA/XLI. Sales, service and support of Aviv Spectrometers, Aviv Spectrophotometers and Aviv Fluorometers. BioLegend Booth # 210 9727 Pacific Heights Blvd San Diego, CA 92121 Phone Fax Email Web

858-455-9588 877-455-9587 [email protected]

World-Class Antibodies, Proteins, Assays and Research Solutions. Complete Brilliant Violet™ Antibody Conjugates for the Violet Laser: BV510™, BV711™, BV785™. Personalized Multicolor Flow Cytometry Panel Design. New LEGENDScreen™ Human Cell Screening (PE) Kits. Request Bulk Cytokines & Chemokines for Bioassay. Ultra-LEAF™ (Low Endotoxin, Azide-Free) Antibodies. New ELISA Kits: IL-35, Active TGF- β1.


EXHIBITOR DIRECTORY Brookhaven Instruments Corporation 750 Blue Point Road Holtsville, NY 11742 Phone Fax Email Web

Booth # 212

631-758-3200 631-758-3255 [email protected]

Brookhaven pioneered modern techniques in characterizing nanoparticles, proteins, and polymers using light scattering for particle sizing, zeta potential, and absolute molecular weight analysis. New SEC/GPC software, ParSEC, works with almost any detector on the market. New particle characterization software, Particle Solutions, is a unique, database approach with unsurpassed search features. Cayman Chemical Company 1180 East Ellsworth Rd Ann Arbor, MI, 48108

Booth # 105

Phone 734 971-3335 Fax 734 971-3420 Our scientists are experts in the synthesis, purification, and characterization of biochemicals ranging from small drug-like heterocycles to complex biolipids, fatty acids, and many others. We are also highly skilled in all aspects of assay and antibody development, protein expression, crystallization, and structure determination. Our organic and analytical chemists specialize in the rapid development of manufacturing processes and analytical methods to carry out clinical and commercial GMP-API production. Pre-clinical drug discovery efforts are currently underway in the areas of bone restoration and repair, muscular dystrophy, oncology, and inflammation. A separate group of Ph.D.-level scientists are dedicated to offering Hit-to-Lead Discovery and Profiling Services for epigenetic targets. Our knowledgeable chemists can be contracted to perform complete sample analysis for analytes measured by the majority of our assays. We also offer a wide range of analytical services using LC-MS/MS, HPLC, GC, and many other techniques. Cayman is a leader in the field of emerging drugs of abuse, providing highpurity Schedule I-V Controlled Substances to federally-licensed laboratories and qualified academic research institutions for forensic analyses. We are certified by ACLASS Accreditation Services with dual accreditation to ISO/IEC 17025:2005 and ISO Guide 34:2009. FASEB MARC Program 9650 Rockville Pike Bethesda, MD 20814 Phone Fax Email Web

Booth #205

301-634-7930 301-634-7353 [email protected]


EXHIBITOR DIRECTORY The FASEC MARC (Maximizing Access to Research Careers) Program provides a variety of activities to support the training of students, postdoctorates, faculty and researchers from underrepresented groups who are engaged in the biomedical and behavioral sciences research and training. We offer faculty/mentor with students and poster/platform presenter travel awards for scientific meetings (national and regional) and FASEB Science Research Conferences. We also sponsor career/leadership development and grantsmanship training seminars and workshops. GE Healthcare Booth # 221 800 Centennial Avenue – PO Box 1327 Piscataway, NJ 08855-1327 Phone Fax Email Web

800-526-3593 877-295-8102 [email protected]

GE Healthcare provides tools for drug discovery, biopharmaceutical manufacturing and cellular technologies so research scientists worldwide can be more productive, effective, and creative. Our focus is to support the bioprocess researcher from idea to result and be the partner of choice in cell and protein research. HORIBA Scientific Booth # 201 3880 Park Avenue Edison, NJ 08820 Phone Fax Email Web

732-494-8660 732-549-5125 [email protected]

HORIBA Scientific’s SPR Imaging (SPRi) systems utilize SPR to perform multiplexed binding kinetics measurements of up to 400 proteins, antibodies or DNA in one experiment. Our Fluorescence line, with the addition of the Photon Technology International (PTI) products, offers the most extensive array of steady-state, lifetime and microscopy-based fluorometers. JASCO Booth # 107 28600 Mary’s Court Easton, MD 21601 Phone 800-333-5272 Email [email protected] Web The new J-1000 Series CD Spectrometers offer unparalleled sensitivity, with reach from the vacuum UV to the NIR wavelengths. Also, introducing Simultaneous Multi-Probe Spectroscopy (SMP) which consists of three modes (CD, LD and Absorbance) running concurrently with the option to run Fluorescence, Anisotropy, ORD, Temperature, Kinetics and more!


EXHIBITOR DIRECTORY JASCO has been the leading manufacturer of CD, VCD and other chiro-optical instrumentation since the 1960’s. With offices and dealers worldwide we are able to provide the research community with quality products and experienced support for any application. Kinnakeet Biotechnology 2336 Colony Crossing Pl Midlothian, VA 23112 Phone Fax Email Web

Booth # 206

866-522-5338 804-506-4414 [email protected]

Since 2003 Kinnakeet Biotechnology has specialized in recombinant viral construction, amplification, protein expression and purification using the baculovirus protein expression system. Outstanding customer service and extremely competitive pricing for both small and large scale production (from 1L to >100L) have assured that >90% of our business comes from repeat customers. Malvern Instruments 117 Flanders Road Westborough, MA 01581 Phone Fax Email Web

Booth # 207

508-768-6400 508-768-6403 [email protected]

Malvern supports better characterization/control of proteins/macromolecules. Charge, size, mass, molecular weight, polydispersity are critical parameters, measured using: Zetasizer, dynamic/static light scattering for size, molecular weight, protein charge; NanoSight Nanoparticle Tracking Analysis, particle-by-particle characterization; Viscotek SEC, molecular weight/structure; Sysmex FPIA-3000, size/shape analysis of aggregates/subvisible particles; Viscosizer 200, particle size, concentration, formulation viscosity. Molecular Dimensions 849 Sunshine Lane Altamonte Springs, FL 32714 Phone Fax Email Web

Booth # 211

407-886-6901 321-972-8896 [email protected]

Molecular Dimensions founded in 1998 is a world leading supplier of screens, reagents and instrumentation for protein structure determination by X-ray crystallography. We have over 100 intelligent solutions for… Protein Expression, crystal growth screening, custom screens and reagents, crystal growth plates, cryocrystallography, crystal growth storage, analytical instrumentation, working with crystallographers for crystallographers.


EXHIBITOR DIRECTORY NACALAI USA, INC. Booth #102 10225 Barnes Canyon Road, Suite A 103 San Diego, CA 92121 Phone Fax Email Web

858-404-0403 858-404-0408 [email protected]

Nacalai USA, Inc. exhibits the COSMOCORE (Core-Shell particle) and COSMOSIL HPLC columns, which include analytical and preparative reversed-phase columns, HILIC columns, as well as unique stationary phases such as the Cholester (cholesteryl group bonded) and pi-NAP (naphtylethyl group bonded). We also provide high performance magnetic nanoparticles for chemical biology. NanoTemper Technologies, Inc. 395 Oyster Point Blvd., Suite 135 South San Francisco, CA 94080 Phone Fax Email Web

Booth # 200

650-763-1658 650-350-4390 [email protected]

NanoTemper Technologies develops, produces and markets innovative, high quality instruments for biomedical research. The products are based on NanoTemper's unique and proprietary technology, Microscale Thermophoresis (MST), used for the analysis of biomolecular interactions. NanoTemper offers the Monolith NT series instruments for MST measurements with fluorescent label and label-free. PeproTech, Inc. Booth # 220 5 Crescent Avenue Rocky Hill, NJ 08553-0275 Phone Fax Email Web

800-436-9910 609-497-0321 [email protected]

Over the past 25 years, PeproTech has grown into a global enterprise manufacturing an extensive line of Recombinant Human, Murine and Rat Proteins, Animal-Free Recombinant Proteins, Monoclonal Antibodies, Affinity Purified Polyclonal Antibodies, Affinity Purified Biotinylated Polyclonal Antibodies, ELISA Development Kits, Cytokine Packages and Cell Culture Media Products. Pressure BioSciences, Inc. 14 Norfolk Avenue South Easton, MA 02375

Booth # 104

Phone 508-230-1828 51

EXHIBITOR DIRECTORY Fax 508-230-1829 Email [email protected] Web PBI develops and sells instrument systems for proteomic, genomic, and lipidomic sample preparation based on our pressure cycling technology (PCT) platform. PBI is also a distributor of Constant Systems. (CS) cell disruption equipment for the extraction of proteins and other biomolecules from organisms such as yeast and bacteria. Two instrument systems will be showcased (1) PBI’s Barocycler HUB440, capable of reaching 58,000 psi (ideal for protein extraction, accelerated enzymatic digestion, and protein structure studies), and the CS MultiShot Cell Disrupter, designed for processing samples between 1-80 mL. St. Jude Children’s Research Hospital 262 Danny Thomas Place Memphis, TN 38105 Phone Fax Email Web

Booth # 106

901-595-2750 901-595-5376 [email protected]

St Jude Children’s Research Hospital is a non-profit biomedical research institution in Memphis, TN, where 190 basic science and clinical researchers investigate the molecular basis of both normal cellular and diseased processes. Visit our booth to meet with dedicated postdoctoral recruiters and to discuss and apply for postdoctoral fellowship positions. TA Instruments 159 Lukens Drive New Castle, DE 19720 Phone Fax Email Web

Booth # 208

302-427-4000 302-427-4001 [email protected]

TA Instruments is the world leader in protein characterization by microcalorimetry. Our Nano ITC Standard and Low Volume and our Nano DSC with Autosampler are powerful tools for measuring small-molecule-protein interactions, protein-protein interactions, drug-target binding, and biomolecular structure and stability. Visit to learn more about the latest measurement technology. The Protein Society Booth # 213 BWTech South Campus 1450 S. Rolling Rd, Ste 3.007 Baltimore, MD 21227 Phone Fax Email Web

443-543-5450 443-543-5453 [email protected] 52

EXHIBITOR DIRECTORY TTP Labtech Booth # 101 Melbourn Science Park, Melbourn Royston, Herts, SG8 6EE Phone +44 1763 262626 Email [email protected] Web TTP Labtech’s dragonfly screen optimiser is a liquid handler for simple, fast and accurate protein crystallisation screen optimisation. dragonfly provides the freedom to use any liquid type, regardless of viscosity, due to its positive displacement, non-contact dispensing. Each disposable can dispense any volume, from 0.5 µL upwards, into any well. WILEY 350 Main Street Malden, MA 02148 Phone Fax Email Web

Booth # 202

781-388-8200 781-338-8212 [email protected]

Wiley is the leading society publisher. Our scientific, technical, medical and scholarly business publishes on behalf of more societies and membership associations than anybody else, and offers libraries and individuals 1250 online journals, thousands of books and e-books, reviews, reference works, databases, and more. For more information, visit, or our online resource:


EXHIBITOR WORKSHOPS Workshop #1 - GE Healthcare

Sunday, July 27th | Noon - 1 PM | Gaslamp A NEW Amersham™ 600 Imager Series (CCD-based Camera Systems)

Liz Pita, GE Healthcare, Piscataway, New Jersey, US Join us to learn more about the newest generation of imaging systems! The Amersham™ 600 series builds upon the robust legacy of ImageQuant™ LAS4000 series that provided high quality, publication-ready images and reliable research data. Amersham 600 portfolio includes four systems for routine research applications in pharmaceutical, biotechnology and academic research including Chemiluminescent and Fluorescent Western blotting, Gel documentation, DNA and RNA analysis, 2D gels and Colorimetric detection for quantitative and QC analysis. The new platform complements our Western blotting detection reagents, such as our legacy Amersham™ ECL as well as Amersham ECL Prime, ECL Select and ECL Plex to provide a complete Western blotting solution. Please join us to learn more about how our new CCD-based camera systems can help you in your research!

Workshop #2 – NanoTemper Technologies

Monday, July 28th | Noon - 1 PM | Gaslamp A Some Like it Hot: Determination of Biomolecular Interactions Using MicroScale Thermophoresis

Christine Crosby, Ana Lazic, NanoTemper Technologies, S. San Francisco, California, US This workshop will give an overview of MicroScale Thermophoresis (MST), a technology for used for the analysis of biomolecule interactions. MST is the directed movement of molecules in temperature gradients. This thermophoretic movement is determined by the entropy of the hydration shell around the molecules. Almost all interactions and also any biochemical process relating to a change in size, charge and conformation of molecules alters this hydration shell and is thus detectable by MST. In this workshop we will describe the technical details and the benefits of the MicroScale Thermophoresis technology platform. We will show examples for interaction measurements ranging from protein – ribosome, protein – protein, small molecule – receptor down to protein – ion binding studies to experiments where the interactions between receptors incorporated in vesicles and soluble proteins are analyzed. During the workshop measurement of interactions will be demonstrated on NanoTemper Technologies’ NT.115 and NT.LabelFree.

Workshop #3 - GE Healthcare

Tuesday, July 29th | Noon - 1 PM | Gaslamp A Multimodal Chromatography: A New Generation of High Selectivity Media for Challenging Purifications

Felix Solamo, GE Healthcare Life Sciences, Piscataway, New Jersey, US Multimodal chromatography is a powerful tool for difficult separation challenges, including MAb-aggregate removal. New multimodal resins were developed to address this challenge. The performance of a multimodal anion exchanger in polishing is shown for two MAb runs in bind/elute and flow through modes. A multimodal cation exchanger for Mab purification model was used to choose conditions for the desired purification.


INVITED SPEAKER ABSTRACTS Symposium #1 – Translation & Folding SYMP 01-21 Policing Secretion: How Cells Enforce Protein Quality Control Within The Endoplasmic Reticulum

Liz Miller, Columbia University, New York, New York, US

The mechanistic basis for quality control surveillance within the secretory pathway remains poorly understood. We have used a systematic approach to discover proteins that influence the biogenesis of a model misfolded protein, the yeast ABC transporter, Yor1. Broadly analogous to human CFTR, mutations in which cause cystic fibrosis, Yor1 acts at the plasma membrane as a drug pump to confer resistance to oligomycin. Misfolding mutations cause ER retention and proteasomal degradation, leading to oligomycin sensitivity. This phenotype affords a rapid and robust screen for mutations that enhance or suppress the ability of cells to tolerate increasing concentrations of drug. Using a high throughput genetic screen, we measured the effect of each non-essential yeast gene on growth conferred by Yor1-ΛF, equivalent to the predominant disease-causing allele in CFTR. We discovered novel regulators of membrane protein biogenesis, including a pathway that seems to detect protein misfolding at the earliest stages of protein synthesis. We propose misfolding events can generate feedback to the ribosome that halts or slows translation, serving to either promote folding or prevent aberrant proteins from entering the secretory pathway. We also discovered an unexpected role for an ER export receptor, Erv14, which seems to increase the affinity of cargo proteins for the vesicle coat proteins, thereby enhancing ER egress. Genetic and physical interactions between the various regulators we identified suggest that these events are coordinated to promote efficient protein synthesis, folding and forward traffic. SYMP 01-22 Continuous Tracking Of Protein Folding At Microsecond Resolution By A Line Confocal Detection Of Single Molecule Fluorescence

Satoshi Takahashi, Tohoku University, Japan

Single molecule fluorescence spectroscopy (SMFS) is expected to reveal detailed dynamics involved in protein folding; however, the time resolution of previous methods of SMFS is typically limited to a few milliseconds. We recently developed a line-confocal microscope combined with fast sample flow (Oikawa et al., Sci. Rep. 3, 2151 (2013)), and achieved the time resolution of 20 microsecond in obtaining time series of FRET efficiency from single molecules. Using the system, we investigated the B domain of protein A (BdpA), a three-helix bundle, doubly labeled with donor and acceptor fluorophores. We assigned traces having high and low FRET efficiencies to the native and unfolded states, respectively, confirming the twostate transition of BdpA. The traces assigned to the unfolded state showed a significant fluctuation occurring in the submillisecond time region. The traces assigned to the native state showed the gradual shift in the FRET efficiency as the changes in the denaturant concentration. Similar data were obtained for BdpA labeled at different sites, suggesting the melting of the native state structure. We are currently improving the system to achieve the better time resolution and the longer observation time. The line confocal detection of SMFS will become a powerful tool to understand the mechanism of protein folding and other dynamics as an experimental counterpart of molecular dynamics calculations.


INVITED SPEAKER ABSTRACTS SYMP 01-23 Intrinsically Disordered Proteins: Kinetics And Mechanism

Jane Clarke, University of Cambridge, Cambridge, UK (Chair)

Many intrinsically disordered proteins function by folding upon binding to a target protein. It is often said that IDPs provide high specificity with low affinity, but kinetic analysis of a number of systems suggests that this is not universally correct. Why then are disordered proteins so ubiquitous? Is disorder in the IDP important for the function? I will discuss some of our recent kinetic and mechanistic studies of a number of IDPs that fold upon binding.

Symposium #2 – Bacterial Interactions SYMP 02-40 The Gram-Negative Cell Envelope as Seen by Protein Antibiotics

Colin Kleanthous, University of Oxford, Oxford, UK

Protein antibiotics are species-specific antimicrobials deployed by Gram-negative bacteria to kill their neighbours during competition for resources. They are implicated in the stable co-existence of bacteria within microbiomes and are known to give pathogenic organisms an advantage against commensal competitors during gut inflammation. My laboratory is exploring the import mechanism of nuclease colicins, protein antibiotics specific for the model organism Escherichia coli that target its genome, ribosome or tRNAs. Our aim is to understand how these folded, 60-kDa proteins traverse the entirety of the Gram-negative cell envelope to deliver their toxic payload. My talk will focus on recent work where we have been using colicins as probes of fundamental processes in the outer membrane, including demonstrating how these toxins use an intrinsically disordered domain to pass an epitope signal to the periplasm through the porin OmpF. SYMP 02-41 Bacterial Proteins That Modulate Host Membrane Transport Pathways

Craig Roy, Yale School of Medicine, New Haven, Connecticut, US

Bacteria that replicate inside eukaryotic cells typically produce effector proteins having biochemical activities that manipulate host functions. Many of these bacterial effector proteins are delivered into the cytosol of host cells and modulate processes important for creating a vacuole that supports intracellular replication. The biochemical functions of effector proteins from the intracellular pathogens Legionella pneumophila and Coxiella burnetii that play specific roles in controlling host membrane transport will be described. These studies reveal new enzymatic activities and protein structures that demonstrate these pathogens encodes novel effector proteins that can manipulate evolutionarily conserved host proteins that control membrane transport processes, which provide insight into how bacterial pathogens are able to construct a unique vacuole inside host cells. SYMP 02-42 Protein Interactions Regulating Self/Non-Self Recognition in Bacterial Contactdependent Growth Inhibition

David Low, University of California, Santa Barbara, California, US (Chair)

Bacteria that express contact-dependent growth inhibition systems (CDI) outcompete siblings that lack immunity, suggesting that these systems mediate intercellular competition. Notably, CDI systems appear to be restricted to members of the same or closely related species. Analysis of the CDI system from E. coli


INVITED SPEAKER ABSTRACTS EC93, which interacts with β-barrel assembly protein BamA, revealed the molecular basis of this speciesrestriction. The predicted membrane topology of BamA indicates that three of its extracellular loops vary considerably between species, suggesting that loop heterogeneity may control CDI specificity. Our data indicate that BamA loops 6 and 7 form the CdiAEC93- binding epitope and their variation between species restricts CDIEC93 target cell selection. Although BamA loops 6 and 7 vary dramatically between species, these regions are identical in hundreds of E. coli strains, suggesting that BamAE.coli and CdiAEC93 play a role in self/non-self discrimination. Self/non-self discrimination further effected once toxins are injected into neighboring bacterial cells via specific immunity proteins which specifically bind to their cognate toxins. Structural analysis of toxin-immunity complexes reveals similar protein folds for two nuclease toxins with little homology between them. Notably, the cognate immunity proteins bind to different toxin regions to block activity. The self/non-self recognition orchestrated by CDI systems likely plays an important role in the biology of bacterial communities.

Symposium #3 – Frontier High-Throughput Techniques SYMP 03-01 Probing Meiotic Specializations to Genome Decoding by Ribosome Profiling

Gloria Brar, University of California, Berkeley, California, US

Meiosis is a complex and well-conserved program of cellular differentiation. We performed genome-wide measurements of mRNA abundances and new translation using ribosome profiling through yeast meiosis to better understand the molecular basis for the full cellular restructuring that accompanies meiotic chromosome segregation. This quantitative and global view of translation through a developmental process revealed great complexity to the protein complement of these differentiating cells, both in the number and the structure of expressed genes. Nearly every gene in the yeast genome was translated in meiotic cells in a strongly stage-specific manner, including disparate and regulated induction of conserved stress pathways. Translational regulation contributed broadly to this temporal control of protein synthesis timing through several mechanisms, including the use of competitive upstream Open Reading Frames (uORFs) on regulated 5’ leader sequences. Additionally, meiotic translation of thousands of novel short ORFs was observed, expanding our view of what constitutes a coding region even in the most well annotated eukaryotic genome. SYMP 03-02 Visualizing Transcription Pausing and Backtracking Genome-Wide at Nucleotide Resolution

L. Stirling Churchman, Harvard Medical School, Boston, Massachusetts, US

It is now clear that transcription is heavily regulated after RNA polymerase has escaped from promoter regions, mediated largely through transcriptional pausing. These events influence the abundance, covalent composition and cellular fate of the resulting transcript. Dissecting the pathways and genomic elements that control transcriptional pausing requires strategies for following in vivo transcription elongation at similar precision as afforded by in vitro transcriptional assays. Native elongating transcript sequencing - NET-seq, accomplishes this goal by exploiting the extraordinary stability of the DNA-RNARNA polymerase ternary complex to capture nascent transcripts directly from live cells without crosslinking. The identity and abundance of the 3’ end of purified transcripts are revealed by deep sequencing thus providing a quantitative measure of RNAP density with single nucleotide precision. Application of NET-seq in Saccharomyces cerevisiae reveals pervasive polymerase pausing and backtracking throughout the body of transcripts. While similar pause frequencies have been observed on bare DNA, an understanding of their molecular origin has been constrained by the handful of DNA


INVITED SPEAKER ABSTRACTS templates that have been studied in vitro. Analysis of pause sites across the yeast genome uncovers key molecular interactions within the DNA-RNA-RNAP ternary complex that dictate much of the observed pausing and backtracking dynamics. These interactions and their energetics are at the core of regulatory processes that use transcriptional pausing to control gene expression. SYMP 03-03 Structural Analysis of Proteins in Native Environments, Fact or Fiction?

Juri Rappsilber, University of Edinburgh, Scotland, UK No abstract provided.

SYMP 03-04 A Mass-Spectrometry-Based Map of Universally-Shared Animal Protein Complexes

Ed Marcotte,University of Texas - Austin, Austin, Texas, US (Chair)

An important aspect of a protein’s function is its assembly with other proteins into higher molecular weight complexes that are typically the active species in the cell. Knowledge of protein complexes often reveals proteins’ functions, especially when some members of the complex are better characterized than others. This problem is critically important for the core set of proteins shared by every animal cell, which form the basic machinery common to every human cell and the cells of many major model organisms; more than 1/3 of these key proteins are still largely uncharacterized. More generally, maps of protein complexes provide the mechanistic foundations for understanding diverse human traits and diseases. One approach that is proving remarkably powerful is based on systematically mapping protein complexes by native biochemical fractionation and high-throughput mass spectrometry. In this paradigm, protein complexes are computationally inferred from the separation behavior of proteins across many, independent biochemical fractionations. We have launched a major effort to apply this strategy to define the set of major, stable protein complexes shared across animal cells. In initial studies, we separated cultured human cell extracts into >2,000 biochemical fractions, subsequently analyzed by tandem mass spectrometry (nearly 9,000 hours of instrument time), thereby enriching and systematically identifying 622 putative native soluble protein complexes and documenting ~14,000 protein interactions. We have now extended these studies to samples from 7 animal lineages, analyzing >70 biochemical fractionations comprising >7,000 distinct biochemical fractions, in all capturing the biochemical separation behavior of ~12,000 animal proteins. I’ll describe our progress analyzing these data and our efforts to define the core set of stable protein complexes conserved across metazoa, as well to measure the evolutionary conservation, divergence, and rewiring of protein complexes across full animal proteomes.

Symposium #4 – Proteins in Altered States SYMP 04-05 Protein Aggregation Done Right: The Biogenesis of Functional Amyloids

Matthew Chapman, University of Michigan, Ann Arbor, Michigan, US

Organisms that span nearly every facet of cellular life produce functional amyloids. Microbial functional amyloids fulfill essential physiological roles for the bug, while also providing a sophisticated suite of genetic and biochemical tools for understanding how cells coordinate and control amyloid formation. Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. Such controlled amyloidogenesis requires that the fiber subunit, CsgA, be chaperoned prior to secretion to avoid premature oligomerization and associated toxicity. We found that


INVITED SPEAKER ABSTRACTS CsgC is a highly potent inhibitor of CsgA amyloid formation. The loss of CsgC resulted in the accumulation of toxic intracellular CsgA amyloid in vivo. Furthermore, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations by preventing β-sheet-rich oligomerization in vitro. CsgC also displayed amyloid inhibitory activity against related bacterial client proteins such as CsgA-derived peptides and sequence homologues. Interestingly, CsgC inhibited amyloid assembly of α-synuclein, but not Aβ42. We identified a common Q-X-G-X1/2-N-X5-Q motif in CsgC client proteins that is not found Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids. SYMP 04-06 Structural Malleability of Intrinsically Disordered Proteins Underlying Alternative Functional States

Peter Tompa, Vrije Universiteit Brussel, Brussels, Belgium

Intrinsically disordered proteins (IDPs) and complex multidomain proteins are characterized by a dynamic ensemble of conformations that cannot be unequivocally described by traditional static terms of structural biology (1). Their structural ensemble is dynamic and malleable, enabling them to adapt to a wide variety of regulatory signals. The quantitative description of structural ensembles has just started (2) and here we will show that it holds the promise to elucidate complex protein regulatory phenomena (3), such as moonlighting (4) and allostery (5) in the “supertertiary” structure (6) of proteins. 1) Tompa, P. (2011) Unstructural biology coming of age. Curr. Opin. Struct. Biol. 21: 419. 2) Varadi, M. et al. (2014) pE-DB, a database of protein structural ensembles. Nucl. Acids. Res. Nucleic Acids Res. 42: D326. 3) Tompa, P., Varadi, M. (2014) Predicting the predictive power of IDP ensembles. Structure 22: 177. 4) Tompa, P., Szász, Cs. and Buday, L. (2005) Structural disorder throws new light on moonlighting. Trends Biochem. Sci. 30, 484-489 5) Tompa, P. (2014) Multisteric regulation by structural disorder in modular signaling proteins: an extension of the concept of allostery. Chem. Rev. (Epub ehaed of print) 6) Tompa, P. (2012) On the supertertiary structure of proteins. Nature Chem. Biol. 18, 597. SYMP 04-07 Amyloid Assemblies and Their Interactions With Cellular Components

Helen Saibil, Birkbeck College, London, UK

Protein misfolding into amyloid is closely associated with progressive, fatal and as yet incurable neurodegenerative diseases. We are beginning to understand some of the interaction partners in the protein quality control systems whose failure leads to aggregation and toxicity, but a general principle is lacking for the mechanisms and targets of toxicity. We have been using 3D electron microscopy to study these processes in vitro and in cells. In vitro model of membrane damage: Misfolded oligomers and ends of fragmented amyloid fibrils have been implicated in damage to cellular membranes. Cryo-electron tomography of liposomes being attacked by short fibrils of beta-2 microglobulin reveals that binding of fibril ends to the liposome surface results in distortion and breakage of the membranes, by creating pointed extrusions and removal of the outer leaflet of the lipid bilayer (Milanesi et al, PNAS 109, 20455, 2012). Structure of toxic Abeta oligomers: Abeta protofibrils, precursors to mature amyloid fibrils, are correlated with inhibition of synaptic long term potentiation via binding to cellular PrP (Nicoll et al, Nature Comms 4:2416, 2013). We have used single particle EM and tomography to show that these assemblies have an unexpected distinctive, flexible helical nanotube structure. Effects of chaperone-disaggregase systems on yeast prions: Deposition of amyloid aggregates in cells is thought to result from the balance between protein misfolding/aggregation and the actions of molecular chaperones and disaggregases in preventing or reversing these processes. In order to examine this balance in vivo, we are studying the three-dimensional arrangement of model prion aggregates in yeast cells. [PSI+] and [RNQ+] prions are deposited as clusters of fibrils in distinct, but not membrane-bound, assemblies in the yeast cytoplasm.


INVITED SPEAKER ABSTRACTS The polymerization of amyloid forming regions and fibril breakdown by disaggregases also results in transmission of the prion phenotype to daughter cells and mating partners. Using electron tomography of yeast cell sections containing prions, prepared by high pressure freezing and freeze-substitution or cryo sectioning, we observe changes in the assembly state of the fibril clusters when levels of the Hsp70 system chaperones or the disaggregase Hsp104 are altered. SYMP 04-08 Dynamic Complexes, Folding And Phase Separation Of Disordered Proteins In Biological Regulation

Julie Forman-Kay, The Hospital for Sick Children, Toronto, Ontario, Canada (Chair)

Intrinsically disordered proteins (IDPs) and regions play important biological roles, including in translation and RNA processing. They lack stable structure and can adopt different structural states. Many IDPs are monomeric or engage in discrete interactions, folding upon binding or retaining significant disorder in the bound state. The disordered 4E-BP2 interacts tightly with eIF4E to suppress cap-dependent translation initiation. Binding involves the canonical YXXXXLΦ motif, which undergoes a disorder-to-helix transition, as well as a secondary site, leading to a dynamic bipartite complex enabling kinase accessibility. Phosphorylation induces folding of part of 4E-BP2, sequestering the YXXXXLΦ motif and reducing eIF4E binding. Phosphorylation-induced folding of 4E-BPs exemplifies a new paradigm for IDP-mediated regulation. Other IDPs are involved in large-scale association having different degrees of order, from more defined fibers to disordered liquid states. These latter can provide the matrix for cellular membrane-less organelles. The Ddx4 RNA DEAD-box helicase is involved in one such organelle, germ granules, functioning in RNA processing in spermatogenesis. When expressed in cells, the protein forms micronsized organelles. In vitro, the disordered N-terminal 250 residues phase separate to form droplets with similar properties. Perturbations of Ddx4 Phe or Arg residues disrupt phase separation, suggesting a role for multi-valent cation-pi interactions. Ongoing biophysical studies of Ddx4 will illuminate the mechanisms of the biogenesis and disassembly of membrane-less organelles. Studies of the variety of IDP accessible conformational states and their regulated transitions will be critical for understanding translation and RNA processing, as well as many other cellular functions.

Symposium #5 – Protein Evolution SYMP 05-24 Evolution in a Test Tube Yields De Novo Enzymes With Unusual Structure and Dynamics

Burckhard Seelig, University of Minnesota, St. Paul, Minnesota, US

Billions of years of natural evolution yielded enzymes that catalyze a wide range of chemical reactions facilitated by their intricate three-dimensional structures. Despite the rapidly increasing wealth of information on protein structures we currently have only a limited understanding of how new protein folds emerge in nature. At the same time, the engineering of entirely new protein scaffolds that are able to carry out chemical catalysis remains a major challenge in enzyme design. We developed a general strategy to create artificial enzymes by harnessing the functional diversity of very large libraries of randomized proteins. We isolated enzymes that catalyze a ligation reaction for which no natural enzymes are known. The starting library of 4 trillion mutants was based on a non-catalytic zinc finger scaffold. To our surprise, the new enzymes isolated by in vitro selection and evolution had entirely lost the starting scaffold and adopted a new fold instead. Different from classic natural proteins, the enzyme lacks secondary structural motifs and shows high conformational dynamics. Contrary to common belief, the unique properties of this novel fold demonstrate that a small protein structure with suitable flexibility is


INVITED SPEAKER ABSTRACTS sufficient to carry out enzymatic function. This example emphasizes the power of an evolutionary approach, which can yield useful novel enzymes that nature has never seen before. SYMP 05-25 Sequence-Function-Fitness Landscapes Viewed by Massively Parallel Sequencing Approaches

Dan Bolon, University of Massachusetts Medical School, Worcester, Massachusetts, US

Current sequencing technology enables experimental quantification of mutational landscapes that describe the functional or fitness effects of all possible point mutations in a gene. These mutational landscapes provide insights into biochemical and biophysical mechanisms that govern protein evolution. This presentation will include recent studies of fitness landscapes in yeast, mammalian cells in culture and influenza virus. SYMP 05-26 Evolution of Novel Components of the Bacterial Flagellar Motor

Morgan Beeby, Imperial College London, London, UK

Bacteria propel themselves in beneficial directions using cell wall-embedded rotary motors that spin helical propellers called flagella. Studies of the Escherichia coli motor have revealed a large nanomachine composed of hundreds of proteins — from approximately twenty protein families — that harness protonmotive force to produce torque. Using electron cryo-tomography to perform 3D in situ imaging we recently discovered that many bacteria have motors considerably more complex than the ‘normal’ core motor found in E. coli. Why have some bacteria recruited additional proteins to a core that is already fully functional? I will describe phylogenetic and structural work to understand this question. We are currently focusing on a family of large novel structural components found in a variety of proteobacteria including the Vibrio, Campylobacter, and Helicobacter genera. By combining bacterial genetics, phylogenetics and electron cryo-tomography we have identified the proteins that form these large structures. Combining these results we have developed a model for the recruitment of these additional proteins, and have been able to speculate on the effect of these proteins on motor mechanics, in turn highlighting possible evolutionary driving forces behind protein recruitment. SYMP 05-27 The Remarkable Pliability and Promiscuity of Specialized Metabolism

Joseph P. Noel, Salk Institute for Biological Studies/HHMI, La Jolla, California, US (Chair) No abstract provided.

Symposium #6 – Proteins in Disease & Therapeutics Sponsored by Bristol-Myers Squibb

SYMP 06-43 Structural Basis of Broad Neutralization of Viral Pathogens

Ian Wilson, The Scripps Research Institute, La Jolla, California, US

Influenza, Hepatitis C, and HIV-1 continue to constitute significant threats to global health. We have structurally and functionally characterized their viral antigens and interaction with broadly neutralizing antibodies (bnAbs). A number of antibodies have recently been identified that are extremely potent and neutralize across multiple subtypes and types of these viruses through binding to functionally conserved sites, such as the receptor binding site or the fusion domain. For HIV-1, novel epitopes involve glycans.


INVITED SPEAKER ABSTRACTS The glycan-dependent Abs have unique features that enable them to penetrate the glycan shield on the viral Env protein and bind extensive and complex epitopes that consist of sugars and underlying protein segments on gp120 on HIV-1 Env. This structural information is now being used to aid in vaccine design for HIV-1, HCV and for a more universal flu vaccine. IAW is supported by NIH grants AI100663, AI082362, AI84817, AI099275 and GM094586 and the Crucell Vaccine Institute. SYMP 06-44 The HIV-1 Viral Spike: Conformational Machine for Entry and Evasion

Peter Kwong, NAIAD - NIH, Bethesda, Maryland, US

The HIV-1 envelope (Env) glycoprotein spike, which comprises three gp120 and three gp41 subunits, is a type 1 fusion machine, which uses the energy stored in its metastable fold to facilitate virus-host entry by fusing viral and cellular membranes. The prefusion spike exists in at least two conformations: a neutralization-resistant ground state and a CD4 receptor-bound intermediate state, while the postfusion spike consists of only the gp41 subunit, with the gp120 subunit having been shed from the virion surface during the fusion process. Over the past 20 years, substantial structural information has been obtained on both gp120 and gp41, in pre- and postfusion conformations. However the prefusion ground state has resisted atomic-level analysis. Here we used neutralizing antibodies PGT122 and 35O22 to capture the viral spike in its ground state. Crystals were obtained of these two antibodies in complex with a soluble Env trimer construct (BG505.SOSIP. 664), and the structure determined to 3.5 Å resolution. Prefusion gp41 folds around extended N- and C-terminal beta-strands of gp120. Comparison of ground state and CD4bound conformations of gp120 reveal the structural rearrangements induced by receptor. In addition to a description of the trimeric ground-state structure of the HIV-1 Env ectodomain, implications for HIV-1 vaccine design will be discussed. SYMP 06-45 Immune Sensing of Vitamin B Metabolites

Jamie Rossjohn, Monash University, Clayton, Victoria, Australia

The T cell receptor complex, expressed on the surface of T-cells, comprises the antigen-specific heterodimeric αβ T-cell receptor (TCR) that is associated with the CD3 complex. Ligation of the TCR by an Antigen (Ag) presenting molecule initiates T-cell signalling. While is conventionally considered that TCRs interacts with peptides bound to the Major Histocompatibility Complex (MHC), TCRs can also bind lipidbased Ags bound by CD1 family members. Moreover, we have recently established how T-cells, namely Mucosal-associated invariant T-cells, can recognise microbial-based vitamin B metabolites when bound to the MHC-I-like molecule, MR1 (refs 1,2) . I shall discuss the structural basis of vitamin B metabolite presentation and recognition of this MAIT TCR-MR1 system. SYMP 06-46 Engineering Improved Antibodies Against HIV

Pamela Bjorkman, California Institute of Technology/HHMI, Pasadena, California, US

Over 30 years after the emergence of HIV-1, there is no effective vaccine, and AIDS remains an important threat to global public health. Following infection by HIV-1, the host immune response is unable to clear the virus due to a variety of factors, including rapid viral mutation and the establishment of latent reservoirs. The only target of neutralizing antibodies is the trimeric envelope (Env) spike complex, but HIV1 can usually evade anti-spike antibodies due to rapid mutation of its two spike glycoproteins, gp120 and gp41, and structural features that allow the spike to hide conserved epitopes. Because a completely protective vaccine against HIV has not been found, possible prevention/treatment options involving delivery of broadly neutralizing antibodies (bNAbs) identified in a minority of HIV-infected individuals are


INVITED SPEAKER ABSTRACTS being considered. bNAbs that target conserved epitopes on the HIV envelope spike can prevent infection in animal models, delay rebound of HIV after cessation of anti-retroviral drugs, and treat an ongoing infection. Enhancing the efficacy of bNAbs; in particular, designing bNAbs that retain potency against escape mutants selected during exposure to bNAbs, would facilitate their use as therapeutics. We previously used structure-based design to create NIH45-46G54W, a CD4-binding site (CD4bs) antibody with superior potency and/or breadth compared with other bNAbs. Here we report even more effective variants of NIH45-46G54W designed using analyses of the NIH45-46/gp120 complex structure and sequences of antibody-resistant HIV clones. One mutant, 45-46m2, neutralizes 96% of HIV strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti-HIV antibody to date. A detailed description of its mechanism is presented based on a 45-46m2/gp120 crystal structure. A second mutant, 45-46m7, designed to thwart resistance from NIH45-46G54W due to mutations in a V5/loop D gp120 consensus sequence, restores neutralization of HIV consensus sequence mutants, thus effectively targeting a common route of HIV escape. In combination, almost all HIV isolates are effectively neutralized, reducing the possible routes for the evolution of fit viral escape mutants.

Symposium #7 – Chemical Biology & Enzymology SYMP 07-09 Spatially-Resolved Proteomic Mapping of Mitochondria in Living Cells Using an Engineered Peroxidase Reporter

Alice Ting, Massachusetts Institute of Technology, Cambridge, Massachusetts, US No abstract provided. SYMP 07-10 2014 P ROTEIN SCIENCE BEST PAPER AWARD WINNER TALK 1

Mark Landau, Yale University, New Haven, Connecticut, US Mark J. Landau, Hitesh Sharma, Karen S. Anderson (2013)

Selective Peptide Inhibitors of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase From Toxoplasma Gondii Provide Insights Into Domain-Domain Communication and Allosteric Regulation. Protein Sci. 22:1161-1173. SYMP 07-11 Structure-Based Screens for Protein De-orphanization

Brian Shoichet1, 2, 1University of Toronto, Toronto, Ontario, Canada, 2University of California-San Francisco, San Francisco, California, US

For only small fraction of sequenced genes is the function of their encoded proteins known, or even predicted with confidence. Here we investigate a structure-based approach to predicting enzyme and receptor activity. Libraries of metabolites or drug-like molecules are screened against the structures of enzymes or G Protein-Coupled Receptors (GPCRs), respectively, and high-scoring molecules are tested experimentally as either substrates or agonists/antagonists/allosteric modulators. This approach has discovered substrates against enzymes of the amidohydrolase and enolase superfamilies. Very recently we have expanded this work to orphan GPCRs, in collaboration with Bryan Roth’s laboratory. A combined experimental and computational strategy to deorphanize GPR68 will be discussed, with applications to the role of this orphan GPCR in vivo, in a mouse model.


INVITED SPEAKER ABSTRACTS SYMP 07-12 Molecular DNA Devices in Living Systems

Yamuna Krishnan, National Center for Biological Sciences, Bangalore, India

Due to its nanoscale dimensions and ability to self-assemble via specific base pairing, DNA is rapidly taking on a new aspect where it is finding use as a construction element for architecture on the nanoscale.1 Structural DNA nanotechnology has yielded architectures of exquisite complexity and functionality in vitro. However, till 2009, the functionality of such synthetic DNA-based devices in living organisms remained elusive. Work from my group the last few years has bridged this gap where, we have chosen architecturally simple, DNA-based molecular devices and shown their functionality in complex living environments. Using two examples, from our lab, one of a rigid, DNA polyhedron2 and the other a molecular switch3 that functions as a pH sensor I will illustrate the potential of DNA based molecular devices as unique tools with which to interrogate living systems. SYMP 07-13 Activity-based Proteomics - Applications for Enzyme and Inhibitor Discovery

Benjamin Cravatt, III, The Scripps Research Institute, La Jolla, California, US (Chair)

Genome sequencing projects have revealed that eukaryotic and prokaryotic organisms universally possess a huge number of uncharacterized enzymes. The functional annotation of enzymatic pathways thus represents a grand challenge for researchers in the genome era. To address this problem, we have introduced chemical proteomic and metabolomic technologies that globally profile enzyme activities in complex biological systems. These methods include activity-based protein profiling (ABPP), which utilizes active site-directed chemical probes to determine the functional state of large numbers of enzymes in native proteomes. In this lecture, I will describe the application of ABPP and complementary proteomic methods to discover and functionally annotate enzyme activities in mammalian physiology and disease. I will also present competitive ABPP platforms for developing selective inhibitors for poorly characterized enzymes and discuss ongoing challenges that face researchers interested in assigning protein function using chemoproteomic methods.

Symposium # 8 – Cellular Structures SYMP 08-14 New Insights Into Microtubule Mechanics

Manuel Théry1, 2, 3, 1LPCV, Grenoble, France, 2iRTSV, Grenoble, France, 3DSV / CEA, Grenoble, France

Tubulin dimers self-organize into hollow tubes called microtubules. These microtubules support numerous cellular functions such as intra-cellular transport or mitotic spindle assembly. Therefore the architecture of the microtubule network is essential in the regulation of cell physiology. Unfortunately our understanding of microtubule mechanical properties is surprisingly limited. Indeed the lack of appropriated experimental approaches has hampered our capacity to investigate these properties. The thermal fluctuations of microtubules in cell-free extracts have shown that they were extremely rigid, with a persistent length a thousand times larger than cell size. However they appear highly curved in cells which somehow contradict in vitro measurements. We developed a new microfluidic device to apply controled constraint on microtubules and observed their bending properties. Thereby we revealed new and unexpected features of microtubule mechanics.


INVITED SPEAKER ABSTRACTS SYMP 08-15 LORNE CONFERENCE ON PROTEIN STRUCTURE AND FUNCTION EXCHANGE SPEAKER Protein Degradation in Bacteria and Mitochondria: When the N-Terminus Signals the End

Kaye Truscott, La Trobe Institute for Molecular Science, Melbourne, Victoria, Australia No abstract provided.

SYMP 08-16 A Protein Interaction Network That Directs Human Cytoplasmic Dynein to Microtubule Ends

Thomas Surrey1, 2 , 1London Research Institute, London, United Kingdom, 2Cancer Research UK, London, United Kingdom

Growing microtubule ends recruit several proteins collectively called +TIPs which confer local functions to the microtubule cytoskeleton. These +TIPs form dynamic protein interaction networks with competitive and hierarchical interactions. The rules that determine which of the multiple +TIPs bind to the limited number of available binding sites in microtubule end regions are only poorly understood. Here we investigated how the major human minus end directed motor dynein that is also an important +TIP, is targeted to growing microtubule ends in the presence of competitors. Using a TIRF microscopy-based in vitro reconstitution assay, we found that a hierarchical recruitment mode targets the large dynactin subunit p150glued to growing microtubule ends via EB1 and CLIP-170 in the presence of competing SxIP motif-containing peptides. The human dynein complex is then targeted to growing microtubule ends through an interaction of the tail domain of dynein with p150glued. Our results show how the connectivity and hierarchy within dynamic +TIP networks are orchestrated. SYMP 08-17 Mechanisms of Mitosis and Intracellular Scaling in Xenopus

Rebecca Heald, University of California-Berkeley, Berkeley, California, US (Chair)

The goal of my laboratory is to elucidate the molecular mechanisms of cell division and morphogenesis. We use cytoplasmic extracts prepared from eggs of the frog Xenopus laevis to reconstitute and study mitotic chromosome condensation and spindle assembly and function in vitro, applying imaging, biochemical, and biophysical approaches. To study mechanisms of spindle and organelle size control, we take advantage of a smaller, related frog, Xenopus tropicalis, to investigate interspecies scaling, and extracts prepared from fertilized eggs at different stages of embryogenesis to study developmental scaling. Our research will provide novel insight into how cell/organelle scaling contributes to intracellular morphogenesis and cell division, processes essential for viability and development, and defective in human diseases including cancer.

Symposium #9 – Protein Engineering & Synthetic Biology SYMP 09-28 No title provided Christopher A. Voigt, Massachusetts Institute of Technology, Cambridge, Massachusetts, US

No abstract provided.

SYMP 09-29


INVITED SPEAKER ABSTRACTS Sustaining Life With Proteins Designed De Novo

Michael Hecht, Princeton University, Cambridge, Massachusetts, US

The collection of all protein sequences that ever existed on earth represents a tiny fraction of the sequence space that is possible. From the enormous diversity of possible sequences, nature has selected very small collections of 'molecular parts' to sustain life: Only ~4,000 genes in E. coli and ~23,000 in humans. These considerations may lead to the assumption that natural sequences are somehow special. Is this true? Or can sequences designed ‘from scratch’ also provide essential functions necessary to sustain life? To address these questions, we designed and constructed millions of artificial proteins encoded by a library of synthetic genes. Structural studies show that many of our novel proteins fold into stable 3dimensional structures. Biochemical assays demonstrate that many of them bind biologically relevant molecules, and genetic studies show that several of these novel proteins function in vivo to provide functions necessary to sustain the growth of E. coli. These results suggest that (i) the molecular toolkit for life need not be limited to genes and proteins that already exist in nature; (ii) artificial genomes and proteomes can be built from non-natural sequences; and (iii) synthetic organisms relying on de novo designed proteins may be possible. SYMP 09-30 Metal-Directed Protein Evolution

Akif Tezcan, University of California-San Diego, San Diego, California, US

Metals, especially those of the d-block (i.e., transition metals), play irreplaceable roles in biology, stabilizing protein structures and enabling biochemical transformations that would be impossible in their absence. Given their dual, structure- and function-building power, it is natural to ask whether transition metals can also accelerate/catalyze the evolution of proteins. In this presentation, I will talk about our group's efforts in exploiting metal coordination chemistry for building and evolving new biological structures and functions. SYMP 09-31 De Novo Protein Structures and Assemblies by Design

Dek Woolfson, University of Bristol, Clifton, United Kingdom (Chair)

We have developed a toolkit of de novo peptides (1). These can be used as building blocks for the rapid construction of new protein-like structures and supramolecular assemblies. This talk will illustrate the utility of this approach with two examples for making nanoscale peptide-based pores (2) and selfassembled cages (3). Potential applications of the structures and materials achieved span nanoscience, synthetic biology and biotechnology. 1 .A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology JM Fletcher et al. ACS Synthetic Biology 6, 240-250 (2012) 2 .A de novo peptide hexamer with a mutable channel NR Zaccai et al. Nature Chemical Biology 7, 935-941 (2011) 3. Self-assembling cages from coiledcoil peptide modules. JM Fletcher et al. Science 340, 595-599 (2013)


INVITED SPEAKER ABSTRACTS Symposium #10 – Proteins in Dynamic & Driven Processes SYMP 10-32 Deciphering Protein Dynamics During Endocytic Budding by Time-Resolved Electron Microscopy

Maria Isabel Geli Fernandez-Penaflor, Molecular Biology Institute of Barcelona, Barcelona, Spain

Endocytic membrane budding from the plasma membrane requires the coordinated recruitment and function of more than 50 proteins which concentrate cargo, deform the lipid bilayer and effect fission to generate the primary endocytic vesicle. Molecular models explaining the process are mainly based on the information derived from the live-cell fluorescence microscopy, which offers a regular resolution of about 200 nm, and the structural and biochemical information of the purified components involved. However, understanding how endocytic proteins really effect membrane deformation within the cells would require the dynamic view of the process at nearly atomic resolution. Even though this goal is far from reachable at the moment, we have made an effort to fill the resolution GAP between the fluorescence microscopy and the structural biology by developing a methodology that integrates our knowledge derived from the livecell imaging with the statistical processing of parameters describing the shape of endocytic invaginations and the position of gold particles labelling 18 different endocytic proteins, on ultrathin section of the yeast S. cerevisiae. This approach has allowed us to describe the dynamics of the endocytic machinery coupled to the deformation of the lipid bilayer with a resolution down to 7 nm, solving important issues in the field such as the point of emergence of membrane curvature relative to the recruitment of the endocytic proteins or the exact function of actin polymerization, the myosin motor activity or the BAR proteins during vesicle budding. SYMP 10-33 2014 P ROTEIN SCIENCE BEST PAPER AWARD WINNER TALK 2

Brian Ziemba, University of Colorado, Boulder, Colorado, US Brian P. Ziemba, Emma J. Murphy, Hanna T. Edlin, David N.M. Jones (2013) A Novel Mechanism of Ligand Binding and Release in the Odorant Binding Protein 20 From the Malaria Mosquito Anopheles Gambiae. Protein Sci. 22:11-21.

SYMP 10-34 Spatial Regulation of Molecular Motors

Samara Reck-Petersen, Harvard Medical School, Boston, Massachusetts, US

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. In this work we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins - homologs of Lis1 and Clip170 – are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the microtubule plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion though its microtubule-binding domain. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein’s intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track.


INVITED SPEAKER ABSTRACTS SYMP 10-35 The Bacterial Magnesium Channel CorA – Dynamic Ways to Translocate Divalent Cations

Emil F. Pai, University of Toronto, Toronto, Ontario, Canada

The first crystal structure of Thermotoga maritima CorA (TmCorA) determined in the presence of high concentrations of divalent ions revealed a rotationally symmetric pentamer [Lunin et al., Nature, 440, 833; Eshagi et al., Science, 313, 354; Payandeh & Pai, EMBO J, 25, 3762, 2006]. Although amino acids crucial in gating and regulation could be identified [Payandeh et al., JBC, 283, 11721, 2008] the detailed molecular mechanism of channel opening remains unclear. Initial MD calculations pointed to an iris-like movement [Chakrabarti et al., Biophys J, 98, 784, 2010] upon channel opening. In the crystal structure of a mutant CorA, a hydrated Mg2+ ion binds to the periplasmic GMN motif, revealing clues of selectivity. Without Mg2+, TmCorA displays radial and lateral tilts of protomers that lead to an asymmetric arrangement of subunits and bending of the central, pore-lining helix. MD simulations support these movements, including a most unusual bell-like deflection. Mass spectrometric analysis confirms that major proteolytic cleavage and disulfide formation occur within a region that is selectively exposed by such a motion [Pfoh et al., PNAS, 109, 18809, 2012]. A recent crystal structure of a pentameric arrangement of the cytosolic part of TmCorA suggests counterbalancing forces located in the trans-membrane part of the channel helices and the binding sites of divalent ions between the subunits. Our results indicate a sequential allosteric model of regulation; intracellular Mg2+ binding locks TmCorA in a symmetric, transportincompetent conformation and loss of intracellular divalent ions causes an asymmetric, potentially influxcompetent conformation. SYMP 10-36 Engineering Proteins for Visualization and Control of Signaling Networks In Vivo

Klaus Hahn, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, US (Chair)

Cell motility requires the orchestration of many dynamic cellular systems, which can only be fully understood through quantitation of protein activity in living cells and animals. This talk will describe new tools to visualize and manipulate signaling in vivo, using Rho family GTPase networks and cell motility as test beds. Multiplexed imaging of biosensors and computational image analysis will be used to examine the coordination of GTPases. The role of specific protein activation events regulating coordination will be probed using engineered allosteric switches and different methods to control the dynamics of protein activity with light. New technologies will include methods to direct activated kinases to specific targets, broadly applicable methods to control sequestration of proteins at intracellular membranes with light, and improvements in biosensor design for applications in animals.

Symposium #11 – Protein Degradation SYMP 11-18 Ubiquitin-Dependent Regulation of Proliferation and Differentiation

Michael Rape, University of California-Berkeley, Berkeley, California, US Abstract not provided. SYMP 11-19 Investigating the Mechanisms of the Proteasome by Cryo-EM

Paula da Fonseca, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom


INVITED SPEAKER ABSTRACTS In eukaryotes the ubiquitin/proteasome pathway is responsible for the controlled targeting and degradation of a wide range of proteins, including key cellular regulators such as those controlling cell cycle progression and apoptosis. The 26S proteasome is a large ATP-dependent protease responsible for the highly regulated degradation of proteins specifically targeted by ubiquitin. It comprises a proteolytic 20S core associated to 19S regulatory particles, which specifically recognise, unfold and translocate the substrate proteins into the proteolytic chamber. The 26S proteasome is a well-recognised target for cancer therapy and its deregulation is associated with neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. However, its functional mechanisms are still significantly elusive. We determined the structure of the human 26S proteasome by electron cryo-microscopy (cryo-EM) and single particle analysis where secondary structure elements are clearly identified. By combining our cryo-EM map with data from x-ray crystallography and structural modelling we revealed the organisation of the 19S regulatory particle subunits and presented a molecular model for the complete human 26S proteasome. Through recent developments in the field of structural electron microscopy, triggered by the availability of improved electron microscopes and direct detectors, it is now possible to achieve resolutions that used to be attainable only by crystallography or NMR methods. Such developments are now being used to explore the functional mechanisms of the proteasome. SYMP 11-20 Application of Small Molecule Probes to the Study of Protease Function

Matthew Bogyo, Stanford University, Stanford, California, US

Proteases are enzymes that primarily function by degrading protein substrates. Since this process is irreversible, proteases must be carefully regulated within cells and organisms in order to prevent undesired consequences. Furthermore, proteases often play pathogenic roles in common human diseases such as cancer, asthma, arthritis and atherosclerosis. Over the past decade, my laboratory has developed a series of small molecule activity-based probes (ABPs) that specifically bind to the active form of protease targets through an enzyme catalyzed chemical reaction. These reagents can be used to enrich complex proteomic samples for monitoring of global patterns of protease activity as well as to directly image protease activity in live cells and whole animals. They can also be used to monitor the efficacy and selectivity of small molecule drugs. We are currently applying these probes to study the role of specific proteases in tumor growth and metastasis in mouse models of cancer as well as during the process of inflammation in mouse models of atherosclerosis, asthma and pulmonary fibrosis. In addition, we have applied covalent small molecules to identify important regulators of parasite pathogenesis. Recent advances in these projects will be presented.

Symposium #12 – Membrane Proteins & Receptors SYMP 12-37 Mechanism-based Tuning of Cytokine Receptor Signaling

Chris Garcia, Stanford University School of Medicine/HHMI, Stanford, California, US

In my lecture I will address a fundamental question in receptor biology: Does the dimeric geometry of a ligand-receptor complex influence signaling ? or is receptor dimerization a signaling “on/off” switch ? In particular, Cytokines are known to dimerize their receptor ectodomains to initiate signaling, but it is unclear how receptor dimer architecture impacts signaling, or if dimer geometry can be manipulated to ‘tune’ signaling. In order to directly interrogate this question, we have modulated cytokine receptor signaling by altering the orientation and proximity of a cytokine receptor dimer using diabodies as


INVITED SPEAKER ABSTRACTS surrogate ligands. We directly correlate the intact receptor dimer structures, determined crystallographically, with functional outcome and we find that indeed such systems are ‘tunable’ by remodeling dimer topology and distance. Furthermore, we show that these extracellular-directed diabodies can overcome intracellular oncogenic mutations in Jak2. Thus, induced alterations in receptor dimer geometry can ‘tune’ signaling and extracellular ligands that enforce large receptor inter-subunit distances can counteract intracellular oncogenic ligand-independent receptor activation. SYMP 12-38 From Liposomes to Fliposomes: In Vitro Reconstitution of Lipid-Dependent Dual Topology and Post-Assembly Topological Switching of a Membrane Protein

William Dowhan, University of Texas Houston Medical School, Houston, Texas, US

The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. Systematic alteration of membrane lipid composition uncovered a role for direct lipid-protein interactions in determining initial topogenesis of and dynamic post-assembly topological changes in protein transmembrane domains (TMDs). Using an in vitro proteoliposome system in which lipid composition can be systematically controlled before (liposomes) and after (fliposomes) protein reconstitution, we determined the minimum and sufficient requirements for a membrane protein to reorganize between topologically distinct states. Increasing or decreasing phosphatidylethanolamine levels after initial protein reconstitution resulted in re-orientation of the original mixture of native and inverted topological conformers to favor the native or inverted protein conformer, respectively. Therefore, reversible “flipping” of TMDs is dependent on direct lipid-protein interactions independent of molecular chaperone or translocon involvement. Using Förster resonance energy transfer methods, we determined that the rates of TMD flipping in both directions were on a second scale and occurred rapidly after proteoliposome lipid composition was changed. These observations demonstrate a potential thermodynamically driven biological switch for generating dynamic structural and functional heterogeneity for a protein within cells dependent only on the properties of the protein and its lipid environment. Funded by NIH grant GM20478 (WD). SYMP 12-39 Structural and Mechanistic Diversity of ABC Transporters

Douglas Rees, California Institute of Technology/HHMI, Pasadena, California, US

ATP Binding Cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins responsible for the ATP powered membrane translocation of a wide variety of substrates. The highly conserved ABC domains defining the superfamily provide the nucleotide-powered engine that drives transport. In contrast, the transmembrane domains creating the translocation pathway are more variable, with three distinct folds currently recognized. Structural analyses of the high affinity methionine MetNI importer and of a bacterial homologue of the mitochondrial Atm1 exporter will be discussed within the mechanistic framework of the alternating access model. The interconversion of outward and inward facing conformations of the translocation pathway is coupled to the switching between open and closed interfaces of the ABC subunits that are associated with distinct nucleotide states. As observed for MetNI, additional domains may be present that can regulate transport activity. Building on this qualitative molecular framework for deciphering the transport cycle, an important goal is to develop quantitative models that detail the kinetic and molecular mechanisms by which ABC transporters utilize the binding and hydrolysis of ATP to power substrate translocation.


POSTER ABSTRACTS Poster Session: Proteins in Disease & Therapeutics POST 10-50 Coupling the Antimicrobial Action of a Novel Un-natural Cationic Amphiphilic Polyproline Helix with its Cell Penetrating Ability to Target Intracellular Bacteria

Manish Nepal , Jean Chmielewski, Purdue University, West Lafayette, Indiana, US

The rise and emergence of multidrug resistance bacteria requires advancements in novel anti-microbial agent design. An additional challenge to development of anti-microbial agent is that bacteria such as Salmonella, Listeria, and Brucella reproduce inside mammalian cells and are inaccessible to many antimicrobial drugs (β-lactams and animoglycosides). Herein we present a cationic amphiphilic polyproline helix (CAPH), Fl-PLPRPR-5, that exhibits antibacterial action against a broad spectrum of pathogenic Gram positive (Listeria monocytogenes; MIC 8 µM) and Gram negative (Acinetobacter baumannii; MIC 2 µM) bacteria. Additionally, the agent also displays superior cell penetration with the macrophage cells; the “safe haven” for some of the intracellular resistant bacterial strains. Preliminary studies have confirmed the internalization of Fl-PLPRPR-5 in E. coli and its non-membrane lytic mode of action. Fl-PLPRPR-5 demonstrates minimal hemolysis of human erythrocyte cells and also resists enzymatic degradation against trypsin. Fl-PLPRPR-5 rescued J774A. POST 10-51 Rational Design of Thefurin Cleavage Site of an Anti-CD22 Recombinant Immunotoxin Based on Pseudomonas Exotoxin A

John E. Weldon1, 2, Ira Pastan2 1 Department of Biological Sciences, Towson University, Towson, Maryland, US, 2Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, US

Recombinant immunotoxins (RITs) are fusion proteins that combine antibodies with protein toxins in order to selectively target and kill cells. RITs that join antibodies against tumor-associated antigens with the bacterial toxin Pseudomonas exotoxin A (PE) are currently undergoing clinical trials for the treatment of cancers, and have achieved some notable successes. The general utility of RITs, however, is limited by several factors that include the requirement for a suitable antigen target, the formation of neutralizing antibodies in patients, and the necessity for potent targeted activity on tumor cells. These and other limitations are areas of active exploration to improve the efficacy of PE-based RITs. One potential enhancement strategy involves rational engineering of RITs utilizing our understanding of the PE intoxication pathway. During intoxication PE is internalized by receptor-mediated endocytosis, traffics through the endolysosomal system to the Golgi, and undergoes retrograde transport to the endoplasmic reticulum. PE is subsequently exported into the cytoplasm, where it encounters and ADP-ribosylates elongation factor 2, halting protein synthesis and leading to cell death. A step in the pathway that may be amenable to protein engineering is the cleavage of PE by the intracellular protease furin during endolysosomal trafficking. We suspect that furin cleavage may be a rate-limiting step in the intoxication pathway of PE, and have targeted the furin cleavage site for engineering in an effort to enhance the cytotoxicity of PE-based RITs. Our findings suggest that furin cleavage efficiency and RIT cytotoxicity are not directly correlated, but overall cytotoxicity improvements warrant further investigation. POST 10-52 Structural and Functional Analysis of Lysosomal Phospholipase A2, a Close Homolog of LecithinCholesterol Acyltransferase 3, 1 2 2 2, 4 4, 1, 5 Alisa Glukhova , Robert J. Kelly , Vania Hinkovska-Galcheva , James A. Shayman , John J. Tesmer



Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, US, 2Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, US, 3Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan, US, 4Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, US, 5Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, US

Lysosomal phospholipase A2 (LPLA2, PLA2 group XV) is a recently discovered acyltransferase that catabolizes glycerophospholipids and breaks down lung surfactants. Inhibition of LPLA2 may be the basis for the phospholipidosis observed with long-term administration of amiodarone, a treatment for ventricular arrhythmia. LPLA2 has 50% sequence identity with lecithin-cholesterol acyltransferase (LCAT), an HDL-associated plasma protein that regulates reverse cholesterol transport. Here we present the 1.83 Å structure of human apo-LPLA2 and a 2.3 Å structure of LPLA2 treated with an irreversible inhibitor isopropyl dodec-11-enylfluorophosphonate, which represents an acyl transfer intermediate. LPLA2 is an αhydrolase with additional lid domains responsible for substrate recognition and specificity. LPLA2 also possesses a membrane binding motif, consisting of a hydrophobic patch surrounded by positively charged region. The wide opening of the LPLA2 active site faces the membrane for an easy access to glycerophospholipids and lipophilic alcohols, its preferred substrates. Based on these structures, substrate modeling, and the position of disease-causing mutations in LCAT, we propose that orientation of the bound phospholipid in the active site underlies the specificity of LPLA2 for fatty acids in the sn-2 vs. sn-1 position, and that preference for length of the acyl chains is dictated by two hydrophobic grooves leading away from the catalytic triad of the enzyme. We modelled the structure of LCAT and mapped genetic mutations leading to either fish eye disease (FED) or familial LCAT deficiency (FLD). We propose that total loss of enzyme activity in most FLD cases is caused by mutations in residues playing a structural role. On the other hand, FED mutations mostly cluster on the surface of the enzyme and presumably affect its activation by HDL particles. POST 10-53 Structural Basis for Antigen Recognition of a Tumor Specific Therapeutic Antibody

Reza Movahedin, Teresa M. Brooks, Cory L. Brooks Chemistry , California State University, Fresno, California, US

Antibody mediated immunotherapy of tumors has the potential to revolutionize cancer treatment.Specificity of monoclonal antibodies (MAbs) allows them to specifically target tumor cells.A universal feature of cancer cells is aberrant protein glycosylation, results in exposure of tumor specific and new epitopes that are normally masked that are attractive targets for therapeutic MAbs. MUC1 is a membrane glycoprotein found in epithelial cells.The with a large extracellular domain called the VNTR (variable number of tandem repeats) composed of a 20-120 repeating segments of 20 amino acids.The VNTR region is heavily O-glycosylated in healthy tissues.during neoplastic transformation the VNTR region of MUC1 exhibits truncated glycosylation, exposing the peptide and truncated carbohydrate .The aberrantly glycosylated MUC1 is overexpressed in the majority of adenocarcinomas.These features render MUC1 an ideal target for antibody-mediated immunotherapy. MAb-AR20.5 is a therapeutic antibody currently undergoing clinical development for treatment of MUC1 positive pancreatic cancer.The antibody was generated by immunization with tumor-derived MUC1 and exhibits potent antitumor activity. The antibody binds to a peptide epitope within the VNTR region of MUC1, molecular details of this interaction are uncharacterized and it is currently unknown what role antigen, glycosylation plays in antibody binding. To further our understanding of how this antibody interacts with antigen, we have purified and crystallized the Fab fragments of AR20.5 in complex with a synthetic MUC1 VNTR peptide. We have cultured the AR20.5 hybridoma in a bioreactor producing large quantities of IgG. Using papain digestion and cation exchange chromatography we have purified Fab fragments from AR20.5 IgG, for a combination of binding and structural studies. We have grown crystals of AR20.5 Fab in complex with VNTR peptide


POSTER ABSTRACTS that diffract to 2.2 Å. The Fab structures reveal the nature of the interaction between AR20.5 and cancer associated MUC1 furthering our understanding of how VNTR peptide glycosylation may influence binding. Ultimately by understanding epitope recognition and the effects of antigen glycosylation we seek to improve antibody affinity and specificity for development of the next generation of therapeutics antibodies. Our results have the potential to have direct translational benefits for the improvement of treatment outcomes in pancreatic cancer. POST 10-54 Neutralization of Listeria Monocytogenes by Single Domain Antibodies 2 1 1 2 Ian Huh , Robert Gene , Jyothi Kumaran , Cory Brooks 1

Human Health Therapeutics, Biologics, National Research Council of Canada, Ottawa, Ontario, Canada, 2California State University, Fresno, Fresno, California, US Listeriosis is a serious food-borne disease caused by the Gram-positive bacteria Listeria monocytogenes.

The bacteria are of particular concern for pregnant women, as even in asymptomatic cases the bacteria can cross the placental barrier to cause abortion. Although the bacteria can be treated with antibiotics, inclusion of a prophylactic that can prevent Listeria colonization as a component of pre-natal care would greatly reduce the risk of Listeriosis and abortion of the fetus during pregnancy. Bacterial invasion of epithelial cells is mediated by coupled-bindings of the virulence factors Internalin A (InlA) and Internalin B (InlB) to receptors on the target cell surface. InlA binds the E-Cadherin while InlB binds the hepatocyte growth factor receptor (c-Met) to trigger endocytosis of Listeria. Therefore, inhibition of internalin interactions with their cognate receptors may inhibit Listeria from invading a cell, opening a new therapeutic avenue. Single domain antibodies (sdAb) are the smallest known antibody fragments that retain binding function. They are derived from the unique heavy chain antibodies found in camels, alpacas and llamas. Due to the convex architecture of sdAb binding sites, they can access epitopes unavailable to conventional antibodies, such as enzyme active sites and protein-protein interaction sites. Using gentamicin protection assays and flow cytometry we have demonstrated that InlB specific sdAb are capable of inhibiting Listeria invasion of HeLa cells in vitro. Furthermore, we have discerned the molecular mechanism behind the capability of the sdAb to inhibit Listeria colonization. We have obtained a high resolution X-ray crystal structure of InlB in complex with an sdAb. The structure revealed that the sdAb binds in a negatively charged cavity on the surface of InlB. Comparison of our structure with a structure of InlB in complex with c-Met revealed that the sdAb directly competes for the same binding site as the Listeria cell surface receptor, leading to the invasion inhibition. Our results demonstrate the potential of sdAb as a new class of therapeutics to protect women from Listeria during pregnancy. POST 10-55 Understanding Regulation of P-Rex1, an Enhancer of Metastatic Potential 1, 2, 3 1, 2, 3 1, 2, 3 Jennifer Cash , Ellen Davis , John J. Tesmer 1

Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, US, 2Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, US, 3Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, US

Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guaninenucleotide exchange factor (RhoGEF) that regulates cell motility and is strongly associated with cancer metastasis. P-Rex1 is synergistically recruited to the cell membrane and activated by PIP3 and heterotrimeric G protein βγ (Gβγ) subunits, positioning the enzyme downstream of multiple classes of cell surface receptors that control processes such as cytoskeleton rearrangement and cell migration. P-Rex1 has thus become an attractive therapeutic target for the suppression of cancer metastasis. However, development of inhibitors against P-Rex1 is hindered by the fact that its structure and regulatory


POSTER ABSTRACTS mechanisms are poorly understood. My long-term goal is to define the molecular basis for regulation of P-Rex1 by PIP3 and Gβγ, principally using the technique of X-ray crystallography. The resulting structures are expected to define the surfaces and residues of P-Rex1 that are important for its interaction with its regulators, information that will facilitate the rational design of therapeutics that could be used to treat PRex1-associated cancer. Towards this goal, we have determined crystal structures of the Dbl homology/pleckstrin homology (PH) catalytic core of P-Rex1 in complex with its substrate small GTPases Cdc42 and Rac1. In addition, we have also determined high-resolution structures for the independent PH domain in complex with IP4, a soluble analogue of PIP3. These atomic structures have likely defined the key site responsible for regulation of P-Rex1 by PIP3 and reveal several potential protein−protein interaction motifs that may serve as Gβγ binding sites or structural elements contributing to autoinhibition by other domains within the enzyme. POST 10-56 Expression and Purification of Functionally Active Recombinant Plasmepsin 9 from Plasmodium falciparum 2, 1 Folasade M. Olajuyigbe 1

Department of Biochemistry, Federal University of Technology Akure, Akure, Nigeria, 2Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, US

Plasmepsins, a group of homologous aspartic proteinases are attractive drug targets against malaria. Plasmepsin 9 (PM9) expressed in the blood stage of malaria life cycle with unknown function has been strongly considered as a potential target. However, recombinant expression of active PM9 for biochemical and structure-activity analysis of the enzyme has been very challenging. Here, the preliminary report on the expression and purification of active recombinant plasmepsin 9 fromPlasmodium falciparum is presented. The plasmid of truncated proenzyme (proPfPM9) from Plasmodium falciparum was expressed in Escherichia coli Rosetta 2(DE3)pLysS competent cells. The protein was purified from inclusion bodies using combination of cation exchange and gel filtration chromatography. The expression and purification fractions were subjected to SDS-PAGE. The zymogen was activated to produce mature form of active recombinant PfPM9. Catalytic activity test of PfPM9 was determined using a chromogenic substrate, LysPro-Ile-Glu-Phe-Nph*Arg-Leu (RS6). Substrate hydrolysis was examined from substrate cleavage scan. SDS-PAGE confirmed the expression and purification of truncated proenzyme (proPfPM9) with molecular weight of approximately 54 kD. Kinetic assays showed that the purified PfPM9 was active. The hydrolytic activity of PfPM9 on RS6 was confirmed by a shift in absorbance peak from 280 nm to 272 nm at the end of assay reactions. Inhibition studies on the mature PfPM9 showed that the activity of the enzyme was inhibited by pepstatin A. Protocols for successful expression and purification of active recombinant plasmepsin 9 in E. coli have been established in this study. These will aid production of sufficient yield of PfPM9 for detailed kinetic and structural characterization of the enzyme and development of future inhibitors against malaria. POST 10-57 Characterizing HER2 Gene Variation at the Protein Level to Address Racial Disparities in Breast Cancer Mortality

Wei He1, 4, Matthew Saldana2, Tiffany Scharadin3, Steven Hoang-Phou1, Denise Trans3, Dennis Chang2, Kermit Carraway2, Paul Henderson3, Matthew A. Coleman5, 1 1 Radiation Oncology, UC Davis, Sacramento, California, US, 2Department of Biochemistry and Molecular Medicine, UC Davis, Sacramento, California, US, 3Hematology Oncology, UC Davis, Sacramento, California, US,4NSF Center for Biophotonics, Sacramento, California, US, 5Lawrence Livermore National Laboratory, Livermore, California, US 74

POSTER ABSTRACTS African-American women have a much higher rate of breast cancer mortality than Caucasian women. Notably, African-American women are also found to be at greater risk for more aggressive forms of breast cancer known as triple negative. Recently, a single nucleotide polymorphism (SNP) of HER2, W452C, which occurs predominantly in African-American women, was reported significantly correlated with breast cancer. This study reported that tumors from patients harboring W452C do not amplify the erbb2 (HER2) gene or overexpress the protein, suggesting that this variant may contribute to breast cancer development through a novel mechanism. To study the structural and functional differences associated with W452C, first we looked at over-expression of HER2 harboring W452C mutation in cells, which resulted in increased disulfide dimer formation, likely caused by the extra cysteine in extracellular domain. Expression of HER2-W452C also increased basal ATK signaling, which could lead to enhanced tumorigenecity. To address the biochemical role of the W452C SNP in breast cancer we applied a cell-free in vitro reconstitution system that uses nanolipoprotein particles (NLPs) to solubilize and support functional membrane proteins. Cell-free produced wild type HER2 and W452C were tested for tyrosine phosphorylation as well as specific binding to therapeutic anti-HER2 monoclonal antibodies trastuzumab and pertuzumab. Our results showed comparable tyrosine phosphorylation levels for both wild type and W452C HER2, suggesting that the variant itself might not alone be a driver of cancer. We also observed a decreased binding affinity of trastuzumab for W452C compared to wild type HER2, indicating that W452positive patients might not respond to trastuzumab. On the other hand, W452C had a higher affinity for pertuzumab. Overall, our studies suggest that the W452C variant may account for a cancer cell phenotype through a distinct signal pathway. Breast tumors bearing W452C variant may be differentially sensitive to clinically pertinent therapeutic agents. Further characterization of this variant will be important for the development of more effective and precise therapeutic intervention treatment regime. This in turn could lead to better diagnostic decisions to identify the W452C variant and use of targeted measures that better help address racial disparity problems in breast cancer mortality. POST 10-58 The Interaction Between the H. Pylori Oncoprotein Caga and Human Pro-Apoptotic ASPP2 is Distributed Throughout Both Proteins and Involves Intrinsically Disordered Regions 1 2 1 2 1, 3 Tali H. Reingewertz , Anat Iosub-Amir , Daniel A. Bonsor , Assaf Friedler , Eric J. Sundberg 1

Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, US, 2Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel, 3Microbiology and Immunology, University of Maryland, School of Medicine, Baltimore, Maryland, US

The leading risk factor for gastric cancer in humans is infection by Helicobacter pylori strains that express and translocate CagA, which interacts with numerous host cell proteins, dysregulating cellular signaling and causing transformation of gastric epithelial cells. CagA interacts with the tumor suppressor ASPP2, a pro-apoptotic protein that specifically stimulates p53-mediated apoptosis, reversing its pro-apoptotic function and promoting p53 degradation. The structure of a complex between the N-terminal domain of CagA and a 56-residue fragment of ASPP2, of which only 22 residues were resolved, was recently described. Here, we present structural and biophysical analyses of the interaction between CagA and ASPP2. We mapped the binding regions between ASPP2 and CagA using peptide arrays, demonstrating extensive interactions between CagA and numerous peptides distributed throughout the ASPP2 protein sequence. Using size exclusion chromatography, as well as circular dichroism and nuclear magnetic resonance spectroscopy, we found that the ASPP2 region spanning residues 331-692 is intrinsically disordered in its unbound state. A fragment of ASPP2 within this region, spanning residues 448-692, and the previously characterized intrinsically disordered proline-rich domain of ASPP2 spanning residues 693918 interact with CagA, which we quantitatively characterized by surface plasmon resonance analysis. We also observed a weak interaction between CagA and the extreme C-terminal Ank-SH3 domain of ASPP2


POSTER ABSTRACTS (residues 893-1128). Additionally, we found that the presence of the C-terminal region of CagA was dispensable for the interaction with the ASPP2 region spanning residues 331-692, but contributed to the interaction with the ASPP2 region spanning residues 692-1128. These data implicate numerous previously uncharacterized regions distributed throughout the protein sequences of both CagA and ASPP2 as determinants of this protein-protein interaction, providing novel targets for disruption of an important complex for H. pylori-mediated gastric cancer. POST 10-59 Is There a Common Structural Basis for Amyloidosis Toxicity? A New Receptor-Mediated Mechanism of Pancreatic Islet Amyloidosis-Induced Beta-Cell Toxicity in Type 2 Diabetes

Andisheh Abedini1, Annette Plesner3, Ping Cao2, Jinghua Zhang1, Fanling Meng2, Chris T. Middleton4, Ling-Hsien Tu2, Hui Wang2, Fei Song1, Rosa Rosario1, Martin T. Zanni4, Bruce Verchere3, Daniel P. Raleigh2, Ann Marie Schmidt1 1 School of Medicine, New York University Medical Center, New York, New York, US, 2Department of Chemistry, Stony Brook University, Stony Brook, New York, US, 3Child & Family Research Institute and Department of Surgery and Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, 4Department of Chemistry, University of Wisconsin-Madison, Wisconsin, US

Amyloid formation by the hormone islet amyloid polypeptide (IAPP or amylin), causes pancreatic beta-cell death, consequences of which include diabetes and islet transplant failure. The nature of the toxic species produced during IAPP amyloid formation and the cellular mechanisms by which it elicits cell death are unknown. We define the toxic entity produced during IAPP amyloid formation: transient, soluble, loosely packed, pre-fibrillar, oligomeric lag phase intermediates with modest beta-sheet structure, which appear to be on-pathway for amyloid formation. The properties of the toxic entities are considerably different from those proposed for other amyloidogenic proteins, calling into question the hypothesis that toxic species from different proteins share common features. The toxic intermediates are ligands of the receptor for advanced glycation end products (RAGE). In a murine model of islet amyloidosis, pancreatic RAGE protein expression is upregulated and co-localizes with islet amyloid deposits, along with the apoptosis marker, cleaved caspase 3. Dominant negative inhibition of cellular RAGE via soluble RAGE, genetic deletion of RAGE or addition of a RAGE-blocking antibody prevents IAPP-induced beta-cell toxicity. These findings establish that IAPP induced pancreatic beta-cell death is mediated via a novel receptor-mediated mechanism, and have implications for the treatment of diabetes and for the broader field of amyloidosis diseases. POST 10-60 SUMO4 C438T Polymorphism is Associated With Papulopustular Skin Lesion in Korean Patients with Behçet's Disease

Hyun-Sook Kim Soonchunhyang University Seoul Hospital, Seoul, Korea

Objects: Small ubiquitin-like modifier 4 (SUMO4) is involved in a range of autoimmune diseases and is known to down-regulate the transcription activity of nuclear factor kappa B (NF-kB). Our objective was to investigate the association of a certain polymorphism (C438T) of the SUMO4 gene with Behçet’s disease (BD) in terms of its incidence and clinical features in Korean patients. Methods: We consecutively enrolled 83 patients with BD and 120 healthy controls. Genomic DNA was extracted from whole blood samples. We identified a single nucleotide change (C438T) in the SUMO4 gene using an amplification refractory mutation system (ARMS) technique. To validate the ARMS technique, we compared its results to the results of direct sequencing in 20 subjects. HLA-B51 status was determined by polymerase chain reaction


POSTER ABSTRACTS sequence-specific primers. Results: The presence of papulopustular lesions (p = 0.006) and vascular involvement (p = 0.045) was significantly different between C438T genotypes in HLA-B51-positive patients with BD. There were no differences in alleleic or genotypic frequencies of the SUMO4 C438T polymorphism between patients with BD and controls (p = 0.567 and p = 0.818, respectively). The difference in papulopustular skin lesions between CC and CT+TT genotypes in HLA-B51-positive patients with BD was also statistically significant (p = 0.002, OR = 23.40, 95% CI: 2.33 – 235.54). Conclusion: The C438T polymorphism in the SUMO4 gene is associated with significantly increased risk of papulopustular skin lesions in HLA-B51-positive patients. POST 10-61 Calcium-Dependent Proteases and Their Proteinaceous Inhibitor in the Brain Regions Affected by Neurodegeneration in Rats

Liudmila A. Lysenko1, Nadezda P. Kantserova1, Nikolay L. Rendakov1, Khristina N. Prokopenko2, Nina N. Nemova1 1 Lab of Environmental Biochemistry, Institute of Biology, Karelian Research Centre of RAS, Petrozavodsk, Russian Federation, 2Dept of Organic and Biological Chemistry and Molecular Biology, Petrozavodsk State University, Petrozavodsk, Russian Federation

Neurodegeneration both pathology- and age-associated is followed by a disturbance in protein turnover and imbalance in main proteolytic systems in nervous tissue. In order to determine the contribution of intracellular proteases in neurodegeneration we study proteasome, lysosomal, and calpain-calpastatine systems in animal models of Alzheimer disease and at induced excitotoxicity. There were shown (i) the ineffectiveness of ubiquitin-proteasome system to control a quality of cellular proteins, (ii) the suppression in autophagic degradation of damaged proteins, organelles or cells, (iii) the activation of proteases promoted cell death by apoptosis or necrosis (caspases, calpains). Calpains are calcium-activated proteases tightly regulated by specific proteinaceous inhibitor, calpastatine. Calpain regulation in damaged brain regions depends on multiple factors such as (i) calcium dysregulation, (ii) selective calpain and calpastatine mRNA expression, and (iii) spatial distribution of proteases and their inhibitor. First of all a rise in intracellular Ca2+ both from intracellular stores and passive currents led to increase in proteolytic and autocatalytic calpain activity. Secondly the mRNA content of main calpain forms – Capn1 and Capn2 – is higher while calpastatine level is constant in degenerating brain regions. Thirdly calpains and calpastatine normally colocalized in neurons are found to be spatially separated in damaged nervous cells. The resulting effect – deregulated calpain activity – is a common characteristic of degeneration-affected brain regions; calpain hyperactivation is accounted as a key moment, or point-of-no-return, in neurodegeneration. However having regard to incomplete information on triggering mechanisms of neuronal loss and new data obtained on different models of neuropathology (amyloid- and glutamateinduced) we can suppose a phenomenon of calcium signaling deficit on the early stage of pathological process. In the issue of searching of rational therapy approaches enable to delay neurodegeneration we evaluate some selective calpain regulators as well as exogenous agents with pleiotropic effects targeted to stimulate endogenous neuroprotective mechanisms. The work was carried out using technical facilities of IB KarRC RAS Sharing Equipment Centre and financially supported by RFBR grant 12-04-01597 and the program «Leader Scientific Schools», progect 1410.2014.4. POST 10-62 Monomeric IKK: Probing Activation and Specificty

William E. Rogers Chemistry and Biochemistry, San Diego State University, San Diego, California, US 77

POSTER ABSTRACTS Inhibitor of KappaB Kinase (IKK) is a major regulation point on the NF-κB activation pathway. Recently we and other published crystal structures in three states of kinase activation; fully active, partially active, and inactive. These three structures reveal that the dimer unit of IKK gains increasing distance between the two kinase domains as it becomes active. This movement occurs within in the structural and dimerization domain(SDD). From this data we engineered a protein construct that stopped at this point. Here we investigate how this monomeric version of IKK compares to the the native dimeric, and how this can provide clues to the kinase's activation and specificity. POST 10-63 An Ultrasensitive Platform for the Detection of Protein Biomarkers in Spiked Human Serum

Stephen Vance, Marinella Sandros Nanoscience, University of North Carolina at Greensboro, Greensboro, North Carolina, US

Most biomarkers in the early stage of a disease such as cancer, neurological disorders and atherosclerosis −16 −12 are present at very low concentrations in serum (10 to 10 M ) amongst a diverse mixture of other biomolecules. In the case of cancer, apparent symptoms occur after it has metastasized through the body limiting opportunities for different interventions and therapies. Since the chemical variations that occur during tumor development can take place over several years, biomarkers could be used to detect cancer early. The availability of an ultrasensitive detection platform that can profile multiple biomarkers simultaneously is potentially a powerful method for the diagnosis of diseases and monitoring of subsequent therapeutic treatments. In the present work, we have developed an ultrasensitive Surface Plasmon Resonance imaging (SPRi)-based nano-aptasensor for the detection of an inflammatory biomarker at 5 fg/ml (attomolar) in spiked human serum. This ultrasensitive system was engineered through the unique integration and combination of the SPRi platform with microwave-assisted surface chemistry, aptamer technology and nanoparticles, to create a clinically relevant biosensor. The preliminary results are encouraging and show promise in extending the platform to detect an array of biomarkers in complex biological fluids that are indicative and even predictive, of disease onset and disease progression. POST 10-64 Regulation of Calpain Activity in Fish Brain by Weak Low-Frequency Magnetic Fields

Nadezda P. Kantserova1, Liudmila A. Lysenko1, Natalia V. Ushakova2, Vyacheslav V. Krylov2, Nina N. Nemova1, 1Institute of Biology, Karelian Research Centre of RAS, Petrozavodsk, Russian Federation, 2I.D. Papanin Institute for Biology of Inland Waters of RAS, Borok, Yaroslavl' region, Russian Federation

Calpains are calcium-regulated proteases found in the cytosol of all eukaryotic cells; they are involved in various cellular functions including survival and death. It is known that fish calpain system is similar to that of mammals and includes two ubiquitous forms (μ- and m-calpains) and their proteinaceous inhibitor, calpastatin. Dysregulation of the calpain system was found to mediate a variety of pathological conditions including muscular dystrophy, cancer, inflammation, and neurodegenerative diseases. Nevertheless the number of therapeutic approaches to regulate calpain activity in vivo through calpain inhibition is limited due to the low selectivity of synthetic calpain inhibitors and the unsatisfactory pharmacological properties of developed inhibitors. After a series of experimental works by Liboff and Lednev it was found out that the immediate target for weak low-frequency magnetic fields in biological systems could be calcium ions. Because of absolute calcium dependency of calpain-calpastatine system it becomes a suitable model to study calcium-related mechanism of magnetic field effects on living organisms. In order to demonstrate how weak low-frequency magnetic fields affect calpains on fish brain there was a series of in vivo and in vitro experiments carried out. It has been found that intravital effect of indicated factor led to a significant decrease in calpain activity in fish brain. It was also shown that purified enzymes have also been


POSTER ABSTRACTS substantially inactivated under the effect of weak low-frequency magnetic fields. The results (i) contribute in our understanding the phenomena of magnetic field effects on living matter, (ii) add the new information on calpain proteolytic system biology, and (iii) support fundamental basis for the development of the alternative (non-invasive and non-pharmaceutical) methods targeting calpain activity. This work was carried out using IB KarRC RAS Sharing Equipment Centre facilities and financially supported by RFBR grant No. 12-04-01597 and the program «Leader Scientific Schools» (No. 1410.2014.4). POST 10-65 Amyloid Beta Peptide Aβ40 and Aβ42 Form Separate Fibrils in Binary Mixtures

Xiaoting Yang1, Risto Cukalevski1, Georg Meisl2, Birgitta Frohm1, Tuomas Knowles2, Sara Linse1 1 Biochemistry and Structural Biology, Lund University, Lund, Sweden, 2Department of Chemistry, University of Cambridge, Cambridge, United Kingdom

Many studies show that the aggregation of amyloid beta (Aβ) peptides lies behind Alzheimer’s disease. Several isoforms of Aβ have been identified. Aβ40 and Aβ42 are the main species of Aβ peptide and confirmed to co-exist in the extracellular amyloid plaques. Amyloid fibril formation is a highly specific event which is very unlikely to form mixed fibrils. Previous studies have found that the aggregation of pure Aβ peptides displays one transition. This study indicates aggregation of equimolar mixtures of Aβ42 and Aβ40 has two transitions. Aggregation kinetics is followed by thioflavin T (ThT) assay and circular dichroism spectroscopy. Mass spectrometry is used to detect the Aβ42/Aβ40 monomer ratio as a function of aggregation time using isotope labels and trypsin digestion. The Aβ42/Aβ40 monomer ratio decreases from1 at t0 to nearly 0 at the end of the first transition which indicates the first transition is mainly the aggregation of Aβ42. Complete consumption of both Aβ42 and Aβ40 at the end of the second transition implies this transition is mainly the aggregation of Aβ40. This is supported by the short half-time, lower ThT fluorescence intensity of Aβ42 and longer half-time, higher ThT fluorescence intensity of Aβ40 when aggregating separately. Fibril morphology and node-to-node distance are studied by cryo-transmission electron microscopy. Fibrils that formed after the first transition display similar morphology to Aβ42 while after the second transitions it shows co-existence of Aβ42 and Aβ40 fibrils. By comparing the aggregation kinetics of pure peptides, Aβ42 is found to accelerate Aβ40 aggregation in a concentration-dependent way, while Aβ40 has a slight retardation effect on Aβ42. Aggregation of each peptide can be catalyzed by low concentrations of the fibrils formed from the same Aβ peptides by enhancing secondary nucleation. However, no catalysis is observed when Aβ42 fibrils are added at low concentration to Aβ40 monomer or vice versa. Surface catalysis and elongation are thus specific events and microscopic processes of the aggregation in the mixture are mainly affected during the primary nucleation. Aβ42 and Aβ40 do not form joint fibrils and the mechanism is related to more favorable interactions in the highly organized in register packing in pure fibrils that dominate over the unfavorable de-mixing entropy. POST 10-66 Molecular Engineering of L-Asparaginases as Therapeutic Enzymes for the Treatment of Leukemia

Manfred W. Konrad, Christos S. Karamitros Enzyme Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany

L-asparaginases (L-ASNases) catalyze the deamidation of the amino acid L-asparagine (L-Asn) to Laspartate (L-Asp) and ammonia. Bacterial L-ASNases are approved therapeutic enzymes for use in the treatment of various blood cancers to deplete serum L-Asn levels. Their efficiency as protein drugs is based on the fact that several hematological malignancies depend for growth on the extracellular supply of the non-essential amino acid L-Asn. To avoid the immune response and other side reactions inherent to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. One human


POSTER ABSTRACTS isoform, hASNase3, belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily where the protein is synthesized as a single polypeptide chain that is devoid of catalytic activity. Increased expression of hASNase3 was observed in several tumors, but its functional relevance is unknown. Autoproteolytic cleavage of this protein into two tightly associated subunits α and β releases the catalytically critical amino group of Thr168 at the N-terminus of the β-subunit. Recombinant hASNase3 purifies as the uncleaved, ASNase-inactive form, and undergoes self-cleavage at a very slow rate. We have found that the free amino acid glycine very selectively acts to accelerate intramolecular processing of hASNase3 both in vitro and in human cells. The dependence of hASNase3 activation on glycine may be related to the altered metabolic profile of cancer cells. The search for biochemical strategies to increase the serum half-life of the enzyme has prompted considerable efforts to enhance the stability of L-ASNase, by protecting it through e.g. polyethyleneglycol modification, and packaging into nanocapsules. Encapsulation enhances stability and potentially prevents exposure of the enzyme to the immune system. We capitalized on the use of the Layer-by-Layer (LbL) method of biocompatible microcapsule formation, using calcium carbonate particles as core templates for protein adsorption, which subsequently were coated with poly-L-arginine and dextran sulfate layers. Our work suggests that efficient encapsulation in combination with successful enzyme engineering will set the basis for novel ways to treat blood cancers. POST 10-67 Tweaking the Spines of Kinase Structures

Lalima G. Ahuja1, Jiancheng Hu3, Alexandr P. Kornev1, 5, Andrey S. Shaw3, 4, Susan S. Taylor1, 5, 2 1 Pharmacology, University of California San Diego, La Jolla, California, US, 2Chemistry and Biochemistry, University of California San Diego, La Jolla, California, US, 3Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, US, 4Howard Hughes Medical Institute, St. Louis, Missouri, US, 5Howard Hughes Medical Institute, La Jolla, California, US

The Protein kinase superfamily controls signaling pathways by phosphorylating downstream proteins. The expertise of these enzymes lies in their ability to work as an ON/OFF molecular switches to regulate signal transduction depending on the need of the cell. Perturbations in this switch are implicated in various cancers and diseases. Recently, the ON/OFF states of Kinases have been linked to the assembly/disassembly of spatially conserved hydrophobic motifs at the kinase core called the ‘Spines’. These are the Regulatory (R) and the Catalytic (C) spines that run through the kinase domain. In the present study, using the RAFs as a model system, we show how selectively trapping the assembly of R-or C-spines allows us to lock a kinase in ON or OFF state. These locked activation states serve as tools to study aspects of kinase mechanism not elucidated so far. The R-spine Phe mutants are constitutively active and locked in the ON state. Phe mutations in the R-spine promote dynamic assembly and folding of an active kinase domain such that a BRAF kinase bypasses all regulatory mechanisms required for activation. These mutants are hence constitutively active and are immune to activation-loop phosphorylation and dimerization. These mutations hence explain the unregulated activation of RAF kinases as seen in various cancers. In contrast to the R-spine mutants the C-spine Phe mutants are locked in the OFF state. These BRAF mutants are ‘pseudo-kinases’ that are unable to catalyze any phosphotransfer. However, these catalytically-dead kinases are able to activate their cognate partners by dimerization; elucidating catalysis-independent functions of canonical kinases. These pseudo-kinases bind their cognate partners as scaffolds and allow for their activation. The C-spine mutants hence explain the role of dead kinases in the activation of other kinases as seen in some cancers.



Correlation Between Aβ (1-40) aggregation in E.coli and in vitro Amyloid Fibril Formation Kalyani Sanagavarapu, Irem Nasir, Celia Cabaleiro-Lago, Sara Linse Biochemistry and Structural Biology, Lund University, Lund, Sweden

Amyloid fibrils are formed by ordered aggregation of polypeptides and proteins. Many amyloidogenic proteins are associated with neurodegenerative diseases including Alzheimer’s disease. In bacteria, inclusion bodies are often formed from the aggregation of overexpressed recombinant proteins. Inclusion bodies share many characteristics with amyloids. . In fact, similar driving forces are behind the formation of amyloids and inclusion proteins from partial unfolded proteins QUESTION ADDRESSED Is it possible to predict the amyloid fibril formation propensity by analyzing the propensity to form inclusion bodies during expression? METHODS AND MATERIALS A set of 28 single amino acid mutants of Aβ (1-40) were screened for a change in aggregation behavior on cell cultural level. SDS-PAGE analysis was used to assay the expression pattern of soluble and insoluble fractions after cell disruption. In vitro fibrillation studies of seven mutants using thioflavin-T assay were used to investigate how the fibrillation kinetics correlates with the inclusion body formation. RESULTS A reproducible level of expression of each mutant is achieved. Expression pattern is greatly variable over the peptide variants. We explore the correlation of inclusion body formation level with properties such as the peptide net charge and the overall hydrophobicity of the peptide. The fibrillation kinetics of mutants correlates well with the inclusion body formation. Analysis of the expression profile allows prediction of fibril formation propensity for mutants that exhibit strong changes in solubility compared to Aβ (1-40). POST 10-69 Structure-based Design of Dual Small-Molecule Inhibitors of Mdm2/Mdmx for Efficiently Reactivating P53 in Cancer Cells

Zhengding Su1, 2, David Duda2, Lingyun Qin1, Yao Chen1, Huashan Zhang1, Weiping Wang1, Brenda Schulman2 1 Hubei U of Technology, Wuhan, Hubei, China, 2St. Jude Children's Research Hospital , Memphis, Tennessee, US

Inactivation of p53 functions is an almost universal feature of human cancer cells. Overexpressed oncoproteins, Mdm2 and MdmX, ultimately impairs p53 function in nearly half of human tumors. By virtue of its crucial role in cancer cell development, the aberrant interaction of Mdm2/MdmX-p53 represents an attractive target for cancer therapy. Recent studies have proven that inhibition of both Mdm2 and MdmX is more efficient than single target to promote cellular apoptosis in cancers. Here, based on segmental mutational analysis and a high-resolution crystal structure of the N-terminal domain of Mdm2 in complex with nutlin-3a, we have rationally designed dual small-molecule antagonists of Mmd2/MdmX, with affinity binding to Mdm2/MdmX in sub-micromolar concentration. One potent dual inhibitor has been confirmed to enable activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis and growth inhibition of human tumors. Our work demonstrates the feasibility of rational design of multiple functional small molecules targeting aberrant protein-protein interactions for cancer therapy.\ POST 10-70 Subtle Differences Between NOS Active Sites Lends Towards the Development of a Bacterial NOS Specific Inhibitor

Jeffrey K. Holden1, Soosung Kang3, Richard B. Silverman3, Thomas L. Poulos1, 2 1 Molecular Biology and Biochemistry, University of California-Irvine, Irvine, California, US, 2Pharmaceutical Sciences, University of California-Irvine, Irvine, California, US, 3Departments of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois, US


POSTER ABSTRACTS Nitric oxide synthase (NOS) catalyzes the oxidation of L-Arg into nitric oxide (NO) and is found in both mammals and select bacteria. Previous work by our group and others has established the bacterial NOS as an excellent antibacterial target for pathogens Staphylococcus aureus and Bacillus anthracis. Since all currently known NOS’ share a near identical active site architecture (composed of a heme prosthetic group, substrate binding site and co-substrate binding site) the design of an isoform specific inhibitor design is not a trivial task. Recently, we designed a series of inhibitors that take advantage of the bacterial NOS Ile218 and His128 residues; mammalian equivalents are Val and Ser, respectively. From x-ray crystal structures we have found the binding mode of these inhibitors to both be constrained sterically and favored by the noncovalent interactions afforded by the bulkier Ile218 and His128 active site residues. Moreover, kinetic analyses of these inhibitors reveal them to not only be more potent against the bacterial form of NOS then the mammalian forms but also to have antibiotic-like properties. POST 10-71 The Role of Extracellular EMMPRIN and its Glycosylation in Modulating Cancerous Phenotypes

Agnieszka A. Kendrick, Elan Z. Eisenmesser BMG, Univeristy of Colorado Denver, Aurora, Colorado, US

Extracelluar Matrix Metalloproteinase Inducer (EMMPRIN) is a type I transmembrane glycoprotein implicated in a variety of conditions including: inflammatory diseases, breast, pancreatic, colorectal and lung cancers. In pancreatic cancer, antibody therapies are now being developed to specifically target EMMPRIN and subsequently suppress the detrimental effects of EMMPRIN activity in order to increase the chances of survival for pancreatic cancer patients. Thus far, such therapies have not been fully effective, presumably due to the lack of information on the molecular details of EMMPRIN activity and its specific interactions. EMMPRIN is highly upregulated in several different carcinoma tissues and recent data designates EMMPRIN to be a marker of pancreatic cancer progression. EMMPRIN’s potent stimulation of both matrix metalloproteinases (MMPs) and cytokines has also been implicated in the progression of cancer metastasis, specifically by modulating the tumor environment surrounding cancer cells. Another factor contributing to EMMPRIN’s function is its high level of glycosylation (10-30 kDa). The described study here aims to directly characterize glycosylated extracellular EMMPRIN in regard to its stimulatory activity and target interactions, thereby providing basis for the development of potential EMMPRIN targeting therapies. We have optimized a mammalian expression systems to purify different glycosylation forms of extracellular EMMPRIN at milligram quantities. Using biological studies we assessed the difference between unglycosylated and glycosylated extracellular EMMPRIN activity in cancer cell lines. Additionally, we performed crosslinking pull-down experiments, followed by mass spectrometry to identify the specific extracellular EMMPRIN interacting partners and we have begun characterizing potential interactions. Finally, our studies have determined the functional influence of glycosylation on the activity of extracellular EMMPRIN and begun to unravel the specifics of extracellular EMMPRIN interactions with other proteins.


POSTER ABSTRACTS POST 10-72 Structural Insights Into the Role of the Smoothened Cysteine Rich Domain in Hedgehog Signalling

Rajashree Rana1, 2, Candace Carroll3, 1, Ho-Jin Lee1, Ju Bao1, Suresh Marada1, Grace Royappa1, Stacey Ogden1, 2, Jie Zheng1, 2 1 St. Jude Children's Research Hospital , Memphis, Tennessee, US, 2University of Tennessee Health Science Center, Memphis, Tennessee, US, 3Hardin Simmons University, Abilene, Texas, US

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled-receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from bothDrosophila and human. Our results highlight a novel function of the Smo CRD, demonstrating its role in binding to small molecule modulators. POST 10-73 CRAMP 16-33 Inhibits the Assembly and Gtpase Activity of Ftsz and Perturbs the Formation of The Cytokinetic Z-Ring in Bacteria

Shashikant Ray, Dulal Panda Departement of Biosciences and Bioengineering, Indian Institute of Technology Bombay, India, Mumbai, Maharashtra, India

Cathelin related antimicrobial peptide (CRAMP), a cationic peptide containing 37 amino acids, is thought to regulate innate immunity and to provide host defense mechanism in mammals. CRAMP 16-33 (GEKLKKIGQKIKNFFQKL), a part of the CRAMP peptide, has been reported to display antimicrobial activity.

In this study, we found that CRAMP 16-33 inhibited the growth of B. subtilis and E.

coli cells. CRAMP 16-33 (10 µM) treatment increased the length of bacterial cells by 4.7 folds. Confocal microscopic images showed that CRAMP 16-33 treatment perturbed the localization of GFP-FtsZ at the midcell of bacteria. However, it did not damage the membrane structures of B. subtilis cells. In vitro, CRAMP 16-33 directly interacted with B. subtilis FtsZ and induced conformational changes in FtsZ. Light scattering and electron microscopic analysis of the assembly of FtsZ indicated that CRAMP 16-33 inhibited the assembly of purified FtsZ. In vitro, the peptide suppressed the GTPase activity of FtsZ but did not inhibit the binding of GTP to FtsZ. Though CRAMP 16-33 displays sequence similarity with MciZ, a negative regulator of FtsZ assembly, it could not inhibit the binding of MciZ to FtsZ. Further, CRAMP 1633 did not appear to inhibit tubulin polymerization. The results together suggested that CRAMP 16-33 inhibits bacterial proliferation by inhibiting FtsZ assembly.

POST 10-74 Dimerized Translationally Controlled Tumor Protein (TCTP) induces Interleukin-8 secretion in human bronchial epithelial cells via MAPK and NF-κB pathway

Heewon Lee, Haejun Pyun, Jeehye Maeng, Kyunglim Lee† Graduate School of Pharmaceutical Sciences, Colledge of Pharmacy, Ewha Womans University, Seoul, Korea, Republic of


POSTER ABSTRACTS Histamine-releasing factor (HRF) is being extensively studied, because of its physiological importance to airway inflammation and tumorigenesis. HRF amplifies allergic reactions by promoting mediator release from basophils, eosinophils and epithelial cells during late phase reactions. Previously we demonstrated that NH2-terminal truncated Del-N11TCTP forms dimers through intermolecular disulfide bonds and is a potent secretagogue of interleukin-8 (IL-8). In this report, using human epithelial cell line BEAS-2B, we examined the signal transduction events which mediate IL-8 release in response to Del-N11TCTP. We found that staurosporine (a protein kinase inhibitor), ET-18-OCH3 (a PI-spcific PLC inhibitor), BAY11-7082 (an NF-κB inhibitor), PD98059 (a MEK1/2 inhibitor), SB203580 (a p38 MAPK inhibitor) and SP600125 (a JNK inhibitor), all blocked Del-N11TCTP induced IL-8 secretion in a dose-dependent manner. We found from western blot studies that phosphorylation of MEK1/2, MKK3/6, ERK and JNK, as well as, IκBα degradation, followed by p65 translocation to the nucleus, occurred during treatment of BEAS-2B cells with Del-N11TCTP. The translocation of p65 subunit was confirmed using confocal microscopy. AP-1 transactivation increased 1.5-fold when BEAS-2B cells were incubated with Del-N11TCTP for 18 hours; and the transactivation rate of NF-κB was elevated in a dose- and time-dependent manner. Del-N11TCTPinduced IL-8 secretion was inhibited significantly when the cells were treated with p38 MAPK- or NF-κBinhibitors. Based on these results, we conclude that IL-8 secretion induced by dimerized Del-N11TCTP is regulated by MAPKs and NF-κB in BEAS-2B cells. POST 10-75 Lysolipids Modulate Aggregation of the Repeat Domain of a Human Functional Amyloid, Pmel17

Zhiping Jiang, Jennifer C. Lee Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute , Bethesda, Maryland, US Pmel17, an essential protein for pigmentation in human skin and eyes, is trafficked to and proteolytically processed in the melanosome, where it forms fibrous striations to which melanin is deposited. Previously, we established that the repeat domain (RPT) derived from Mα, the luminal region of Pmel17, is sufficient to form fibrils under mildy acidic melanosomal conditions. Here, we report the effect of lipids on RPT aggregation to explore whether intramelanosomal vesicles and/or melanosomal membrane can initiate and facilitate fibrillogenesis. Membrane mimics, vesicles and micelles, formed from phospholipids and lysolipids were employed. Along with circular dichroism spectroscopy, we exploited single-Trp containing variants to gain site-specific information on protein-lipid interaction. We find that RPT aggregation is strongly influenced and accelerated by the presence of lysolipids, especially negatively charged lysolipids such as lysophosphatidylglycerol (LPG) whereas two-chained phospholipids have minimal effects. While LPG stimulates RPT amyloid formation at submicellar concentrations (optimal LPG-to-protein ratio ~ 15 – 30), LPG micelles decelerate RPT fibril formation. In contrast to LPG, zwitterionic lysophosphatidylcholine (LPC) promotes RPT aggregation regardless of its concentration. Our data suggest that the formation of a helical intermediate in LPG by its amyloidogenic core residues is the molecular basis for LPG modulated RPT aggregation whereas LPC affects RPT aggregation by forming lipid-protein mixed micelles. Although our data do not preclude the participation of other factors such as protein-protein interactions on the membrane, the specific protein-lipid interactions can regulate amyloid formation in vivo as melanosomes are enriched in lysolipids.


POSTER ABSTRACTS POST 10-76 Extracellular Clusterin Suppresses the Formation of Cytotoxic & α-synuclein Species by Interacting With Prefibrillar Species and Facilitates Their Lysosomal Degradation: Implications in Parkinson’s Disease

Abdullah Sultan, Bakthisaran Raman, Ch M. Rao, Ramakrishna Tangirala CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500 007, India, Hyderabad, andhra pradesh, India

Extracellular deposition of misfolded proteins is involved in diseases such as Parkinson’s, Alzheimer’s, Creutzfeldt-Jakob and dialysis-related amyloidosis. The deposition of these misfolded proteins is associated with inflammation and local acidosis. Clusterin is an extracellular protein whose levels increase several-fold during inflammation. In addition to the extracellular form, clusterin also exists in various intracellular forms. We have investigated the effect of various forms of clusterin on the amyloid fibril formation of α-synuclein. At neutral pH, extracellular clusterin prevents the amyloid fibril formation of αsynuclein at sub-stoichiometric concentrations. However, intracellular forms of clusterin lack chaperone activity against α-synuclein amyloidosis. Under conditions of local acidosis (pH 6.0), which occurs at the sites of inflammation, clusterin exhibits significantly more exposed hydrophobic surfaces and dissociates into smaller oligomers that show enhanced chaperone activity against α-synuclein amyloidosis. The amyloid fibrils of α-synuclein are cytotoxic and are resistant to intracellular degradation. Using confocal live cell imaging and flow cytometry, we found that the species of α-synuclein formed in the presence of clusterin are not cytotoxic, localize to lysosomes and readily undergo lysosomal degradation. Analytical ultracentrifugation and dot blot studies revealed that clusterin interacts with the prefibrillar oligomeric species formed during the course of α-synuclein amyloid fibril formation. Thus, extracellular clusterin is a potential extracellular chaperone even under conditions of physiological acidosis relevant during inflammation, with promising therapeutic implications in amyloid-related diseases such as Parkinson’s. POST 10-77 The Adhesion Regions Of gingipains From P. gingivalis Are Composed Of At Least Two Distinct Types Of Domains

Charles A. Collyer1, Jinlong Gao2, Daniele Vicari1, Nan Li3 1 Molecular Bioscience, University of Sydney, Sydney, New South Wales, Australia, 2Faculty of Dentistry, University of Sydney, Sydney, New South Wales, Australia, 3School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia

Gingipains are multi-domain peptidases that are critical virulence factors expressed by the keystone periodontal disease pathogen P. gingivalis. The adhesion regions of these proteases are are principally comprised of a number of modules that belong to the type 1 (T1) family of gingipain adhesins (also known as cleaved adhesin domains). We have previously reported the first crystal structures of three homologous variants of these 19 kDa T1 domains and shown that they recognise a number of host target proteins. We predicted from sequence analysis and binding data that in addition to the T1 domains the adhesion regions also contain a number of other unrelated adhesins (referred to here as type 2 or T2 gingpain adhesins) and synergistically contribute to the virulence of P. gingivalis. We have recombinantly expressed and crystallized the first example of an 17.5 kDA T2 adhesin coded for by a fragment of the gene for the lysine specific gingipain (kgp). We report here the structure refined at 1.05 angstroms resolution and thereby confirm the existence of the T2 domain family. This structure represents a new fold family which is distantly similar in topology to that of the plastocyanin/azurin family of proteins but with no copper binding sites. Like the T1 adhesins it contains a structural binding site for calcium. We observe that the recombinant T2 adhesin also binds directly to host target proteins but importantly can also inhibit bacterial invasion in cell based assays. The role of specific binding activity in the cell invasion


POSTER ABSTRACTS mechanism conferred by T2 adhesins is likely to be a critical component the overall virulence of this pathogen. References [1] N. Li et al. N. Hunter & C. Collyer, Mol Microbiol., 76(4), 861–873 (2010) [2] N. Li and C. A. Collyer Eur J Microbiol & Immunol., 1, pp. 41–58 (2011) [3] Li, N. et al., N. Hunter & Collyer C.A. Mol Microbiol., 81(5) 1358-1373 (2011) [4] P. Yun, N. Li, C.A. Collyer & N. Hunter. Infect & Immun., 80 (10) 3733-3741 (2012) [5] Ganuelas, L.A., Li, N., et al., Collyer, C.A. Eur J Microbiol & Immunol., 3 (3), pp. 152–162 (2013) POST 10-78 Alzheimer’s Disease and Cerebral Amyloid Angiopathy, Doppelgangers?

Rabia Sarroukh2, Ellen Hubin3, 4, 1, Louise C. Serpell5, Nico A. van Nuland3, 4, Kerensa Broersen1, Vincent Raussens2 1 Nanobiophysics Group, Universiteit Twente, Enschede, Twente, Netherlands, 2Laboratory of Structure and Function of Biological Membranes, Universite Libre Bruxelles, Brussels, Brussels, Belgium, 3Structural Biology Brussels, VUB, Brussels, Brussels, Belgium, 4Department of Structural Biology, VIB, Brussels, Brussels, Belgium, 5School of Life Sciences, University of Sussex, Brighton, Sussex,

United Kingdom Amyloid-b (Ab) is the component of neuritic plaques in brains of subjects with Alzheimer’s disease (AD) and Lewy body dementia. Similar plaques are also related to cerebral amyloid angiopathy (CAA) when coating cerebral blood vessels as well as stated in muscle disorder inclusion body myositis. Ab Plaques are marshaled in tangles of repeated filamentous entities: fibrils. Connection of distinct fibrils with different clinical phenotypes has already been clarified for prion diseases through the existence of distinct strains. Even if recent findings suggest that Ab plaques spread in a prion-like fashion, it is not clear whether different Ab fibrils may be related to diverse illnesses. About 80% of AD patients also suffer from CAA but the link between the two diseases remains elusive. Mutations in the APP gene are one of these connections. We found experimental conditions to produce two different stable polymorphs with the Ab Italian mutant and demonstrated that AbE22K can form respectively antiparallel and parallel cross-b fibrils. Further biophysical characterization demonstrates that those two ‘strains’ differ in the accessibility of the repeating unit. Consequently, they exhibit different predisposition to bind fluorescent probes (Thioflavin T and bis-ANS). More interestingly, antiparallel fibrils interconvert into parallel ones under environmental pressure. We suggest that the ability of Ab to populate stable conformations, propagating

distinct structural features and their interconversion may be closely related to the development of CAA and/or AD. In therapy conceptualization against Ab related illnesses; tropism is an important phenomenon to be reckoned with. POST 10-79 Investigating Of The Immunomodulatory Function Ofplasmodium Falciparumhsp70 Expressed From Various LPS Minus Bacterial Strains

Ofentse J. Pooe2, Gabriele Köllisch3, Holger Heine4, Addmore Shonhai1, 2 1 Biochemistry, School of Mathematics and Natural Science, University of Venda, Thoyandou, South Africa, 2Biochemistry and Microbiology, University of Zululand, Kwa-Dlangezwa, KZN, South Africa, 3Parasitology, Philipps University Marburg, Marburg, Germany, 4Division of Innate Immunity, Priority Area Asthma and Allergies, Research Center Borstel, Borstel, Germany

Heat shock proteins (Hsps) are conserved molecules that constitute a major part of the cell’s molecular chaperone system (protein folding machinery). Plasmodium falciparum Hsps play an important cytoprotective role ensuring that the malaria parasite survives under the harsh conditions that prevail in the host environment. P. falciparum Hsp70-1 (PfHsp70-1) is a ubiquitous, cytosol-localised Hsp70 that is essential for parasite survival. Apart from their role as molecular chaperones, it is believed that some Hsps


POSTER ABSTRACTS of parasitic origin are capable of modulating host immunity through signal transduction (chaperokine role). Most investigations focusing on the chaperokine functions of Hsps use recombinant forms of the proteins produced in E. coli. The main drawback of this expression system is that the produced recombinant protein co-purifies with lipopolysaccharides (LPS). Although LPS removal techniques have been developed, they do not completely remove these contaminants, leading to confounding data as LPS is a strong immune activator. In the current study, we sought to investigate the immunomodulatory function of PfHsp70-1. The recombinant form of the protein was produced in three bacterial expression hosts (E. coli XL1 Blue, E. coli ClearColi BL21 and Brevibacillus choshinensis). The protein was expressed with an N-terminal polyhistidine tag to facilitate purification by nickel affinity chromatography. PfHsp70-1 produced using the E. coli ClearColi andBrevibacillus expression systems was associated with no detectable traces of LPS. The protein exhibited no immunomodulatory function when it was exposed to macrophage cells and cultured in vitro. However, PfHsp70-1 expressed in E. coli XL1 Blue was tainted with LPS contaminants and this protein preparation exhibited immunomodulatory function suggesting that the LPS background was responsible for the signal. Our findings suggest that PfHsp70-1 does not possess immunomdulatory function. Furthermore our study confirms that E. coli ClearColi and Brevibacillus expression are reliable expression hosts for the production of recombinant protein for use in immunomodulatory studies. POST 10-80 Near Infrared Spectral Monitoring Reveals Water Molecular System Dynamics During The Amyloidogenic Nucleation

Eri Chatani, Yutaro Tsuchisaka, Yuuki Masuda, Roumiana Tsenkova Kobe University, Kobe, Japan

Amyloid fibrils are protein supramolecular assemblies, the deposition of which is associated with numerous serious diseases. The formation of amyloid fibrils generally proceeds through nucleation and growth phases, and the nucleation is one of the most important phases controlling the overall fibrillation kinetics and final structure of amyloid fibrils formed. However, little is known about the details of how proteins and their surrounding water molecules vary. In this study, we have performed in-situ near infrared (NIR) spectral monitoring of the heat-induced spontaneous fibrillation reaction of human insulin. When absorption spectrum in a region of the first overtone of water OH bonds over 1300-1600 nm interval was monitored in real time and multivariate spectral data analyses was applied for unraveling immense information in the spectra, characteristic spectral changes related to the fibrillation reaction were detected. By combining additional evaluation of water absorbance pattern called Aquagram, we have newly proposed unique water molecular system dynamics, i.e., transient dissociation and subsequent development of hydrogen-bonded water networks during the nucleation phase of fibrillation. The specific transformations of water spectral pattern observed by the NIR analysis shed light on the role of water molecules in the formation of amyloid fibrils, and furthermore, could be used as a new biomarker for early non-invasive diagnosis of amyloid-related diseases. POST 10-81 Liver Internalization of Cholesterol Decoded by X-rays

Hay Dvir1, 2 1 Biology, Technion - Israel Institute of Technology, Haifa, Israel, 2Cell Biology, La Jolla Institute for Allergy & Immunology, La Jolla, California, US

Hypercholesterolemia, high levels of plasma LDL-cholesterol, is a major risk factor for atherosclerosis and coronary heart disease, a leading cause of death in developed countries. The clearance of LDL from the


POSTER ABSTRACTS circulation occurs via endocytosis with the LDL-receptor (LDLR) mainly by liver cells. This hepatic recruitment of the LDL-LDLR complex to the clathrin-coated pits is mediated by the endocytic adaptor protein, autosomal recessive hypercholesterolemia (ARH), which binds the NPxY internalization signal at the cytoplasmic tail of LDLR. Mutations in either the LDLR tail or in ARH lead to familial hypercholesterolemia and early onset of atherosclerosis. Since the NPxY sequence motif occurs in many proteins and there are nearly 60 proteins in human that can bind such signals, the apparent exclusive role of ARH in cholesterol homeostasis has not been understood. We have deciphered this unique internalization code by crystallographic analysis of the structure of the LDLR-ARH interface. POST 10-82 RNA Structural Elements and Protein Interactionsthat Regulate HIV Genome Splicing

Blanton S. Tolbert Chemistry, Case Western Reserve University, Cleveland, Ohio, US

Alternative splicing is a key event of the HIV replication cycle; however, little is known about the RNA structures and protein interactions that regulate splice site selection. Acceptor site A7 is one of the bettercharacterized sites, where its activity along with donor site D4 is required to remove the Rev Responsive Element and produce multiply spliced transcripts encoding tat, rev, and nef. The activity of A7 is suppressed by an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and activated by an exonic splicing enhancer (ESE3). The hnRNP A1 protein binds the silencer elements to effectively block ASF-ESE3 interaction, thereby inhibiting A7 usage. Available splicing models assert hnRNP A1 disrupts RNA secondary structure through 3’-5’ cooperative assembly; however, footprinting studies show discrete protection of the RNA structure within the hnRNP A1-ssA7 complex. To gain insight into the molecular mechanisms that regulate ssA7, we have investigated the structural, biophysical, and biochemical basis of hnRNP A1 recognition of the ISS and ESS3 elements. Here, we present solution NMR structures of the isolated ISS stem loop (55-nt) and ESS3 stem loop (27-nt). We’ve also used calorimetric and NMR titrations to determine binding surfaces. Lastly, we present a structural model of the UP1-ESS3 complex that is derived from NMR-STD, mutagenesis, and crystallographic studies. The structural model offers insight into how hnRNP A1 binds RNA stem loop structures, which is fundamentally different models used to interpret it’s alternative splicing functions. POST 10-83 Molecular Mechanism by Which an Intrinsically Disordered Region in Non-Segmented Negative Strand RNA Virus Phosphoprotein Acts as a Chaperone Of Unassembled Viral Nucleoprotein

Cédric Leyrat, Filip Yabukarski, Malene R. Jensen, Rob Ruigrok, Martin Blackledge, Marc Jamin Université Grenoble Alpes, Grenoble, France

The genomic RNA of all non-segmented negative-strand RNA viruses (NNV), including numerous human pathogens (e.g. rabies virus, measles virus, Nipah virus, Ebola virus, …), is condensed by a homopolymer of nucleoprotein (N), forming long helical ribonucleoprotein complexes named nucleocapsids (NCs) (Figure 1). These NCs are the biologically active templates used for RNA synthesis by the viral polymerase, and 0 thus the replication of these viruses requires the supply of unassembled N molecules (N ) to encapsidate the progeny RNA molecules. The phosphoprotein (P) is another essential component of the viral replication machinery of NNV. It forms homo-oligomers and possesses a modular organization with two stable, well-structured domains concatenated with two intrinsically disordered regions. P connects the 0 0 viral polymerase to NCs and acts as a chaperone of unassembled N (N ) by forming an N -P complex that prevents nonspecific encapsidation of cellular RNAs. In paramyxoviruses and rhabdoviruses, the Nterminal intrinsically disordered region of P comprises an α-MoRE (Molecular Recognition Element) that is 0 sufficient to keep unassembled N in a soluble RNA-free form (N ). In our recent works, we used a


POSTER ABSTRACTS combination of NMR spectroscopy and SAXS to describe the isolated α-MoRE (Leyrat et al., (2011a) Protein Science) and full-length P (Leyrat et al., (2012) J. Mol. Biol.) as ensembles of continuously exchanging conformers that captures their dynamic character. We showed that the α-MoRE has overall molecular dimensions and a dynamic behavior characteristic of a disordered protein but transiently populates conformers containing α-helices (Leyrat et al., (2011a) Protein Science). This region of P 0 0 undergoes a disorder-to-order transition upon binding to N , and the crystal structure of the N -P core complex showed how the MoRE folds upon binding to N and revealed the molecular mechanisms of its 0 chaperone activities. In N , the MoRE of P occupies the binding site that in NCs is occupied by an exchanging sub-domain from the neighboring N protomer, thus preventing N polymerization and thereby RNA binding (Leyrat et al., (2011b) PLOS Ptahogens). We will discuss the implications of the dynamics and of the large conformational space sampled by P in the assembly of new NCs and its role in encapsidating newly synthesized RNA and the possibility to target this essential complex for blocking viral replication. POST 10-84 Substrate and Drug Induced Conformational Heterogeneity in CETP

Revathi Sankar, Sanjib Senapati Indian Institute of Technology Madras, Chennai, India

Background Low levels of High Density Lipoprotein-Cholesterol (HDL-C) are a leading cause of cardiovascular disease world-wide. Recent studies have shown that Cholesteryl Ester Transfer Protein (CETP), whose main function is the bidirectional exchange of cholesteryl ester and triglycerides between HDL and LDL, is an effective target for dyslipidemia. Many small molecule inhibitors have been developed for CETP, but the molecular mechanism of CETP inhibition still remains elusive. One of the speculations is that, the inhibitor bound CETP possesses greater affinity for HDL, thus resulting in the formation of a nonproductive HDL-CETP complex. Here, we attempt to explore the mechanism of CETP inhibition by a comparative analysis of the 3, 5-bis (tri-fluoromethyl) phenyl derivatives class of CETP inhibitors with the substrate-bound CETP by Protein-Ligand docking and Molecular Dynamics (MD) simulations. Results The docking results were consistent with the in-vitro experimental IC-50 values and these molecules inhibit CETP by physically blocking the hydrophobic tunnel. MD results suggested that the inhibitor binding modulates the overall dynamics of the protein which was validated by the Dynamic Cross Correlation Analysis and the Principal Component Analysis. The inhibitor bound complexes show greater extent of twisting at the distal regions of CETP. This twisting facilitates increased exposure of electropositive sidechains, thus improving the protein's affinity towards the highly electronegative surface of HDL. Conclusion Our work provides a molecular insight on the mode of action of 3, 5-bis (tri-fluoromethyl) phenyl class of CETP inhibitors. Besides occluding the channel, the inhibitor binding exposes the electropositive sidechains of the protein residues at the N-terminal distal region, thereby increasing the sensitivity towards HDL, resulting in the formation of a non-productive HDL-CETP complex. The atomistic details gathered from the simulations would aid in the rational drug discovery of future CETP inhibitors. POST 10-85 ASB9 N-terminus Plays an Important Role in Creatine Kinase Regulation

Deepa Balasubramaniam2, Jamie Schiffer2, Jonathan Parnell1, 2, Stephan Mir2, Elizabeth Komives2 1 UCSD, La JOlla, California, US, 2Chemistry and Biochemistry, University of California San Diego, La Jolla, California, US

Creatine kinases (CKs) regulate ATP levels at sites of fluctuating energy demands. Reduced CK activity has been implicated in diseases such as heart failure and multiple sclerosis, however little is known about CK regulation. Ankyrin repeat and SOCS box-containing protein, ASB9, (part of an E3 ubiquitin ligase) has been shown to bind to CK and promote proteasomal degradation of CK. We hypothesize that ASB9 and


POSTER ABSTRACTS its splice variants control the levels and activity of CK at sites where energy is critically needed. We have focused on the interaction between CKB and splice variant 2 of ASB9 that contains only the ankyrin repeat domain (ARD). Our results show that the ASB9-ARD binds CKB with very high affinity (nM) and a stoichiometry of one ASB9 to one CK dimer. Comparison of binding data from an N-terminal truncation mutant of ASB9 suggests that residues 1-35 of ASB9 contribute to the binding affinity. In addition, the Nterminus of ASB9 seems to be responsible for inhibiting the enzymatic activity of CK when bound to ASB9. Hydrogen deuterium exchange mass spectrometry (HDXMS) data shows that only one region in CKB (residues 182 - 203) which is right in front of the active site, is protected upon binding of ASB9 when comparing the deuterium incorporation of CKB peptides alone and in complex with ASB9. A model of the CK-ASB9 interaction generated using computational docking agrees with the binding and HDXMS data. Analysis of the docked model showed that residue D32 in the ASB9 N-terminus binds to the ATP binding site in CK and probably plays an important role in the CK-ASB9 interaction. Interestingly, a D32A mutant of ASB9 binds CK, however has reduced affinity and has no effect on CK activity revealing the importance of this residue. Overall, the N-terminus of ASB9 contributes to the binding of CK, and plays a role in directly inhibiting the CK activity by altering regions near the active site of the enzyme. We are further characterizing the N-terminus of ASB9 to narrow down the residues that directly bind CK and determine the inhibitory kinetic constants of the interaction. The goal of this project is provide essential information for the development of inhibitors that would interrupt the CK-ASB9 interaction, resulting in increased amounts of active CK to treat diseases in which there is a an increased energy demand and decreased CK function. POST 10-86 Limited Proteolysis And Dissociation Of Trimeric State Of The BRICHOS Domain Increase Its AntiAmyloid Activity

Henrik Biverstål1, 2, Lisa Dolfe1, Erik Hermansson1, Jenny Presto1, Jan Johansson1, 3, 4 1 Center for Alzheimer Research, Karolinska Institutet, Huddinge, Sweden, 2Department of Physical Organic Chemistry, Latvian Institute of Organic Synthesis, Riga, Riga, Latvia, 3Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Uppsala, Sweden, 4Institute of Mathematics and Natural Sciences, Tallinn University, Tallinn, Tallinn, Estonia

The prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size exclusion chromatography and electrospray mass spectrometry. A hypothesis is that the trimeric form of BRICHOS is inactive, and that the putative substrate-binding site then is buried in the subunit interface. Limited in vitro trypsination of proSP-C BRICHOS takes place exclusively in a large loop between helix 1 and 2., and cleavage in the same region takes place during proSP-C biosynthesis in the ER or Golgi apparatus. We show here that limited trypsination of BRICHOS results in increased capacity to delay Aβ42 fibril formation kinetics as measured by thioflavin T flourescence, and that the ability to bind to the surface of Ab42 fibrils is unaltered after trypsination. Moreover, treatments that increase the dissociation of the BRICHOS trimer (addition of the dye bis-ANS, known to bind to the putative substrate-binding site, or detergents) increase the capacity to delay Aβ42 fibril formation, while cross-linking of the BRICHOS trimer with glutaraldehyde renders it unable to affect Ab42 fibril formation. These results indicate that modulation of the accessibility of the putative substrate-binding site is a means to regulate BRICHOS activity during proSP-C biosynhesis, and that it may be possible to enhance the anti-amyloidogenic activity of BRICHOS by targeting its oligomeric state.


POSTER ABSTRACTS POST 10-87 Thermodynamic And Structural Characterization Of The Binding Of Zn(II) And Other Molecules To Human Protein DJ-1

Shinya Tashiro1, Jose Caaveiro2, Chun-Xiang Wu3, Quyen Hoang3, Kouhei Tsumoto1, 2, 4 1 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan, 2Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan, 3Department of Biochemistry and Molecular Biology and Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, US, 4Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Parkinson’s disease (PD) is a progressive and devastating neurological disorder for which there is no cure or effective treatment. Mutations of DJ-1 cause familial PD, although the role of DJ-1 in familial or sporadic PD remains unresolved. Very recently, three separate reports have demonstrated the binding of the metal ions Cu(I) and Cu(II) to human DJ-1. This evidence opens new avenues to understanding the function of DJ-1 and its role in PD. Because the concentration of transition metals is altered in the serum of PD patients with respect to basal levels, we sought to examine the interaction of metals belonging to the Irving–Williams series with DJ-1 using high-resolution techniques. An initial screening using differential scanning fluorimetry and isothermal titration calorimetry reveals that Zn(II) binds to DJ-1 with great selectivity among the other metals examined: Mn(II), Fe(III), Co(II), Ni(II), and Cu(II). High-resolution X-ray crystallography (1.18 Å resolution) shows Zn(II) is coordinated to the protein by the key residues Cys106 and Glu18. The binding of Zn(II) showed sub-micromolar affinity (KD=0.6±0.1 μM) and it is driven -1 -1 by favorable enthalpy (ΔH=-4.7±0.1 kcal mol ) and entropy (-TΔS=-3.8 kcal mol ). Site-directed mutagenesis confirms the importance of the residues ligand Cys106 and Glu18. These results suggest that DJ-1 may be regulated and/or stabilized by Zn(II). POST 10-88 PICH And BEND3 Form A Complex: Potential Role In The Processing Of Ultrafine Anaphase DNA Bridges

Ganesha p. Pitchai1, 2 1 Health and medical sciences, The Novo Nordisk Foundation Center for Protein Research, Copenhagen, Denmark, 2Department of Cellular and Molecular Medicine, University of Copenhagen, Centre for Healthy Ageing, Copenhagen, Denmark

The human genome undergoes various threats that can lead to genomic instability, a known driver of cancer and age-associated diseases. Ultra-fine DNA bridges (UFBs) have been identified as one such threat for genomic stability. Normal, healthy cells can recognize and resolve these bridges, but how cells achieve this is not fully understood. UFBs are thin, thread like structures connecting the separating sister DNAs during anaphase, which can be only visualized by staining for PICH (Plk1-interacting checkpoint helicase) or BLM (Bloom syndrome protein) using antibodies. PICH was identified as a binding partner of Plk1, and is characterized by its DNA dependent ATPase and dsDNA translocase activity. Knockdown of PICH induces chromosome non-disjunction resulting in intertwined regions of stretched DNA. Recently published biophysical data show that the affinity of the PICH-DNA interaction increases under tension, which may highlight the importance of PICH in the recognition of stretched DNA in UFBs. We recently identified the BEN domain-containing protein, BEND3, as a new interaction protein partner of PICH, and we are currently investigating this interaction using various biophysical methods. Our findings show that PICH and BEND3 directly interact with each other. We are currently mapping the minimal region of interaction between these proteins in order to identify key residues involved in the interaction, which may lead to further understanding of PICH-BEND3 complex formation and their role in UFB processing.


POSTER ABSTRACTS Further, we are analyzing the roles of conserved domains in both BEND3 and PICH for their ability to promote protein:protein or protein:DNA interactions. POST 10-89 Computational Docking and Site Directed Mutagenesis to Identify Steroid Binding Sites on Ionotropic Glutamate Receptors

Emily Bartle, Philip Varnes, Carol Parish, Ellis Bell University of Richmond, Richmond, Virginia, US

AMPA, NMDA, and kainate receptors belong to the ionotropic glutamate (iGluR) family of receptors located in the post-synaptic neural membrane. These receptors bind glutamate, a major fast excitatory neurotransmitter, which activates the receptor channels. Consequently, the receptor’s proper function plays an important role in synaptic plasticity, memory and learning. Mis-regulation of these receptors has been linked to many neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. Therefore, our research focuses on understanding the receptors’ activity and regulation to ultimately develop therapeutic applications. The primary aim of the project is to determine the exact locations for the binding sites of two endogenous neurosteroids, pregnenalone sulfate (PS) and pregnanalone sulfate (PREGAS), to the receptors. Using molecular docking studies and other computational methods, several potentially significant residues have been suggested by our collaborator and subsequently tested via sited directed mutagenesis. Because the site directed mutagenesis has yet to produce significant changes in binding affinity, we are attempting to answer the same question through alternative methods, such as limited trypsin proteolysis and hydrogen/deuterium exchange protection assays. This work was supported by NSF Grant MCB-104995 to EB POST 10-90 Effects of Agonist and Regulator Binding on the Structure and Conformational Flexibility of the Ligand Binding Domains of Ionotropic Glutamate Receptors

Forest Barkdoll-Weil, Carol Guzman, Philip Varnes, Ellis Bell University of Richmond, Richmond, Virginia, US

Ionotropic Glutamate receptors (iGluRs) are tetrameric proteins with 4 domains, an amino terminal domain, the ligand binding domain, the transmembrane ion channel and a largely disordered intracellular C terminal region. The ligand binding domain binds glutamate, a major fast excitatory neurotransmitter, which activates the receptor channels. There are three main families of iGluRs, AMPA, NMDA, and kainite which among other things differ in their responses to two endogenous neurosteroids, pregnenalone sulfate (PS) and pregnanalone sulfate (PREGAS). To investigate the role that conformational flexibility of the ligand binding domain plays in response to either glutamate or the neurosteroids (in the presence or absence of glutamate) we have used Multiwavelength Collisional Quenching (MWCQ) which reports on the exposure and charge environment of tyrosines and tryptophans in the protein. While Glutamate has different effects on flexibility depending upon the family of receptor, the use of differently charged quenching molecules shows glutamate induced changes in the charge environment that can be correlated with steroid binding ability.


POSTER ABSTRACTS POST 10-91 A Novel CD4-based Chimeric Antigen Receptor for Functional Cure of HIV

Li Liu1, Bhavik Patel1, Mustafa Ghanem1, Zhilli Zheng2, Virgilio Bundoc1, Richard Morgan2, Steven A. Rosenberg2, Barna Dey1, Edward A. Berger1 1 Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, Maryland, US, 2Surgery Branch, National Cancer Institute/ National Institutes of Health, Bethesda, Maryland, US

Development of an effective vaccine against HIV continues to face challenges thwarted by the surfaceexposed trimeric Envelope glycoprotein (Env). Alternate approaches for prevention, eradication or “functional cure” of HIV are therefore gaining worldwide attention to control spreading of infection, or elimination of persisting viral reservoirs, or at least prolonged cessation of the administration of HAART without viral rebound. In an attempt to develop a potent inhibitor of HIV entry, we had previously designed a bi-functional soluble protein, sCD4-17b, in which the first two extracellular domains of CD4 (the primary receptor for HIV) were attached to the 17b scFv targeting the highly conserved co-receptor binding site on HIV gp120 protein. Compared to a 10 aa short linker, attachment of the CD4 and 17b scFv components via a 35 aa long flexible linker exerted extremely high potency and breadth of HIV1neutralization, due to simultaneous binding of both moieties to the same gp120 subunit of the trimeric Env and thereby preventing Env-mediated HIV entry. Following the success of adoptive transfer of Chimeric Antigen Receptor (CAR)-modified T cells as a treatment for hematological cancers in clinical trials, we have utilized the sCD4-17b moiety as the targeting component of a CAR, with the goal of durable killing of HIV-infected cells as a potential component of a functional cure. We analyzed three recombinant CAR variants, containing either CD4 alone or sCD4-17b with two different linker lengths (10 aa or 35 aa) as the extracellular antigen-binding domain, followed by sequences that encode a transmembrane domain and the intracellular CD28 and CD3ζ signaling domains. Our preclinical results suggest that the targeting moiety CD4-10-17b is superior to CD4-35-17b, consistent with the importance of serial triggering for CAR potency. Moreover, both CD4-17b CARS were devoid of the undesired activity seen with the CD4 CAR of rendering CD8 T cells susceptible to HIV infection.

POST 10-92 Some Like it Hot: Determination of Biomolecular Interactions using MicroScale Thermophoresis

Ana Lazic1, Wyatt Strutz1, Nicole Ford1, Stefan Duhr2 1 NanoTemper Technologies Inc., South San Francisco, California, US, 2NanoTemper Technologies GmbH, Munich, Germany

The analysis of biomolecular interactions is important in the identification of therapeutic targets and development of diagnostics, as well as providing insights into cellular processes. MicroScale Thermophoresis (MST), an immobilization-free technology is used to quantitate biomolecular interactions, ranging from protein-ion to protein-protein affinities. MST, the directed movement of molecules in optically generated microscopic temperature gradients, is monitored by fluorescence. This thermophoretic movement is affected by the entropy of the hydration shell around molecules and is highly sensitive to binding reactions, which affect the size, charge, conformation, and/or hydration shell. We show how MST can be used to probe interactions with previously unmeasurable targets in close-to-native conditions—in vesicles/liposomes, cell lysate, or blood serum. In addition, we demonstrate how interactions with proteins can be quantified in a label-free manner using intrinsic tryptophan fluorescence. POST 10-93


POSTER ABSTRACTS Effects of Regulator Binding on the Structure and Conformational Flexibility of the Amino Terminal Domains of Ionotropic Glutamate Receptors

Carlos Metz, Emily Bartle, Alaina Hyde, Ellis Bell University of Richmond, Richmond, Virginia, US

Ionotropic Glutamate receptors (iGluRs) are tetrameric proteins with 4 domains, an amino terminal domain, the ligand binding domain, the transmembrane ion channel and a largely disordered intracellular C terminal region. While the ligand binding domain binds glutamate, a major fast excitatory neurotransmitter, which activates the receptor channels the amino terminal domains of all three bind endogenous neurosteroids via an unknown mechanism. There are three main families of iGluRs, AMPA, NMDA, and kainite which among other things differ in their responses to two endogenous neurosteroids, pregnenalone sulfate (PS) and pregnanalone sulfate (PREGAS). To investigate the role that conformational flexibility of the amino terminal domains play in response to the neurosteroids we have used Multiwavelength Collisional Quenching (MWCQ) which reports on the exposure and charge environment of tyrosines and tryptophans in the protein and gel filtration to assess the impacts of steroid binding on quaternary structure. Finally CD and thermal melt studies have been used in investigate the effects of steroids on overall secondary structure and stability.The results provide insight into potential mechanisms of steroid regulation of ionotropic glutamate receptors.This work was supported by NSF Grants MCB104995 to EB POST 10-94 The Variant of CREBBP With HAT Domain Deletion Inhibits Hela Cell Proliferation by Downregulating the Expression of TAF1

Zhang Lu-yu1, Li Xin1, Wang Yun-hong1, Wen Quan1, Zhang Jun2 1 Institution of molecular medicine and oncology, Chongqing, China, 2Department of cell biology and genetics, Chongqing, Chongqing, China

CREB binding protein (CREBBP) is an intrinsic acetyltransferase that plays an important role in many vital biological processes. This nuclear protein is ubiquitously expressed and involved in the transcriptional coactivation of many different transcription factors. In the present article, we found that transfection of HAT domain (histone acetyltransferase domain) deleted CREBBP variant into the cervical cancer Hela cells could potently inhibit the cell proliferation. It was uncovered that the acetylation of chromatin histone H3 and H4 would drop to a degree accompanied by the knockdown of EP300 phosphorylation after transfection of the CREBBP-HAT (-) variant. And the expression of TAF1, the largest subunit of the basal transcription factor TFIID, was also accordingly down-regulated. Obviously CREBBP-HAT (-) with no histone acetyltransferase activity could competitively interfere with the functions and interactions of normal CREBBP with other proteins (such as EP300, etc.), which subsequently resulted in decreased phosphorylation level of EP300 probably by preventing the CREBBP protein from regulating the kinase expression in MAPK signaling cascade via acetylation-induced reorganization of chromatin. It has been believed that MAPK/p38 signaling pathway can activate the acetyltransferase activity of EP300 by phosphorylation in many chromatin-remodeling processes. In turn, further descent of histone acetylation catalyzed by EP300 might lower the expression of TAF1 and consequently suppress the transcription activities associated with proliferation in Hela cells. Because, as known, TAF1 generally participates in active transcription in tumor tissues, serves as coactivators, and functions in promoter recognition and transcription initiation. Down-regulation of TAF1 could be responsible for the growth inhibition of Hela cells. The primary investigation suggests an array of new molecular targets relative to transcription regulation in cervical cancer therapy. Key words: CREBBP; EP300: TAF1; phosphorylation; chromatinremodeling


POSTER ABSTRACTS POST 10-95 Effect of Polyethylene Glycol Conjugation on Conformational and Colloidal Stability of a Monoclonal Antibody Antigen Binding Fragment (Fab’)

Cristopher Roque, Anthony Sheung, Nausheen Rahman, Salvador F. Ausar Sanofi Pasteur, Toronto, Ontario, Canada

We have investigated the effects of site specific ‘hinge’ polyethylene glycol conjugation (also referred to as PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen binding fragment (Fab’) using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the thermal and pH stability of the secondary and tertiary structures of the Fab’ do not appear to be significantly affected by PEGylation. Temperature induced aggregation of the Fab’ was evaluated by optical density at 360 nm (OD 360) and by static light scattering. The results indicated that PEGylation was able to slightly increase the transition temperature, as well as prevent the formation of visible and sub-visible aggregates. Mechanical stress results judged by micro-flow imaging (MFI) and measurement of OD 360 showed that the PEGylated Fab’ had significantly higher resistance to surface-induced aggregation compared to the non-PEGylated Fab’. Analysis of the interaction parameter, kD, for the two Fab’ versions showed highly positive values for the PEGylated Fab’, indicative of strong repulsive forces among the molecules. In contrast, negative kD values were observed for the non-PEGylated Fab’, suggestive of attractive forces among the molecules. Altogether, the results indicate that the conjugation of a Fab’ with PEG induces minor changes in the structural stability, while improving colloidal stability and resistance to surface-induced aggregation. POST 10-96 Biochemical And Structural Characterization Of LiaR From Vancomycin-Resistant E. Faecalis: The ‘Master Regulator’ of the Cell-Envelope Stress Response.

Milya Davlieva3, Yiwen Shi3, Michael Zianni5, Troy Johnson1, Paul Leonard1, John Ladbury1, Cesar A. Arias2, 4, Yousif Shamoo3 1

Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, US, 2Department of Internal Medicine, 5Division of Infectious Diseas University of Texas Medical School at Houston, Houston, Texas, US, 3Biochemistry and Cell Biology, Rice University , Houston, Texas, US, 4Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, Texas, US, 5Plant-Microbe Genomics Facility, Ohio State University, Columbus, Ohio, US The CDC estimates there are over 66,000 enterococcus infections per year in the US (CDC, 2013) and has classified vancomycin-resistant enterococci (VRE) as a serious threat. In the absence of a good alternative, the cyclic lipopeptide daptomycin (DAP) has been used “off-label” for the treatment of multidrug-resistant enterococci infections and has become a key “front-line” antimicrobial against these organisms. The emergence of daptomycin resistance during therapy has become increasingly frequent and will increase as DAP is used more broadly. We have shown that DAP resistance in enterococci is linked to mutations in genes that alter the cell envelope stress response (liaFSR). Mutations to genes encoding the proteins comprising the liaFSR two-component signaling pathway occur early in adaptation and are a critical first step toward DAP resistance. LiaR is a ‘response regulator’ in the cell envelope stress response pathway that regulates several downstream operons essential to adaptation. Among the most critical downstream regulatory targets of LiaR is LiaX (formally called Yvlb). In this study, we report the expression, purification, D191N and biophysical characterization of LiaR and an adaptive variant LiaR from E. faecalis that is


POSTER ABSTRACTS associated with DAP resistance. Using MST to measure the binding of LiaR variants to a fluorescently labeled DNA duplex containing the predicted binding site of the liaFSR and liaXYZ operons we demonstrate that the relative affinity of LiaR for the liaFSR operon appears to be much weaker than to liaXYZ. A putative phosphomimetic mutant of LiaR showed increased affinity for both DNA targets suggesting that phosphorylation state is a critical component of LiaR specificity. Moreover, the adaptive D191N mutant LiaR enhanced binding of LiaR to the liaFSR and liaXYZ operons. These data suggest that D191N LiaR mimics some of the properties that would be expected of LiaR in an activated (phosphorylated) state. In order to elucidate the structural basis for LiaR affinity for specific DNA targets, we have solved the wt D191N structure of the DNA binding domain of LiaR and LiaR alone as well as DNA binding domain of D191N LiaR complexed to double-stranded DNA by X-ray crystallography. Taken together with our biochemical studies we have elucidated a mechanistic basis for how mutations in LiaR confer increased resistance to DAP though alterations in signaling. POST 10-97 Potent Inhibition Of α-Synuclein Fibrillization And Toxicity Through An Energy-Independent Chaperone-Like Activity

Jan Bieschke1, 2 1 Biomedical Engineering, Washington University in St. Louis, Saint Louis, Missouri, US, 2Max Delbrueck Center for Molecular Medicine, Berlin, Germany

Alpha-synuclein - a key protein involved in Parkinson's disease (PD) - forms aggregates of amyloid fibrils that are toxic and cause the loss of dopaminergic neurons. Chaperones, such as heat-shock proteins, interact with α-synuclein and interfere with aggregate formation, thereby providing a source for potential neuroprotective intervention. A library of 13,824 proteins was screened in vitro to detect inhibitors of αsynuclein fibrillization in a effort to identify new proteins with anti-amyloid activity, which identified several potent inhibitors of α-synuclein fibrillization, which were then further characterized. The most potent modulator, a cytoskeletal linker protein, inhibited α-synuclein fibril formation at sub-stoichiometric molar ratios of 1:100 and less by an ATP-independent mechanism. Nuclear magnetic resonance (NMR) spectroscopy and biophysical techniques elucidated that the protein specifically interacts with early aggregation intermediates, induces the formation of aggregates trapping the N-terminus of α-synuclein and thus prevents the formation of fibrillization seeds. It significantly reduced α-synuclein-mediated toxicity in primary neurons, while yeast cells overexpressing mutant α-synuclein confirmed that the protein rescued α-synuclein-mediated toxicity and aggregation in vivo, suggesting it to be a promising target for therapeutic intervention.

POST 10-98 Rational Search For A Compound That Selectively Inhibits The Triose Phosphate Isomerase From Trichomonas Vaginalis

José L. Vique1, Luis G. Brieba2, Rossana Arroyo3, Jaime Ortega3, Arturo Rojo4, Ponciano Garcia4, Claudia Benítez1 1 Instituto Politecnico Nacional, Mexico, Mexico, 2cinvestav langebio, irapuato, Mexico, 3cinvestav zacatenco, mexico, Mexico, 4Uam cuajumalpa, Mexico Trichomonas vaginalis is a protozoan, the causal agent of trichomoniasis, the most common non-viral

sexually transmitted infection (STI) spread worldwide. Trichomoniasis is associated with perinatal complications and infections in the genitourinary tract in both sexes. For over 40 years, the treatment against trichomoniasis is the provision of nitroimidazoles, commonly metronidazole and tinidazole. However, 5 to 20% of the patients show no improvement by this treatment. This highlights the need for


POSTER ABSTRACTS new therapeutic regimens against trichomoniasis. Carbohydrates are the main nutrient source for T. vaginalis. Therefore, the enzymes in the glycolytic pathway on T. vaginalis like triose phosphate isomerase (TIM) are potential therapeutic targets. We performed molecular interaction simulations between a set of compounds obtained from libraries and triose phosphate isomerase from T. vaginalis. Subsequently, the compounds with higher probability of interaction were assayed in their ability to inhibit or destabilize the mentioned glycolytic enzyme. Some compounds selected by docking strategies were able to reduce the replication and viability of T. vaginalis cultures. These findings have important implications in the development of new therapeutic strategies against trichomoniasis. POST 10-99 The Level Of Dot1L Recruitment Defines The Degree Of MLL-AF9 Hematopoietic Transformation

Aravinda Kuntimaddi, John Bushweller Molecular Physiology and Biophysics, University of Virginia, Charlottesville, Virginia, US

MLL-AF9 is a chimeric fusion protein arising in mixed lineage leukemia, as a consequence of a chromosomal rearrangement, and MLL-AF9 leukemias have low cure rates and confer a dismal prognosis. AF9 interacts with multiple proteins that have transcriptional regulatory roles, and is an intrinsically disordered protein (IDP), meaning that it is unstructured on its own, but undergoes coupled folding and binding upon interacting with its partners to form structured complexes. One of the AF9 binding partners is Dot1L, the only known Histone 3, Lysine 79 (H3K79) methyltransferase. H3K79 di-methylation (H3K79me2) is widely known to be an epigenetic mark characteristic of transcriptional activation. The mechanism by which Dot1L is recruited by MLL-AF9 and contributes to dysregulated transcription is poorly understood. Our goal is to use structure-function studies to fully understand the importance, mechanism, and the global epigenetic role of the direct recruitment of Dot1L to MLL-AF9. Here we present the NMR solution structure of the Dot1L-AF9 complex, which forms a mixed alpha-beta structure; Dot1L forms a beta strand stabilized by three AF9 helices. The interface between the Dot1L and AF9 proteins is largely hydrophobic. Additionally, we show that Dot1L has multiple interacting sites with AF9 that fold similarly upon binding. We generated several structure-guided point mutations on AF9 that either completely block the Dot1L-AF9 interaction, or have a partial effect by blocking only one Dot1L binding sites at a time. Introduction of these AF9 mutants into MLL-AF9 hematopoietic progenitor cells show that they abrogate the in vitro colony forming capacity of MLL-AF9 in a dose-dependent manner, which is contingent on how many Dot1L binding sites are blocked. To gain an understanding of the global epigenetic effects of blocking the Dot1L-AF9 interaction, we performed ChIP-Seq analysis of H3K79 diand tri- methylation marks. Our data show that the abrogating the Dot1L interaction with MLL-AF9 mutations leads to large scale losses in H3K79me2 and me3 marks within the HOXA cluster, and that of early developmental genes, also in a dose-dependent manner. Thus, we present the mechanism of the Dot1L-AF9 interaction and show that the level of Dot1L recruitment defines the degree of MLL-AF9 hematopoietic transformation. POST 10-100 Long-range Activity Regulation Mechanisms Within The IL-33/ST2/IL-1RAcp Complex

Kendra Hailey1, Bryan E. Jones2, Patricia A. Jennings1 1 UCSD, La Jolla, California, US, 2Eli Lilly & Company, San Diego, California, US

Interleukin-33 is a dual-function cytokine belonging to the IL-1 family of immune system modulators. Extracellular IL-33 acts as a modulator of TH2 response by binding the cell surface receptor ST2 and recruiting a co-receptor, IL-1RAcp. Recent studies identified misregulation of IL-33 can exacerbate breast cancer growth and anaphylactic shock. Therefore, IL-33 regulation has become an emerging target of interest for therapeutics. Currently, the only known naturally-occuring inhibitory mechanism is the release


POSTER ABSTRACTS of soluble ST2 (ST2L). Therefore, it is critical to have a detailed understanding of how the IL-33/ST2/IL1RAcp ligand-receptor interaction works. Our current approach uses a combination of hydrogendeuterium exchange mass spectrometry (DXMS), receptor-binding assays and in vivo activity assays to investigate the molecular determinants of novel allosteric regulation within this subgroup of the IL-1 family. POST 10-101 Modeling and Simulation of Full-length p53 Tetramer Bound to DNA

Ozlem Demir, Pek Ieong Dpt of Chem & Biochem, University of California-San Diego, La Jolla, California, US

p53 was discovered over 30 years ago, was initially ascribed oncogenic activity, but in 1989 recognized as what is considered today the most important tumor suppressor protein in humans. A plethora of cellular and tissue activities are controlled by p53. These processes include induction of cell cycle arrest, apoptosis, senescence, autophagy, angiogenesis, cell migration, suppression of cancer cell specific metabolism, and promotion of anti-tumor microenvironments, which collectively are thought to prevent tumor initiation and maintenance. Understanding the structural details of p53 and its interaction with DNA is of utmost importance. However, the crystal structure of full-length p53 in its functional tetrameric form bound to DNA is not available due to its long disordered regions. There are many partial p53 crystal structures available in PDB. Using the recently elucidated structure of DNA-bound tetrameric p53 DNA-binding domains as a template, we have built an all-atom model of DNA-bound full-length p53 in tetrameric form and explored its dynamics via multiple molecular dynamics(MD) simulations. In addition, 3 different DNA sequences corresponding to p53 target genes that induce different cellular effects are incorporated into this p53 model and explored by MD simulations. This project will be the analysis of these MD simulations focusing on the structural and dynamic aspects of p53 and its interaction with different DNA sequences in atomic detail. POST 10-102 Putative Programmed Cell Death Pathway Of The Malaria Parasite And The Role Of Cytochrome C

Judith H. Prieto Chemistry Department, Western Connecticut State University, Danbury, Connecticut, US Tropical malaria caused by the protozoan Plasmodium falciparum is responsible for more than 200 million

clinical episodes and at least 660,000 human deaths annually. Parasite resistance increases continuously against the presently available drugs. To further understand the biochemical pathways involved in the mechanisms of drug resistance, a quantitative proteomic approach has been developed using stable isotope labeling by amino acids in cell culture (SILAC). In contrast to previous studies with higher drug concentrations, three different sub-lethal dosage amounts of chloroquine were used to treat parasite cultures. The protein abundance was quantified and a list of proteins and their related pathways was collected. Programmed Cell Death (Apoptosis) is one of the control mechanisms used by higher multicellular animals that are crucial for the continuing development of the surviving cells. Surprisingly the causative agent of malaria, a protozoan parasite, shows signs of this programmed “suicide”. DNA fragmentation assays and morphological characterization have been carried out under similar sub-lethal drug concentrations conditions as the proteome samples pointing to an apoptotic phenotype. The proteomic data point to some of the players that might be involved. An up-regulation of cytochrome C suggests it is a protein involved in the apoptosis pathway. We hypothesize that cytochrome C is involved in a pathway similar to the one found in another unicellular organism, yeast, where after binding a target protein a proteolytic cascade is turned on. In order to test this working hypothesis an assay was


POSTER ABSTRACTS developed to confirm the involvement of cytochrome C in proteolysis of downstream targets using a fluorogenic substrate. POST 10-103 Misfolding of the vWF A1 Domain alters the strength of platelet adhesion in Type 2 von Willebrand Disease

Alexander Tischer, Pranathi Madde, Laurie Moon-Tasson, Matthew Auton Mayo Clinic, Rochester, Minnesota, US

We have surveyed the effect of a subset of these mutations most frequently identified in vWD patients on the structure and rheological function of the A1 domain. The mutations in the 2B classification and the 2M classification have a dynamic range of clinical manifestations from a paucity of vWF-platelet interactions to severe thrombocytopenia. To assess function, we have developed a high-speed video microscopy analysis of platelet translocation dynamics under shear flow in a parallel plate microfluidic flow chamber chelated with recombinant A1 domains harboring these mutations. Our analysis of these dynamics results in statistical distributions of pause (residence) times, proportional to the strength of the -1 A1-GPIb interaction. At 1500s shear rate, we find a rank order of mean pause times that parallels known clinical measures of patient vWF activity and platelet counts. Reported patient platelet counts have a linear correlation to our experimental data indicating that the effect of these mutations on the conformational properties of the A1 domain accounts for the properties of multimeric vWF. Furthermore, the severity of thrombocytopenia trends with increasing pause time. To assess structure, we have developed a number of solution biophysical and thermodynamic metrics that classify these mutational variants of the A1 domain as Native (with varying thermodynamic stability), Native-Like (having reduced secondary structure but retaining some thermodynamic stability), and Molten Globule (a complete lack of tertiary structure with residual secondary structure characterized by the absence of a urea and thermal unfolding transition). We find that, with the exception of G1324S (Native) and A1437T (Native-Like), all type 2M mutations induce a molten globule state that abolishes platelet adhesion to the domain. While many of the type 2B mutations retain native structure, the tendency is to adopt a molten globule structure that enhances platelet adhesion to the domain. This feature is particularly evident for V1316M and V1314D that cause severe chronic thrombocytopenia. The results of these studies demonstrate that the majority of these mutations cause the A1 domain to misfold. Whether the misfolding results in loss or gain of function depends on the local secondary structure elements involved. These results demonstrate that subtypes 2M and 2B vWD are categorically protein folding disorders at the A1 domain level. POST 10-104 Predictive And Experimental Approaches For Characterizing Mutations In Proteins

Maria Teresa Buenavista1, 2, 3, Rohanah Hussain3, Ann-Marie Mallon2, David Nutt4, Liam James McGuffin1 1 School of Biological Sciences, University of Reading, Marlborough, Wiltshire, United Kingdom, 2BioComputing, Medical Research Council Harwell, Didcot, Oxfordshire, United Kingdom, 3Beamline B23, Diamond Light Source, Didcot, Oxfordshire, United Kingdom, 4Department of Chemistry, University of Reading , Reading, United Kingdom Introduction Our case study is PKD1L1 with a novel point mutation (rks) screened from a mouse model for ciliopathy. PKD2 and PKD1L1 physically associate in the nodal cilia and this interaction is crucial for sensing fluid flow that drives subsequent events in the left-right patterning of body organs (sidedness). A mutation within the second PKD domain of PKD1L1 (PKDd2) is deduced to likely affect its mechanosensing function and manifest as left-right asymmetry (LRA) disorder. Both predictions and experiments illuminate on how the differences between the variants may account for reduced mechanical strength of the PKDd2 domain manifesting as a sidedness disorder. Methods The 3D models of the 83-


POSTER ABSTRACTS residue PKDd2 were generated using the IntFOLD predictive pipeline; they provided the initial structures for the molecular dynamics simulation simulations. The time-evolved coordinates were studied for their root-mean-square fluctuations and secondary structures. Steered molecular dynamics (SMD) is currently being carried out to obtain force and extension measurements. Using synchrotron radiation circular dichroism, the solution structures of the protein, were compared under three experimental conditions – normal scan, thermal melt and UV denaturation – for protein secondary structure and protein stability. The secondary structures were analysed to compare the structural changes between the variants. Results & Conclusions Under high photon flux UV irradiation, the wild type manifested a slower rate of denaturation. The thermodynamic and kinetic stability yielded by CD values in the far-UV region correlate very well with the hypothesis that the mutant has less stable properties. Through MDS, differences in structural integrity and flexibility of the PKDd2 variants in solution were established. The results showed that the single amino acid change in the mutant promoted local conformational change evident in the first 200ns shown as a β-coil transition. The CD spectral signatures confirm the mainly beta composition of the PKDd2 domain. Likewise, CD results show structural inter-conversions of the domain providing agreement with models. Computational models and experiments differentiated the structure and behaviour of the wild type from the mutant – differences which may underpin the impaired mechanosensing ability of the mutant found in nodal cilia during early development. POST 10-105 Structural Unity In Diversity In Pilins Of Some Enteric Pathogens

Himadri Biswas, Rajagopal Chattopadhyaya Biochemistry, Bose Institute, Calcutta, WB, India

The three dimensional structures of four type IVb pilin globular domains in Vibrio cholerae, Salmonella enteric serovar Typhi, enteropathogenic Escherichia coli (EPEC) and enterotoxigenic Escherichia coli (ETEC)

reported earlier are compared. By superimposing in stereo, it is seen that three of the four pilins possess five super imposable β-strands, four nearly super imposable α-helices and a disulphide bond, while the bundle-forming pilin (BfpA) domain of EPEC does not superpose well, exhibiting a different strand topology, quite different locations for its four helices and several hydrophobic cores instead of a larger one. As a result, it lacks the extensive side chain-side chain interactions of α1 with almost parallel, three central β-strands observed in all other type IV pilins. Further, it cannot form the α2-α4 lateral interactions between consecutive subunits within the left-handed three-start helices forming the fiber. We opine this structure may not be biologically relevant. Our alternative model for BfpA possesses secondary structures almost identical to those in the experimental study but is based on the V. cholerae pilin crystal structure. Structural differences within the type IVb pilins are also discussed.

POST 10-106 Synthesis Of Selectively Functionalized Adiponectin

Andreas Mattern, Annette G. Beck-Sickinger University of Leipzig, Leipzig, Germany

The adipocyte-derived hormone adiponectin has become a key player for the understanding of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. One of its major functions are the insulin sensitizing effects, which are mediated through the activation of AMPK, p38-MAPK and PPARα (1). Next to this, it is involved into glucose regulation and fatty acid oxidation. Recently, three adiponectin receptors AdipoR1, AdipoR2 and T- cadherin have been discovered while an unknown fourth receptor is hypothesized (2). For only two of them (AdipoR1 and AdipoR2) the signaling transduction via adiponectin has been confirmed (3). In order to find new binding partners or co-


POSTER ABSTRACTS receptors, we cloned and expressed full length adiponectin as a fusion protein with a C-terminal intein and a chitin binding domain (CBD) as well as an N-terminal His10-tag. By using the IMPACT-system, the fusion protein was cleaved to form the corresponding thioester. To separate the starting materials as well as the cleaved intein chitin binding domain, the purification was performed with chitin beads. Furthermore, the product was concentrated by Ni-NTA-affinity chromatography. Accordingly, the obtained adiponectin thioester was reacted with a TAMRA-labeled peptide or a biotin labeled peptide, respectively, to receive the corresponding ligation product. Finally the functionalized adiponectin was purified by size exclusion chromatography. Since, the interaction partners for the adiponectin cascade have not yet been studied in detail, it is important to have a selectively functionalized adiponectin (e.g. biotinylated or fluorescent labeled). Due to the difficulty of selective protein labeling, this method provides a good possibility to modify the protein of interest with various functionalities to use it for further interaction studies. (1) Hui et al. (2012) British Journal of Pharmacology 165, 574–590. (2) Awazawa et al. (2011) Cell Metabolism 13, 401–412. (3) Heiker et al. (2010) Biol. Chemistry 39, 1005-1018. POST 10-107 Structural Analysis of the CFA/III Minor Pilin Subunit CofB of Human Enterotoxigenic Escherichia coli

Hiroya Oki1, Kazuki Kawahara1, Shunsuke Fukakusa1, Takuya Yoshida1, Yuji Kobayashi2, Tooru Taniguchi3, Takeshi Honda3, Tetsuya Iida3, Shota Nakamura3, Tadayasu Ohkubo1 1 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan, 2Graduate School of Engineering, Osaka University, Suita, Osaka, Japan, 3Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan Enterotoxigenic Escherichia coli (ETEC) is one of the bacterial causes of diarrhea in children and travelers

in developing countries. Adherence of ETEC to host cell surfaces, colonization of the small intestine, and elaboration of heat-labile and/or heat-stable enterotoxins are essential steps in ETEC pathogenesis. In human ETEC, the adherence and colonizing abilities depend on the presence of colonization factor antigens (CFAs), which form pili (or fimbriae) to attach to the target cell surface. In the determined nucleotide sequence of the whole region for CFA/III formation of human ETEC including a cluster of 14 1 genes, we previously identified the genes encoding major and minor type IVb pilin subunit CofA and CofB, respectively. To understand the structure and assembly mechanisms of the type IVb pilin subunits, we recently determined the crystal structure of CofA and discussed the characteristic mechanism of the 2 filament formation of CFA/III pili of human ETEC. However little is known about the functional role of its minor pilin subunit CofB, which is crucial to understand comprehensively the colonization and adhesive behavior of human ETEC. Therefore we determined here the crystal structure of CofB at 1.88 Å resolution. The structure of CofB showed unique three-domain architecture, distinct from that of other type IV pilins. The central and C-terminal β-sheet-rich domains exhibit no structural homology to any so far characterized protein fold. The N-terminal domain, on the other hand, forms αβ-fold typical of the type IVb pilin with remarkable similarity to that of CofA, and consequently fits very well to the previously proposed filamentous model of CFA/III pili of human ETEC by a simple molecular docking approach. In this presentation, we will report the details of CofB structure and also discuss the functional mechanisms of CFA/III pili of human ETEC based on utilizing the available structural information of both its major and minor pilin subunits. 1. Taniguchi, T., Akeda, Y., Haba, A., Yasuda, Y., Yamamoto, K., Honda, T. & Tochikubo, K. (2001). Infect Immun 69, 5864-5873. 2. Fukakusa, S., Kawahara, K., Nakamura, S., Iwashita, T., Baba, S., Nishimura M., Kobayashi, Y., Honda, T., Iida, T., Taniguchi, T., Ohkubo, T. (2012). Acta Cryst. D68, 1418-1429.


POSTER ABSTRACTS POST 10-108 Analysis Of The Gene Products Related To Osseointegration In The Early Stage Of Titanium Implantation

Masataka Horiuchi1, Rumi Horiuchi2, Masanori Ochiai3, Atsuro Yokoyama2 1 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-gun, Hokkaido, Japan, 2Graduate School of Dental Medicine, Hokkaido University, Sapporo, Hokkaido, Japan, 3Institute of Low Temperature Science, Hokkaido University, Sapporo, Hokkaido, Japan

Dental implant is one of the effective techniques to recover lost teeth. Although its success rate is more than 90%, several percent failure is caused by unknown mechanism. In this study, we have set up the hypothesis that micro-movement caused by the titanium implant induces the expression of genes which prevent osseointegration. In order to identify the genes expressed by micro-movement, we comprehensively evaluated by whole genome microarray analysis of the tissues around the titanium implants in both rat models of acquired and of non-acquired osseointegration. The microarray data suggested the existence of the group of genes involved in bone formation induced from osseointegration, while that of the group of genes involved in non-acquired osseointegration. To further investigate the relationship between those gene products and osseointegration, we are producing Thorombospondin-2 proteins as one of the factors to ensure osseointegration and C1qTNF6 proteins as one of the factors to prevent osseointegration, by using GRP-tag affinity purification system (Horiuchi et al, Protein Eng Des Sel., 2012). In future, we will attempt to directly inject these recombinant proteins to the implant region in the model rats in vivoand validate the effects of those proteins on osseointegration. POST 10-109 Advanced Molecular Tools for Proteomic Analyses of Microvesicles

Masood Kamali-Moghaddam, Liza Löf, Felipe Oliveir, Lotta Wik, Di Wu, Junhong Yan Dept. of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

Despite a large number of protein biomarker candidates presented in the literature, only a small group of proteins have been demonstrated to be clinically useful. Identification of reliable biomarkers in the biological samples requires access to technologies with sufficient specificity and sensitivity to meet the complexity of, for instance, blood proteomic. In addition, the focus on proteins as biomarker has now being expanded to other biomolecules such as high molecular weight microvesicles and exosomes. We have previously described a sensitive and specific assay (4PLA) for detection of complex target structures such as exosomes in which the target is first enriched via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA oligonucleotides. The requirement for coincident binding by five antibodies to generate an amplifiable reporter DNA molecule results in increased specificity and sensitivity. Here, we illustrate the application of different formats of proximity assays for detection and characterization of microvesicles including the surface protein profiling and analyses of the total protein content using large sets of protein panels POST 10-110 Bovine Brain Ribonuclease is the Functional Homolog of Human Ribonuclease 1

Chelcie H. Eller2, Jo E. Lomax1, Ronald T. Raines2, 3 1 Cell and Molecular Biology, Univerisity of Wisconsin-Madison, Madison , Wisconsin, US, 2Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, US, 3Chemistry, Univerisity of WisconsinMadison, Madison , Wisconsin, US Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells.


POSTER ABSTRACTS Understanding these putative roles has relied on continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that protein and human RNase 1. Specifically, we report that human RNase 1 and bovine brain ribonuclease have a similar thermostability, activity against single- and double-stranded RNA substrates, pH–rate profile, affinity for cell-surface glycans, and ability to enter cells. Our results suggest that brain ribonuclease—not RNase A—is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals. POST 10-111 Defect in the Intramolecular and Intermolecular Cross-linking of Collagen Caused by Hcythiolactone

Marta Rusek Department of Patophysiology, Medical University of Lublin, Lublin, Poland Severe hyperhomocysteinemia due to cystathionine-β synthase (CBS) deficiency are associated with

connective tissue (CT) abnormalities in the skeletal system, lung, eye, and vasculature (1), including atherosclerosis and osteoporosis. Homocysteine (Hcy) is metabolized to Hcy-thiolactone (HTL) in an errorediting pathway catalyzed by methionyl-tRNA synthetase (3) and indirect incorporation into protein by a post-translational modification. N-Hcy-protein adducts are formed in chemical reaction of HTL with εamino groups of protein lysine residues, called protein N-homocysteinylation (4). It generates modified proteins and affects in a loss of their enzymatic activities (5). To gain insights into a role of this reaction, we studied Hcy content of collagen, the main component of connective tissue. Collagen Nhomocysteinylation would block the ε-NH2 groups of lysine residues, which would impairs cross-linking (6). A reduced number of cross-links was observed in patients with homocystinuria. Because CT are supramolecular assemblies, even low levels of N-Hcy-lysine in collagen can result in a structural defect. -/We studied Tg-I278T Cbs mice (tHcy=272±50 μM, N-Hcy-protein=16.6±4.1 μM), and Tg-I278T +/+ Cbs mice (tHcy=1.9±1.6 μM, N-Hcy-protein=1.6±0.3 μM) (7). We prepared collagen from bones of these mice using the acetic acid extraction method and analyzed its N-Hcy-collagen content by HPLC -/method. In a Tg-I278T Cbs mouse model, bone N-Hcy-collagen is significantly elevated compared with +/+ Tg-I278T Cbs animals (61.0±41.2 vs. 18.3±5.7 pmol/mg bone, P=0.042. Collagen pyridinoline cross-links -/are significantly decreased in Tg-I278T Cbs mice compared with wild type littermates (20.8±3.5 vs. 15.2±2.7 pmol/mg bone, P=0.022). These findings demonstrate that HTL prevents cross-linking of collagen, which is a target for N-homocysteinylation in vivo in mice and can account for CT abnormalities observed in severe hyperhomocysteinemia. References: 1. Mudd et al. Am J Hum Genet. 1985; 37(1):131. 2. Hamelet et al. Exp Mol Pathol. 2007; 83(2):249-253. 3. Jakubowski, J Biol Chem. 1997;272(3):19351941. 4. Jakubowski, FASEB J. 1999;13(15):2277-2283. 5. Jakubowski, Cell. Mol. Life Sci. 2004;61(4):470-487. 6. Liu et al. J Biol Chem. 1997;272(51):32370-323777. 7. Jakubowski et al. FASEB J. 2009;23(6):1721-1727. POST 10-112 Thermodynamic Analysis of Membrane Interactions with Tau Peptides

Megan Culp, Larry Masterson Hamline University, Lno Lakes, Minnesota, US

The protein tau is abundant in neurons and can become misfolded, which is a hallmark for Alzheimer’s disease. Normally tau stabilizes the cytoskeletal structure through a region in tau called the microtubule binding domain (MBD). The MBD contains four repeat segments referred to as R1, R2, R3 and R4, and


POSTER ABSTRACTS variability in the combination of these segments defines the specific tau isoform. During Alzheimer’s disease tau is misfolded, leading to the aggregation of these repeat segments within the neuronal cell body. Because the fold of a protein dictates its function, a protein that is misfolded will result in a lack of function or a complete loss in its functionality. Previous studies have found that aggregates of tau are localized to the Golgi membrane, but not at the plasma membrane. Other studies have indicated that the introduction of membrane mimics can induce tau aggregation. In this work, the membrane affinity for each repeat segment in tau will be examined by using model membranes of the Golgi and plasma membranes. The thermodynamics of this binding event will be investigated by isothermal calorimetry in order to evaluate the thermodynamic contributions upon binding. In addition to the sequence dependence of the repeat fragments on membrane binding, temperature dependent studies will also be performed to help examine the role of solvent exposure of polar and nonpolar residues. POST 10-113 Molecular Basis Of Heme Capture By Isd System Of Staphylococcus Aureus

Jose M. Caaveiro, Nhuan Vu, Koldo Morante, Yoshitaka Moriwaki, Ryota Abe, Kouhei Tsumoto The University of Tokyo, Minato-ku, Tokyo, Japan

The iron-regulated surface determinant (Isd) is composed by more than ten extracellular and membrane proteins that adquire heme for the human pathogen Staphylococcus aureus. Isd is an attractive target to battle S. aureus because its inactivation would interfere with the ability of this pathogen to grow during bacterial infection. Two key questions relevant to the mechanism of Isd proteins pursued in our laboratory are (1) how Isd receptors extract the heme from human hemoglobin, and (2) what are the basis of heme transfer between Isd proteins. We have addressed these questions from structural, biochemical and biophysical standpoints. With respect to the first question, we report that IsdH-NEAT3 (the first extracellular heme-receptor of the Isd system) binds particular types of metal-porphyrins displaying metals in oxidation state (III) but not in oxidation state (II). This observation may explain the susceptibility of S. aureus, as well as other pathogenic microorganisms, to a novel class of antimicrobial compounds based on a protoporphyrin IX scaffold. With respect to the second question, we have investigated the mechanism by which heme is transferred between Isd transporters. So far this characterization has been frustrated because of the transient nature of the protein-protein interactions involved. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in hetero- and selfdimerization. We show that the IsdC transporter not only passes heme to other Isd transporters, as previously described, but performs self-transfer reactions explaining its central role in Isd system. In addition, we identified structural elements required for the rapid and specific transfer of heme between the couple of transporters IsdC and IsdE. We conclude that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs. POST 10-114 Biologically Responsive Recombinant Protein Anchors For Macromolecular Drug Delivery

Jason S. Buhrman1, 2, Jamie E. Rayahin1, Yu Zhang1, Mary Tang1, Richard A. Gemeinhart1, 3, 4 1 Biopharmaceutical Sciences, University of Illinois, Chicago, Illinois, US, 2Medical Scientist Training Program, University of Illinois, Chicago, Illinois, US, 3Bioengineering, University of Illinois, Chicago, Illinois, US,4Opthalmology and Visual Sciences, University of Illinois, Chicago, Illinois, US

Therapeutic macromolecules present unique delivery challenges that have historically limited their clinical utility. Exogenous macromolecules tend to be unstable in the body and therapeutic doses commonly do not reach disease targets. Polymeric delivery systems have shown great success in protecting macromolecular cargo thereby increasing their duration in the body. Release mechanisms that dissociate macromolecular cargo from a delivery vehicle have largely been based on degradation of the vehicle


POSTER ABSTRACTS and/or simple diffusion of the cargo from the polymer system. Ideally, cargo would only be released in the site of disease that would require lower doses of the macromolecule and would minimize off target effects. We sought to create an anchor system that would immobilize macromolecular therapeutics to a delivery vehicle until they reached a disease site. We used the natural affinity between glutathione Stransferase (GST) and glutathione (GSH) to immobilize recombinant protein and DNA via DNA-binding protein to the surface of poly (ethylene-glycol) diacrylate (PEGDA) microspheres. The anchor was stable for six days in simulated physiologic fluid matching extracellular conditions and for 48 hours after intracerebral implant. Functional macromolecular cargo could be released from the surface of the PEGDA microspheres by enzyme catalyzed proteolysis, and by increased glutathione levels. Environments with activated proteolytic enzymes or high GSH concentrations are common in several disease states including bacterema, cancer, wound healing, and venous thrombosis. Due to the ease of creation, and the already high numbers of therapeutic proteins being purified with GST fusion anchors, we believe the GST/GSH anchor to be a promising component of future protein delivery systems. POST 10-115 X-ray Crystallography & Small Angle X-ray Scattering Studies of Interferon Regulatory Factor 4

Soumya Govinda Remesh, Carlos R. Escalante Virginia Commonwealth University, Richmond, Virginia, US

Transcriptional regulation involves the orchestrated binding of transcription factors to regulatory DNA binding sites known as enhancers. Interferon (IFN) regulatory factor family member (IRF4) is a transcription factor that serves specific roles in transcriptional regulation of IFN responsive genes, is limited to the immune system and is critical in B- & T-cell differentiation. Generally, members of IRF family, like IRF3 and IRF5, have carboxy terminal auto-inhibitory regions that are phosphorylated to generate a transcriptionally active dimer. IRF4 also has a carboxy terminal auto-inhibitory region and it gets activated by binding to multiple different partners presumably through a general mechanism of activation with slight variations with each binding partner. For my graduate work, I am investigating the general mechanism of activation of the auto-inhibited IRF4 to compare it with other members of the IRF family. I have determined the crystal structure of the C-terminal activation domain of IRF4 and carried out small angle X-ray scattering (SAXS) studies to generate ab initio models for the full-length protein and obtain insights into the autoinhibitory mechanism. The data suggests that the putative linker of IRF4 connecting the N- and C-terminus is most likely a folded well-structured domain that interacts with the auto-inhibitory carboxy tail. I am currently working towards structurally characterizing the putative linker region as well using X-ray crystallography, circular dichroism and analytical ultracentrifugation. Information about the mechanism of activation of the auto-inhibited IRF4 highlighting its uniqueness within the IRF family will help in development of novel therapeutics in disease states that are mediated by IRF4 like multiple myeloma, cardiac hypertrophy and certain autoimmune diseases. POST 10-116 Structural Characterization of Protein Aggregates of Wild-Type and Disease Associated Variants of Human γS -crystallin

David M. Montelongo, Chelsea Anorma, Diana Bandak, Rachel W. Martin Department of Chemistry, University of California, Irvine, Irvine, California, US

Protein aggregation has been implicated in numerous diseases as well as in vital cellular processes in organisms ranging from bacteria to humans. Proteins can form aggregates via differing mechanisms according to the folding state of the monomeric building block of the aggregate, which can include native-like structures or alternate stable states. γ-crystallins are a family of structural proteins that maintain the refractive index gradient of the eye lens, and aggregation of these proteins results in cataract


POSTER ABSTRACTS formation. Any of several known point mutations in the primary amino acid sequence of the structural crystallins can result in hereditary early-onset cataract formation, with many examples known for γDcrystallin and the the G18V variant of γS-crystallin. In this study, concentrated samples of wild-type γScrystallin as well as G18V and G106V variants were subjected to a range of solution conditions, which included varying pH (pH 2, 4, 7, 9) and temperature (22°C or 37°C). Each variant produced aggregates under at least one of the conditions tested, with the G18V variant showing increased aggregation propensity compared to the wild-type and G106V variants. Each sample was analyzed using a Thioflavin T fluorescence assay to detect the presence of amyloid-like aggregates against a lysozyme fibril positive control. Thioflavin T fluorescence was detected in wild-type and G106V samples only at elevated temperatures and acidic conditions. Fluorescence was detected in the G18V variant at both temperatures tested and under both acidic and basic conditions, suggesting an increased propensity for this variant to form amyloid fibril structures. Fluorescence was not present in every aggregate sample, suggesting that γS-crystallin may have more than one stable state that results in aggregate formation, and that the protein can form both amyloid and non-amyloid aggregates. The presence or absence of amyloid formation in each sample was verified by imaging techniques, congo red staining, and x-ray powder diffraction. Efforts to understand the structures of these aggregates on a molecular level are also being undertaken with the use of solid-state NMR techniques on isotopically labeled protein aggregate samples. POST 10-117 Insight Into the Catalytic Mechanism of GABA-producing Enzyme: Glutamate Decarboxylase from Sphaerobacter thermophilus

Ruiying Wu1, Shonda Clancy1, Andrzej Joachimiak1, 2 1 Midwest Center for Structural Genomics, Biosciences, Argonne National Laboratory, Argonne, Illinois, US, 2Structural Biology Center, Biosciences , Argonne National Laboratory, Argonne, Illinois, US

Glutamate decarboxylase (GAD; EC is a pyridoxal 5’-phosphate (PLP)-dependent enzyme, which catalyzes the irreversible a-decarboxylation of L-glutamate to g-aminobutyrate (GABA). This enzyme is widely distributed amongst eukaryotes and prokaryotes. The GAD plays a crucial role in the vertebrate central nervous system where it is responsible for the synthesis of GABA, a major inhibitory neurotransmitter. In the current study, we have determined at atomic resolution several crystal structures of GAD from Sphaerobacter thermophilus (StGAD), the apo-form, several mutants and in complex with its cofactor PLP, substrate and product. StGAD has shown high structure similarity with two isoforms of human GAD67 and GAD65. The enzyme contains a flexible loop near the active site. The location of the residues 330-340 in the loop has shown significant difference as observed in wild type and that in three mutant Y333A, Y333Q and Y333N, especially the residues of Tyr333, Met334 and Arg335. Both Lglutamate (substrate) and GABA (product) are found near the PLP-binding domain and the mobile loop, respectively, in the Y333A mutant StGAD structure. The findings reported herein provide evidence for Y333 as the critical residue responsible for the inherent mobility of the catalytic loop and suggest a key role in the catalytic reaction. The accurate molecular detail of StGAD interacting with its substrate, product as well as its PLP cofactor in the atomic structure provides insight into the one-step GABA biosynthesis catalyzed by the glutamate decarboxylase. Detailed information will be presented. This work was supported by National Institutes of Health Grant GM094585 and by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. POST 10-118 Interactions of E. coli Immunoglobulin Binding Protein D and the Fc Part of IgG

Kornelia M. Mikula1, 2, Robert Kolodziejczyk3, Adrian Goldman1, 3



Department of Biochemistry, University of Helsinki, Helsinki, Finland, 2The National Doctoral Program in Informational and Structural Biology, Åbo Academy, Turku, Finland, 3Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom

The discovery of penicillin started antibiotic era, the turning point in the history of medicine. Since then, finding new and more effective drugs has been at the forefront of biomedical research to combat infectious diseases. Bacteria, however, simultaneously have mutated and gained resistance to them. Even though scientists have eliminated some deathly diseases, now bacteria possessing multidrug resistance are again threating humanity. New ways of combating bacteria need to be invented, meaning that our understanding of pathogen-host interactions has to be improved. During infection, gram-negative bacteria express variety of virulence factors that interact with the host organism enabling their survival and progression of host invasion. Among the first and highly expressed are trimeric autotransporter adhesins (TAAs). TAAs comprise large protein family that includes Escherichia coli immunoglobulinbinding proteins (Eibs), such as EibA, C, D, E, F, and G (1, 2). They bind human immunoglobulins and increase serum resistance (3). Additionally, EibD is an autoagglutinin and promotes biofilm formation (1). It is believed that binding of immunoglobulins by bacterial surface proteins allows E. coli to escape host immune surveillance and promote infection (4). Here, we present studies of interactions between EibD and the Fc part of immunoglobulin G. Based on double mutant binding studies, we have identified key amino acid residues involved in EibD - Fc interactions and modeled structure of the EibD - Fc complex. (1) C.H. Sandt, C. W. Hill, Infect Immun 68, 2205-14 (2000). (2) Y. Lu, S. Iyoda, H. Satou, K. Itoh, T. Saitoh, H. Watanabe, Infect Immun 74, 5747-55 (2006). (3) J. C. Leo, A. Goldman, Mol Immunol 46, 1860-66 (2009). (4) J. D. Lambris, D. Ricklin, B. V. Geisbrecht, Nat Rev Microbiol 6(2), 132-142 (2006). POST 10-119 The Dynamic Functional Consequences of the Thrombin-Thrombomodulin Interaction

Lindsey D. Handley, Elizabeth A. Komives UC San Diego, San Diego, California, US

The coagulation cascade is critical to wound healing, but must be well regulated to prevent pathological clot formation. We previously investigated the backbone dynamics of a key component in this cascade, thrombin, and found that the loops surrounding the thrombin active site show dynamics on the ps-ns timescale when the active site is inhibitor-bound, but have dynamics on the μs-ms timescale when the active site is unoccupied. Thrombomodulin (TM) plays a key role in the regulation of the coagulation cascade by switching the function of thrombin from procoagulant to anticoagulant. A recent molecular dynamics study suggests TM does this by influencing the slow timescale dynamics of thrombin, but the actual timescale of these motions is currently unknown. NMR measurements of chemical shift differences (CSDs) can be used to probe changes in the conformational ensemble of thrombin upon TM binding. Other NMR experiments probe the timescales of motion, including R1 and R2 relaxation experiments, which probe the ps-ns time regime, and TROSY Hahn Echo and CPMG experiments, which probe slow (μsms) timescale motions. To perform these NMR studies, our lab has developed a truncation of human TM 15 (TM456m) with complete thrombin-altering activity. We have produced N-labeled human thrombin 15 15 using an E. coli expression system and have collected HSQCs of unbound N-thrombin and N-thrombin in 1:1 complex with unlabeled TM456m. Binding of TM456m induces significant CSDs and differences in peak intensities throughout thrombin. Several residues in the TM binding site, including S36A, L65, I82, M84, and K110, become significantly perturbed upon binding. TM binding also causes significant CSDs in several residues near the active site, including H57 and D102 of the catalytic triad. We hope that a complete understanding of how TM alters the dynamics and function of thrombin will aid in the design of safer anticoagulant drugs.


POSTER ABSTRACTS POST 10-120 A Novel Activator of ATPase Activity of NBD1 Domain of the CFTR

Jay Singh, William Balch Cell & Molecular Biology, The Scripps Research Institute, La Jolla, California, US

The most common Cystic Fibrosis disease is caused by the deletion mutation of phenylalanine at position 508 of CFTR, which is an ATPase super family protein. CFTR protein is made up of five domains; these include two, six trans-membrane spanning domains, two nucleotide binding domains (NBDs) for ATP bindings and hydrolysis, and a regulatory domain. To understand the mechanism of the ATPase activity of NBDs of CFTR, we have developed a highly sensitive and very precise fluorescence based ATPase assay, known as QR Assay. Using the principle that color product quenched the fluorescence signal of the white microplates, by either absorbing excitation or emission signals. QR and phosphate ions (a product of ATP hydrolysis) reaction give an intense red color in the solution that quenched the microplate fluorescence. Interestingly, we found that acetate ions are capable to stimulate the intrinsic ATPase activity of NBD1 domain by through either dimerization or oligomerization of NBD1 domains. Acetate ions promote the cross-linking of NBD1 monomer into dimmer or oligomer of human wild type NBD1 as well as deltaF508 NBD1, which is a sufficient and necessary condition for the enhancement of rate and extent of ATP hydrolysis. Furthermore, we have collected evidence that light scattering of NBD1 domains are substantially increased in the presence of acetate ions indicating the dimerization/oligomerization of NBD1 domains. The associations between the NBD1 domains are ionic because the rate and extent of ATP hydrolysis is decreased in the presence of various salts conditions. Our results strongly suggest that, ATP hydrolysis control the CFTR channel activity through various combinations of salts and acetate ion in the cells. POST 10-121 Targeting the NEET Proteins for Cancer Treatment

Colin H. Lipper1, Mark L. Paddock1, José N. Onuchic2, Ron Mittler3, Rachel Nechushtai4, Emmanuel A. Theodorakis1, Patricia A. Jennings1 1 Department of Chemistry and Biochemistry, University of California San Diego, San Diego, California, US, 2Center for Theoretical Biological Physics and Department of Physics, Rice University, Houston, Texas, US, 3Department of Biology, University of North Texas, Denton, Texas, US, 4Alexander Silberman Institute of Life Science, Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem, Israel

The 2Fe-2S cluster NEET protein family members mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) play key roles in the regulation of mitochondrial iron and reactive oxygen species homeostasis. mNT localizes to the outer-mitochondrial membrane while NAF-1 localizes to the ER and mitochondria-associated ER membranes. Recently, the NEETs were identified as potential targets for cancer treatment. Both proteins were found to be overexpressed in human epithelial breast cancer cells, and decreasing the level of either mNT or NAF-1 by shRNA knockdown to near normal levels significantly reduced cancer cell proliferation and tumor growth and induced mitochondrial dysfunction in an iron dependent manner [1]. The caged Garcinia xanthone derivative cluvenone localizes to the mitochondria and induces apoptosis in cancer cells [2]. Cluvenone binds to both mNT and NAF-1, stabilizing the oxidized 2Fe-2S cluster, and different cluvenone modifications can affect NEET cluster stability by either stabilizing or destabilizing the cluster. We were the first to show that NEETs are cluster transfer proteins [3] and our current findings indicate that the cluvenone compounds alter the transfer of the 2Fe-2S cluster to apo-acceptor proteins. These results are consistent with the NEET proteins as cellular targets of the cluvenone compounds with their effect on the FeS cluster responsible for their selective cytotoxicity. References [1] Sohn YS, et al. (2013) Proc Natl Acad Sci USA 110(36):14676–14681. [2]


POSTER ABSTRACTS Guizzunti G, et al. (2012) Invest New Drugs 30(5):1841-1848. [3] Zuris JA, et al. (2011) Proc Natl Acad Sci USA 108(32):13047-13052. POST 10-122 Understanding the Role of Human DDX21 RNA Helicase in HIV-1 Rev-RNA Assembly in vitro

Li Zhou, James R. Williamson The Scripps Research Institute, La Jolla, California, US

The Rev protein of HIV-1 binds to the Rev Response Element (RRE) located in the env region of incompletely spliced HIV mRNA and promotes nuclear export of intron containing viral transcripts. This step is one of the hallmarks for the transition from early to late viral life cycle and viral infectivity. Several host DEAD/H box RNA helicases are involved in this process. In particular, DDX21 silencing experiments suggested that DDX21 is directly involved in the Rev-RRE dependent nuclear export in the late stage of HIV infection, and it might play a role in the assembly of Rev-RRE complexes. To understand what role DDX21 plays in Rev/RRE functions, we defined the direct interactions between DDX21 and Rev-RRE complexes and explored the functional implications of these interactions. Using full length and truncated recombinant DDX21 and Rev proteins purified from E. coli, we identified the interaction sites between DDX21 and Rev via in vitro pull-down, limited proteolysis and fluorescence polarization assays. Our data suggest that DDX21 associates with Rev in vitro, with the N-terminal DDX21 (the DExD domain) showing similar effects, indicating a possible binding target. Moreover, the N-terminal 64 residues of Rev are sufficient for interaction with DDX21. We also examined the effect of Rev-DDX21 binding on the enzymatic properties of DDX21. Purified recombinant DDX21 displayed robust RNA-dependent ATPase activity and ATP-dependent RNA unwinding activity. We first established the Michaelis-Menten parameters for substrate ATP, and the apparent affinity for stem IIB RNA (high affinity binding site of Rev on RRE RNA), and poly U RNA. We explored the effect of Rev on the RNA unwinding activity of DDX21 via a real-time RNA unwinding assay using fluorescently labeled RNAs. Our results imply that the RNA unwinding activity of DDX21 is not affected by the presence of Rev.

Poster Session: Proteins in Dynamic & Driven Processes POST 11-123 Characterization of a Temperature Responsive Two-component Regulatory System in the Antarctic Methanogen, Methanococcoides burtonii

Tahria Najnin1, Khawar S. Siddiqui1, Paul M. Curmi2, Ricardo Cavicchioli1 1 School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington, New South Wales, Australia, 2School of Physics, The University of New South Wales, Kensington, New South Wales, Australia

Two-component regulatory systems (2CRSs) typically consist of a sensor histidine kinase and a response regulator. Upon receiving a stimulus, the sensor histidine kinase autophosphorylates and transfers the phosphoryl group to the receiver domain of a response regulator. The overall phosphorylation state is governed by a combination of kinase and phosphatase activities mediated by the two proteins. In Bacteria the response regulator tends to contain a helix-turn-helix (HTH) DNA binding output domain, enabling the phosphorylated protein to bind DNA and regulate gene expression. In Archaea, only a minority of response regulators contain DNA binding domains. The Antarctic


POSTER ABSTRACTS archaeon, Methanococcoides butonii, encodes numerous 2CRSs, and only a few response regulators contain predicted HTH output domains [1]. The 2CRS consisting of Mbur_0694 (sensor histidine kinase) and Mbur_0695 (response regulator with a DNA-binding output domain) is transcribed as an operon, and the RNA and protein levels of Mbur_0695 are elevated during growth at low (4°C) vs high (23°C) temperature [2, 3]. The upregulation at low temperature indicates this may represent a temperature responsive 2CRS that is involved in regulating global gene expression in response to growth temperature [3]. In order to characterize the structure, function and activity properties of this 2CRS, Mbur_0694 and Mbur_0695 were cloned, overexpressed and proteins purified by affinity chromatography. The proteins were characterized using size exclusion chromatography, liquid chromatography-mass spectrometry, circular dichroism, differential scanning calorimetry and matrix-assisted laser desorption/ionization techniques. In order to examine phosphate transfer during signal transduction the phosphorylation state and phosphotransfer properties of the proteins were assessed using small phosphate donor molecules including acetyl phosphate and carbamoyl phosphate. To facilitate studies of the response regulator, a mutated version lacking the HTH domain was also examined. This presentation describes progress to date, and represents the first report describing the biochemical properties of any 2CRS in Archaea. POST 11-124 Crystal Structure of the Periplasmic Sensor Domain of Histidine Kinase CusS Bound to Silver

Trisiani Affandi1, Aaron V. Issaian1, Sue A. Roberts1, Megan M. McEvoy1, 2 1 Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, US, 2Soil, Water and Environmental Sciences, University of Arizona, Tucson, Arizona, US

In bacteria, two-component systems act as signaling systems to respond to environmental stimuli. Each two-component system consists of a sensor histidine kinase and response regulator, which work together through histidyl-aspartyl phospho-relay. One of the two-component systems in Escherichia coli, CusSCusR, is known to induce expression of cusCFBA genes under increased periplasmic Cu(I) and Ag(I) concentrations to help maintain metal homeostasis in cells. CusS is a membrane-associated histidine kinase, such that the periplasmic sensor domain is connected with the cytoplasmic domain containing ATP-binding and catalytic kinase domains through two transmembrane helices in the inner membrane. However, the mechanism of how CusS senses increasing metal concentrations and activates CusR is not yet known. Here, we present the crystal structure of the Ag(I)-bound periplasmic sensor domain of CusS at a resolution of 2.15 Å. Two homodimers of the periplasmic sensor domain of CusS were found in the asymmetric unit of a single crystal with four Ag(I) binding sites per homodimer. The crystal structure reveals two symmetric metal binding sites at the dimeric interface between the two subunits that coordinate Ag(I) through a unique site consisting of a His2 motif with a Ag(I)-π interaction between a phenylalanine. Metal binding at the dimer interface is likely relevant to dimerization of periplasmic sensor domain of CusS and activation of the cytoplasmic kinase core of CusS. POST 11-125 Structural Investigation into the Mechanism of the Synthase Subunit of PLPS

Amber M. Smith1, 2, Janet L. Smith 1, 2 1 Biological Chemistry, University of Michigan, Ann Arbor, Michigan, US, 2Life Sciences Institute, University of Michigan , Ann Arbor , Michigan, US PLP synthase (PLPS) from the bacterium Geobacillus stearothermophilus generates pyridoxal 5'-phosphate

(PLP), the active form of vitamin B6, from glutamine, ribose 5-phosphate (R5P) and glyceraldehyde 3phosphate (G3P). PLPS is a D6-symmetric complex of 12 synthase subunits (PdxS) and 12 glutaminase subunits (PdxT). Ammonia generated by glutamine hydrolysis in PdxT is channeled to the PdxS active site where it joins R5P and G3P to form PLP by an unknown mechanism that proceeds through two sequential


POSTER ABSTRACTS covalent intermediates in PdxS. First, R5P and PdxS(Lys81) form a Schiff base imine. Dehydration, incorporation of NH3 from PdxT and loss of inorganic phosphate result in a chromophoric adduct of 1,2 unknown structure . Synthase activity is dependent on a PdxS C-terminal tail, which is disordered in all structures reported to date. To investigate the mechanism of the synthase subunit and the role of the Cterminal tail of PdxS, we obtained three crystal structures of the synthase subunit alone (PdxS/R5P and PdxS/R5P/NH3 complexes) or the intact enzyme (PLPS/R5P/Gln). The structures provide snapshots of PdxS at distinct steps in its complicated catalytic cycle and provide insights into the elusive mechanism and structural elements that drive the conversion of imine to chromophore. PdxS is highly dynamic, alternating between open and closed conformations depending on the presence of PdxT and substrates. The complex of PLPS with its substrates glutamine and R5P is the first structure of intact PLPS with multiple substrates and an ordered C-terminal tail. The C-terminal tail interacts with the body of the synthase subunit, acting as a shield over the R5P site in the closed state of the synthase subunit. The structure of PdxS with R5P in the presence of NH3 captures three distinct states in the 12 active sites of the crystallographic asymmetric unit, providing snapshots of PdxS from imine to chromophore formation. Through biochemical assays and a comparison of the three crystal structures, we identified conserved charged residues that stabilize the closed state that is essential to formation of the chromophore. Supported by NIH grant DK42303. 1.Raschle, T. et al. The Journal of biological chemistry 282, 6098-105 (2007). 2. Hanes, J.W. et al. Journal of the American Chemical Society 130, 3043-52 (2008). POST 11-126 Super Spy Variants Implicate Flexibility In Chaperone Action

Shu Quan2, 1, Lili Wang2, 3, Evgeniy V. Petrotchenko4, Karl A. Makepeace4, Scott Horowitz2, 3, Jianyi Yang5, Yang Zhang5, Christoph H. Borchers4, James C. Bardwell2, 3 1 State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai, China, 2Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, US, 3Howard Hughes Medical Institute, Chevy Chase, Maryland, US, 4Department of Biochemistry and Microbiology-Genome British Columbia Proteomics Centre, University of Victoria , Victoria, China, 5Department of Computational Medicine & Bioinformatics, University of Michigan, Ann Arbor, Michigan, US

Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. Disorder is an emerging theme, not only among many hub proteins but also among molecular chaperones, which in general allow promiscuous substrate binding. We have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. Based on our biochemical and biophysical evaluation of these "Super Spy" variants, there appear to be multiple factors that result in the increased chaperone activity. In addition to increasing binding site hydrophobicity, most of our super-Spy variants show tighter binding to client proteins and are generally more unstable than is wild type Spy. These variants also show increased levels of disorder in the absence of client proteins, indicating increases in apparent flexibility of these variants. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and apparent flexibility in chaperone action. POST 11-127 Elucidation Of The Nonspecific DNA-Binding Mechanism Of The POU Homeodomain Using NMR


POSTER ABSTRACTS Tsuyoshi Konuma1, Erisa Harada1, Takashi Oda2, Mamoru Sato2, Kenji Sugase1 1 Bioorganic Research Institute, SUNTORY foundation for life sciences, Osaka, Japan, 2Graduate school of medical life science, Yokohama city university, Yokohama, Kanagawa, Japan

Transcription factors locate effectively to their target DNA. It is generally accepted that a protein first binds nonspecifically to DNA, and then searches for its specific site. However, the details of the dynamic search process are still largely unknown. In this study, we have investigated dynamics of POU homeodomain (POUHD) of Oct4 upon DNA binding using NMR. Firstly, we titrated 16-bp DNA into POUHD to characterize the conformational changes and binding kinetics upon DNA binding. Interestingly, almost all peaks in HSQC spectra shifted sigmoidally. They could not be fitted to the simple two-state binding model; therefore, nonspecific bound states should be taken into account to explain the titration data. Here, we constructed a new binding model including the nonspecific bound states using the BlochMcConnell equation, which describes the time evolution of magnetization including the chemical exchange effect. The fitting of the chemical shift changes to the new model yielded kinetic parameters for POUHD binding to DNA. Subsequently, to obtain structural information on the nonspecific bound states, CLEANEX-PM, which determines exchange rates between amide and water protons, was applied to POUHD in the presence of excess amounts of POUHD over DNA to increase the population of the nonspecific bound states. As a result, the amide protons of the N-terminal loop are protected from solvent by the bound DNA in the nonspecific binding states, whereas those of the α3-helix remain to be exchangeable with water protons. Therefore, it is suggested that the N-terminal loop first interacts with nonspecific sites of DNA, and then the stable complex with DNA is formed by locating the α3-helix to its specific target site. This mechanism corresponds to the fly-casting model. POST 11-128 Time-lapsing Planaria: Studying Planarian Regeneration by Stop-motion Imaging and by Pulseisotopic Proteome Labeling

Wei Shen1, Karsten Berning2 1 Department of Orthopaedics and Traumatology, The University of Hong Kong 2Department of Biology and Chemistry, City University of Hong Kong, Hong Kong

The unraveled regenerative capacity of planarian makes it a valuable model organism for studying the molecular and cellular basis of tissue repair and regeneration. The combined approach of using time-lapse microscopy and quantitative proteomics has revolutionized modern biology by enabling the characterization of the dynamics of complex biological processes, but is seldom applied to planarian research. One main reason is the strong photophobicity of planarians that makes the imaging of living worms extremely difficult. Many attempts have been made on paralyzing planarians for imaging. However, these methods are tedious and relatively ineffective. Here, we report a new method that involves the use of agarose gel to embed the planarian Dugesia Japonica of typical size (2–6 mm), rendering them immobilized for as long as four days without the use of any chemical or genetic manipulation. Using this method, we documented the first time-lapse movie of the whole regeneration process of an amputated worm. In addition, we incorporated various fluorescent dyes in our imaging, allowing the capture of the single cell movements in planarian epidermis. In parallel, we used quantitative proteomics to characterize the biochemical changes during planarian regeneration during the same time window. Like many other proteomic approaches, the presence of large pools of stable proteins in the planarian proteome often masks the fluctuations of regulatory factors, which are often present in low abundance. To circumvent this obstacle, we fed unperturbed or regenerating planaria with SILAC-encoded human cells, and observed the assimilation of the isotopically labeled amino acids from the human cells into the planarian proteome over the course of four days. We envisage that the rate of assimilation reflects the flux of protein biosynthesis activity required during regeneration. Using this technique, we


POSTER ABSTRACTS identified calponin-2 as a highly-synthesized proteins in head-regenerating, but not in tail-regenerating, planaria, thus confirming the importance of calcium homeostasis in the maintenance of planarian body plan. Taken together, the two techniques developed in this work reveal previously unknown aspects of planarian regeneration, and can potentially extend our understanding of the biology of this fascinating organism. POST 11-129 Hydrogen Deuterium Exchange Mass Spectrometry and Molecular Dynamics Reveal the Interactions of Membrane Phospholipids and Inhibitors with Phospholipases A2

Varnavas Mouchlis, Denis Bucher, J. Andrew McCammon, Edward A. Dennis UC San Diego, La Jolla, California, US

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes which catalyze hydrolysis of the ester bond at the sn-2 position of membrane phospholipids (Chem. Rev. 2011, 111, 6130). The products of PLA2 activity are free fatty acids, predominantly arachidonic acid (AA), and lysophospholipids. The AA is further metabolized by COX-1, COX-2 and 5-LO to form a variety of pro-inflammatory lipid mediators including prostaglandins and leukotrienes. Among the members of the PLA2superfamily, GVIA iPLA2 and GIVA cPLA2 have a similar size and they function through a Ser/Asp catalytic dyad located in an α/βhydrolase domain. However, GIVA cPLA2 shows specificity for phospholipids containing arachidonic acid, while the GVIA iPLA2 shows low specificity. These enzymes have been associated with several inflammatory diseases and thus they are attractive targets for inhibitor development. Both enzymes have been expressed using the Baculovirus expression system. Group-specific assays were developed for testing the inhibitory activity of small compounds against both enzymes. Deuterium exchange mass spectrometry (DXMS) and computer-aided drug design were employed to study the binding interactions of PAPC and inhibitors. DXMS revealed important information about regions of the enzymes interacting with the membrane and inhibitors. The DXMS information and Induced Fit docking have been used to predict the binding mode of PAPC and inhibitors, and to create complexes of the enzymes with a modeled membrane (PLoS Comput. Biol. 2013, 9, 1 and J. Am. Chem. Soc. 2013, 135, 1330). The substrate- and ligand-enzyme complexes revealed key residues that play important roles in binding. Further studies of the complexes with molecular dynamics (MD) simulations have been employed to interpret the DXMS results (J. Biol. Chem. 2013, 288, 1806) and determine the conformational changes resulting from the binding process. Long MD simulations with the Anton supercomputer revealed two states for the enzyme, “closed” and “open”, suggesting that the membrane acts as an allosteric ligand shifting the enzyme to the “open” state. This study provides insight into the interactions of the enzyme with membrane, substrate and inhibitors, and this information will be useful in developing new inhibitors with improved properties. (Supported by NIH RO1 GM 20,501-38; Anton was provided by the National Center for Multiscale Modeling of Biological Systems). POST 11-130 Visualizing the Inter-domain Motions of a Flexible Protein Using Continuous Models

Yang Qi1, Jeffrey W. Martin2, Bruce R. Donald2, 1, Terrence G. Oas1 1 Biochemistry, Duke University, Durham, North Carolina, US, 2Computer Science, Duke University, Durham, North Carolina, US

SpA-N is the N-terminal half of Staphylococcal protein A and it is composed of five protein binding domains. The five domains could bind to antibody, TNFR1 and von Willebrand factor and facilitate the evasion of Staphylococcus aureus into the human immune system. The functional plasticity could result from structural flexibility. We first observed motions of SpA-N in sub-nanosecond timescale using heteronuclear NMR relaxation. The result suggests that the five domains of SpA-N are connected by four


POSTER ABSTRACTS flexible linkers. In order to observe dynamics in a wider range of timescales, we constructed a di-domain mimic of SpA-N with a lanthanide binding tag (LBT) and measured residual dipolar couplings (RDCs). The di-domain construct can bind lanthanide ions and consequently be aligned in the presence of a strong magnetic field. By using different lanthanide ions, we also obtained multiple alignments, which contain additional dynamic information than a single alignment. In addition, we designed a de novo method to extract dynamic information from RDCs. Instead of determining conventional structure ensembles, our method determines an inter-domain orientation distribution to describe the structure of a flexible protein using continuous probability distributions. The method can avoid the over-fitting problem and generate a least biased model. Using the method, we determined the inter-domain orientation distribution of the didomain construct. A strong correlation was observed in the distribution and conformations were well populated in a limited region. Based on the result, we hypothesized that protein A is able to reach important biological conformations with a high probability and avoid other unnecessary conformations. If so, the conformation space of protein A makes protein A a versatile virulent protein by minimizing the entropy cost to bind to a binding partner. POST 11-131 Benchmarking FRET for Live-cell PPI

Tao Lin1, Brandon Scott1, Francisca Essel1, Moul Dey2, Adam Hoppe1, Suvobrata Chakravarty1 1 Chemistry & Biochemistry, South Dakota State University, Brookings, South Dakota, US, 2Health & Nutritional Sciences, South Dakota State University, Brookings, South Dakota, US

In recent years, fluorescence resonance energy transfer (FRET) has become a powerful tool to study molecular interactions, especially protein-protein interactions (PPI). Recombinant fluorescent proteins enable the study of such interaction in living cells. Whilst the dynamics of live cells are known as the ultimate analysis, the limits of the strength of molecular interactions that can be detected by live-cell FRET remain poorly understood as no appropriate indicator is available for this. To meet the demand on FRET reporter molecules for live-cell, we introduce a new facile sensor analysis strategy. Using a test set of 11 pairs of small interacting proteins for whom the respective affinities range between mM – nM, we determine protein concentration in living cells to compute the interaction affinity using the intra-cellular FRET efficiency. Furthermore, we also compare affinity obtained from FRET with that obtained from ITC. All of these results suggest that we establish the limits with live-cell microscopy. This study benchmarks intracellular FRET and is a very useful reference for intracellular PPI methods (BiFC, IDPRIME etc). POST 11-132 Dynamics And Chaperone Function In The Small Heat-Shock Protein αB-Crystallin

Georg Hochberg1, Heath Ecroyd2, Dezerea Cox2, Michael Sawaya3, Cong Liu3, Duilio Cascio3, Miranda Collier1, James Stroud3, John Carver4, Andrew Baldwin1, Carol Robinson1, David Eisenberg3, Justin Benesch1, Arthur Laganowsky1 1 Chemistry Research Laboratory, Oxford University, Oxford, United Kingdom, 2University of Wollongong, Wollongong, New South Wales, Australia, 3University of California, Los Angeles, Los Angeles, California, US, 4The Australian National University, Canberra, Australian Capital Territory, Australia

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, preventing their aggregation into either amorphous deposits or amyloid fibrils. How sHSPs carry out their important function is unknown, but it is generally believed to depend on their complex quaternary dynamics, including the formation of large and heterogeneous oligomers, their inter-conversion via subunit exchange, and the presence of disordered terminal domains. Although these dynamics can now be accurately measured using native mass spectrometry and nuclear magnetic resonance (1), the heterogeneity inherent in this system makes it difficult to test conclusively


POSTER ABSTRACTS which aspects of sHSP assemblies are required for chaperone function. To overcome these challenges, we engineered truncated constructs of the two most abundant sHSPs in human tissue, αB-crystallin and HSP27 in a manner allowing us to carefully control their quaternary dynamics and solve their structures by X-ray crystallography (2). We quantified the quaternary dynamics of these domains using native mass spectrometry, and used engineered cysteines to drive their equilibrium stoichiometries from rapidly interconverting monomers and dimers to conformationally restricted dimers that cannot exchange subunits. Remarkably, we find that the αB-crystallin core domain alone has chaperone activity comparable to that of the full-length protein, despite its inability to form large oligomers and lack of disordered terminal domains and regardless of whether the αB-crystallin core domain is locked into a dimer or predominantly monomeric. Furthermore, it is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-β peptide to cells. Our experiments therefore identify a novel, small and highly structured ‘functional unit’ of the heterogeneous sHSP oligomeric ensemble, potentially enabling more rational design of amyloid inhibitors. 1. Hochberg G & Benesch J (2014) Dynamical structure of αB-crystallin. Prog. Biophys. Mol. Biol. doi: 10.1016/j.pbiomolbio.2014.03.003. 2. Hochberg G, et al. (2014) The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity. Proc. Natl. Acad. Sci. USA 111(16):E1562-E1570. POST 11-133 Protein Flexibility and Gymnastics Drive Robust Clockwise Ticking of a three-protein KaiABC Oscillator

Yonggang Chang, Roger Tseng, Andy LiWang University of California, Merced, Atwater, California, US

Almost all living organisms have evolved endogenous circadian (~24 h) clocks in anticipation of daily environmental changes. The discovery of the cyanobacterial clock has made feasible mechanistic understanding of circadian clocks in that (1) its circadian oscillator is simple, composed of only three proteins KaiA, KaiB, and KaiC; and (2) the oscillator can be reconstituted in a test tube. Our goal is to eventually understand how the three proteins work together to generate sustained rhythms. KaiC displays a 24-h rhythm of phosphorylation under the positive regulator KaiA, which promotes KaiC autophosphorylation, and the negative regulator KaiB, which promotes KaiC auto-dephosphorylation. KaiC is a homohexamer and each subunit has two homologous domains, each of which associates into rings, termed CI and CII, respectively. KaiC has two neighboring phosphorylation sites Ser431 and Thr432 and its phosphorylation cycle occurs in an ordered pattern ST->SpT->pSpT->pST->ST, where S and T stand for S431 and Thr432, respectively, and pS and pT mean that the two sites are phosphorylated. This ordered phosphorylation has remained unclear. Here we show that the phosphorylation on the two sites has distinct effects on the CII ring flexibility, with the Thr432 phosphorylation maintaining a flexible CII ring and the Ser431 phosphorylation rigidifies the CII ring. Upon Ser431 phosphorylation-induced CII ring rigidification, the CII ring stacks with the CI ring, which triggers the exposure of the KaiB-binding site on the CI ring for KaiB to recruit KaiA away from the CII ring to the CI ring, initiating KaiC autodephosphorylation. We conclude that protein flexibility underpins the clockwise directionality of the oscillator. Finally, we show that the robustness of the oscillator hinges on the slow (hours timescale) KaiBKaiC binding, the critical delay for KaiA inhibition. We find that the exceptionally slow binding is in large part due to it that KaiB undergoes a global fold change. Such global fold change could be more widely in other timing-related events such as cell cycle. POST 11-134 Characterization Of Dynamic Processes In Substrate Recognition By Cytochrome P450 Enzymes


POSTER ABSTRACTS Nitin Jain, Nicholas Lopes, Ana Bernal University of Tennessee, Knoxville, Tennessee, US

Apart from structural factors, conformational flexibility of cytochrome P450 enzymes plays a key role in modulating protein-substrate interactions to accommodate substrates of differing physico-chemical properties as well as product specificity. Conformational dynamics of Cytochrome P450cam (CYP101) in various substrate-bound forms have been investigated using a combination of solution NMR spectrocopy and MD simulations to identify key regions undergoing dynamic motions and quantitate the flexibility. Differential dynamics are observed on all time-scales in several regions of CYP101 in presence of various substrates, indicating dynamic selection of CYP101 conformational space by different substrates. Similar conformational dynamic effects have been observed in the complex between CYP101 and its redox binding partner putidaredoxin (Pdx) in terms of substrate turnover. We present the data from these dynamic studies which clearly validate the role of dynamic processes in substrate recognition by CYP101. POST 11-135 Mapping the Interactions Between the Molecular Chaperones Hsp70, Hsp104 and Hsp110

Shankar Shastry, Shannon Doyle, Joel Hoskins, Sue Wickner National Cancer Institute, Bethesda, Maryland, US

Molecular chaperones help maintain cellular protein quality by assisting in the proper folding of nascent and misfolded proteins, preventing protein aggregation, reactivating aggregated proteins and targeting unfolded and misfolded proteins for degradation. Protein aggregation is linked to many diseases including Alzheimer’s disease, Parkinson’s disease, amyloidosis and cancer. In E. coli, the ClpB chaperone collaborates with DnaK and its cochaperones, DnaJ (an Hsp40) and GrpE (a nucleotide exchange factor) to disaggregate and reactivate aggregated proteins. In yeast, Hsp104 disaggregates proteins in conjunction with Ssa1, a yeast Hsp70, and its cochaperones, Ydj1 or Sis1 (Hsp40s) and Sse1 (a nucleotide exchange factor). This collaboration between chaperones is specific: E. coli DnaK only functions with E. coli ClpB and yeast Ssa1 acts with yeast Hsp104. We identified regions on E. coli DnaK important for collaboration with ClpB. These residues reside in a portion of the GrpE binding site on DnaK, as previously observed for the interaction between DnaK and ClpB from Thermus thermophilus (Rozenzweig et al, 2013, Science). Since the interactions between chaperones are specific, we wanted to determine if the residues on Ssa1 that interact with Hsp104 are in the same region as on DnaK. Mutations were generated in Ssa1 and the proteins were purified. When we tested for the ability of the mutants to reactivate inactive protein aggregates with Hsp40 in the presence or absence of Hsp104, we identified Ssa1 mutants that showed defects in collaboration with Hsp104. These mutations were in residues homologous to residues identified as being important for the interaction between DnaK and ClpB. Based on the crystal structure of bovine Hsc70 in complex with Sse1 (Schuerman et al, 2008, Mol. Cell), the residues in Ssa1 that interact with Hsp104 would also be expected to interact with Sse1. [NN2] By uncovering the specifics of the interactions of Hsp70 with Hsp104 and Hsp110, we are able to describe a more comprehensive model for protein disaggregation. POST 11-136 The Role Of Phenylalanine In An Intrinsically Disordered Protein From Yeast Nucleoporins

Korey M. Reid1, Krish Krishnan1, 2 1 Chemistry, California State University Fresno, Fresno, California, US, 2Department of Pathology and Laboratory Medicine, University Of California, Davis, Sacramento, California, US

Intrinsically disordered proteins (IDP) are a class of proteins that do not adopt a well defined threedimensional structure, and are characterized by their minimally folded structure with high intramolecular flexibility under physiological conditions. Their structural disorder directly or indirectly, plays an important


POSTER ABSTRACTS role in many essential cellular and regulatory processes. The nuclear pore complex (NPC) is a large protein complex that is the sole gateway for transport of proteins and RNAs between the nucleus and cytoplasm of a cell. It is composed of approximately 30 nucleoporins, or nups, a third of which are intrinsically disordered and contain a high count of FG-domain repeats (FG-nups). These FG-nups occupy the central pore region of the NPC and play an integral role in the regulation of nucleocytoplasmic transport of macromolecules through the NPC. It is hypothesized that the FG-Nups function in the NPC by forming either cohesive elements mediated through –GLFG- rich motifs or non-cohesive elements mediated through –FG- or –FxFG- motifs. In this study we focus on understanding the role of Phe (F) residues in the structural ensemble formed in a model FG-nup peptide. A 25 AA model peptide (FGnup) is designed using the native sequences of yeast nups containing three FG-domains, two of which are GLFG-motifs. To understand the role of Phe, a variant peptide (AGnup), with a substitution of Phe with Ala is also considered. Molecular conformations of these peptides are determined using modern nuclear magnetic resonance (NMR) methods and all atom molecular dynamics (MD) simulations. Multi-dimensional NMR experiments combined with distance geometry algorithms are used to determine the ensemble of conformations of the two peptides. All atom MD simulations were performed with multiple solvent models to understand residue specific details of intra-molecular interactions. Experimental results suggest that the FGnup is slightly more compact than the AGnup. However, MD results suggest that the average radius of gyration of the FGnup is greater than that of the AGnup. One possible reason for the difference between the NMR and MD simulations could be the time-scale of the dynamics sampled between these two methods. Detailed analysis of the ensemble of conformations obtained experimentally and using simulations will be presented, in addition to the implications of these results on the role of intrinsic disorder in the function of FG-nups. POST 11-137 The Acidic Residues of the IκBα PEST Sequence are Responsible for Actively Dissociating NFκB from DNA

Holly E. Dembinski, Kevin Wismer, Elizabeth Komives Chemistry and Biochemistry, UCSD, La Jolla, California, US

Nuclear factor kappa B (NFκB) transcription factors are responsible for the regulation of hundreds of target genes, their expression is induced by many classes of stimuli, and NFκBs play essential roles in the healthy regulation of cellular development and proliferation in inflammatory and immune responses. Diseases such as cancer, heart disease, Alzheimer’s disease, and AIDS can be attributed to the aberrant regulation of NFκB. The transcriptional activity of NFκB is controlled by its inhibitors, the IκBs. IκBα, in particular, dynamically responds to extracellular stimuli releasing a burst of NFκB that enters the nucleus and activates target genes. The transcriptional activation is short-lived, and our lab has been investigating the mechanism of post-induction repression. We previously showed that IκBα actively dissociates NFκB from DNA. Analysis of the crystal structures of NFκB (RelA/p50) with DNA and with IκBα shows that the IκBα PEST sequence, which is rich in glutamate and aspartate residues, forms similar electrostatic contacts to NFκB as the DNA. Given this, we hypothesized that the IκBα PEST sequence electrostatically repels DNA from NFκB during the dissociation process. Here we present fascinating results that show that the individual and collective, conservative mutation of these acidic residues to their amide counterparts does not affect the binding affinities of these mutants to NFκB; however, the mutant in which all five acidic residues are neutralized is incapable of actively dissociating NFκB from DNA and intriguingly forms a stable IκBα-NFκB-DNA ternary complex. We are currently investigating the structure of this ternary complex via nuclear magnetic resonance, and we are exploring the effects of this mutation and the importance of the accelerated dissociation of NFκB from DNA by IκBα in mouse embryonic fibroblast cells.


POSTER ABSTRACTS POST 11-138 Nanoscale Hydrodynamic Study of Proteins under Thermal Agitation and Electric Field

Yuanming Zhang, Zachary Weiner, Eric Farrell Brookhaven Instruments Corporation, Holtsville, New York, US

Proteins hydrodynamics is characterized at nanometer scale with the temporal analysis of their Rayleigh scattering. The dynamic-structure-factor is experimentally extracted and used to quantify proteins meansquare-displacement under thermal agitation and electrophoretic motion under electric field. Through the subsequent data analysis based on molecular theories of proteins hydrodynamics, it is demonstrated that valuable insights can be obtained regard the physical characteristics of proteins. Hierarchic structures and multiscale dynamics of proteins give rise to their versatile biological functionalities that play vital roles in DNA transcription, mRNA translation, signal transduction, and etc.. These complex biological processes are enacted or controlled through protein transport and interactions. The subject receives increasing attentions not only for understanding of bio-molecular and cellular functions, but also for pharmaceutical development. In order to analyze protein dynamics in a complex biological process, proteins often need to be selectively probed either by immobilization or by labeling. Due to unwanted dynamic alternation and measurement bias caused by immobilization or labeling, labelfree methods are therefore optimal to probe protein dynamics in their native biological environment. Ubiquitous Rayleigh scattering is a viable tool for the purposes of in-situ and label-free probe on proteins hydrodynamics at broad length scale. Protein hydrodynamics can be readily quantified with spectral broadening and shift of the light signal scattered from them. In the present study, using common proteins such as lysozyme and Bovine Serum Albumin (BSA), we demonstrate the sensitivity and precision of the techniques. The hydrodynamic and other physical characteristics of proteins, such as translational diffusion coefficient, charge and electrostatic force/potential range (Debye length), can then be calculated with Stokes-Einstein equation of translational friction force and Debye-Hückel-Henry theory of electrophoretic effect. Furthermore, it is shown that the effects of solvation/suspension media as well as protein-protein interaction can be assessed well within the measurement certainty. POST 11-139 Probing the Clamping Movement of Xylanase B by NMR Spectroscopy 1, 2, 3 1, 2, 3 Nhung T. Nguyen , Nicolas Doucet 1

INRS-Institut Armand-Frappier - University of Quebec, 531 boul. des Prairies, , Laval, H7V 1B7, Quebec, Canada, 2PROTEO, the Québec Network for Research on Protein Function, Structure, and Engineering, 1045 Avenue de la Médecine, Université Laval, Quebec, G1V 0A6, Quebec, Canada, 3GRASP, the Groupe de Recherche Axé sur la Structure des Protéines, 3649 Promenade Sir William Osler, McGill University, Montreal, Quebec, Canada

The present work describes NMR assignments and relaxation dispersion experiments probing the dynamics of apo and ligand-bound xylanase B (XlnB) from Streptomyces lividans. Evidence from mutagenesis, crystal structures and molecular dynamics simulations previously suggested that the 15 “thumb-loop” motion of XlnB might play a major role in substrate binding/catalysis [1,2,3,4,5]. Our NCPMG data show similar millisecond time-scale motions for active-site fingers in the free and xylobiosebound enzyme. However, in the presence of the longer xylopentaose ligand, conformational exchange 1 15 emerges on the thumb loop, along the active-site cleft, and on the opposite side of the fingers. H- N HSQC titration data also indicates the involvement of thumb loop and binding cleft residues in substrate recognition. For the first time, our results illuminate the atomic-scale dynamics of XlnB on the millisecond time-scale, suggesting a global clamping movement during catalysis.References: [1]. Paës G., Berrin J.G., and Beaugrand J. Biotechnology Advances. (2012) 30: 564–592. [2] Pollet A., Lagaert S., Eneyskaya E.,


POSTER ABSTRACTS Kulminskaya A., Delcour J.A., and Courtin C.M. Biochimica et Biophysica Acta (2010) 1804:977–985. [3]. Murakami M.T., Arni R.K., Vieira D.S., Degrève L., Ruller R., and Ward R. FEBS Letter (2005), 579:6505–10. [4]. Vieira D.S, Degreve L., and Ward R.J. Biochimica et Biophysica Acta-Gen Subject (2009); 1790:1301–6. [5]. Hakulinen N., Turunen O., Janne Janis J., Leisola M. and Rouvinen J. European Journal of Biochemistry (2003), 270, 1399–1412.

POST 11-140 Intrinsic GTP Hydrolysis is Observed For a Switch 1 Mutant of Cdc42 in the Presence of a Specific GTPase Inhibitor

Reena Chandrashekar, Kyla Morris, Colin D. Heyes, Paul D. Adams Chemistry and Biochemistry, Univeristy of Arkansas-Fayettevillle, Fayetteville, Arkansas, US

The Ras-related protein Cell division cycle 42 (Cdc42) is important in regulating cell-signaling processes. Cdc42-protein interactions are targeted towards flexible “Switch” regions in the Ras protein that help regulate effector binding. We have studied the kinetics of the intrinsic GTP hydrolysis reaction in the absence and presence of a peptide derivative of a p21-activated kinase effector (PBD46) for a Switch 1 mutant of Cdc42, Cdc42(T35A), and compared it to that of Cdc42 wild type. While the mutation in Cdc42 does not affect GTP hydrolysis, the binding between Cdc42 and PBD46 is disturbed. Moreover, whereas the binding of PBD46 to wild type Cdc42 results in complete inhibition of GTP hydrolysis, the same interaction in Cdc42(T35A) leads to a partial recovery of GTP hydrolysis. The kinetics, together with the concentration dependence of PBD46 on the degree of inhibition of GTP hydrolysis suggest that the dynamics changes in the Switch 1 region of Cdc42 caused by the T35A mutation fosters very slow interconverting conformational states. We rationalize a model where both of these states possess the ability to hydrolyze GTP, but one that has a drastic difference in its ability to interact with PBD46. Our findings suggest that one way to control potentially oncogenic Ras protein over-activity may be facilitated by altering conformational dynamics underlying Ras-protein interactions that can lead to abnormal Rasstimulated cell signaling. POST 11-141 Three-dimensional Structure of the 54 kDa Subunit of the Chloroplast Signal Recognition Particle using Molecular Modeling

Rory Henderson1, Suresh Kumar1, Colin Heyes1, Ralph Henry2 1 Chemistry & Biochemistry, University of Arkansas, Fayetteville, Arkansas, US, 2Biological Sciences, University of Arkansas, Fayetteville, Arkansas, US

The chloroplast signal recognition particle is a heterodimeric complex of the 54kDa cytosolic signal recognition particle homologue, cpSRP54, and a novel 43kDa protein, cpSRP43. While a nearly complete structure for cpSRP43 has been obtained via X-ray crystallography, no structure is yet available for cpsRP54. A structure for this protein could provide valuable information for the rationalization of the extensive information already available regarding its function, and in the understanding of the as yet undetermined mechanism of light harvesting chlorophyll binding protein’s (LHCP) insertion into the thylakoid membrane. In this investigation we developed a structure for cpsRP54 using a combination of homology modeling, de novo structure prediction and molecular dynamics simulation. The resulting structure is consistent with the known properties of the protein and sheds light on some of the mechanistic details of its functioning. POST 11-142 Evidence For Significant Changes In Backbone Motions Between Apo And Gα-Bound Human RGS4


POSTER ABSTRACTS Lusine Simonyan, Shayla A. Brooks, Karin A. Crowhurst Chemistry and Biochemistry, California State University Northridge, Northridge, California, US

RGS proteins act as essential regulators of G protein activity by accelerating GTP hydrolysis by Gα subunits, terminating signaling and ultimately controlling the relay of various neurological signals from outside to inside cells; they therefore play a central role in signal transmission, neuronal development and synaptic plasticity. It is now understood that protein motions, flexibility and conformational changes often have key roles in signal transmission processes. Numerous studies have been performed on the RGS and Gα domains as potential drug targets; consequently, crystal and NMR structures of RGS proteins have been solved. However, despite reports of preliminary evidence for protein flexibility and conformational changes in Gα-RGS interactions, they have not been characterized in detail. One of our project goals has been to compare the internal dynamics of apo versus Gαi1-bound human RGS4 (hRGS4) in order to improve our understanding of the regulation of signal transmission. In the long term we would like to compare these results with data from hRGS7 (which also binds Gαi1 but with lower affinity) in order to gain insight into the role of dynamics on the selectivity of RGS proteins for their Gα targets. In our current phase of the project we have used NMR spectroscopy to record fast and intermediate timescale backbone relaxation as well as hydrogen exchange experiments on apo hRGS4. Results indicate that the N- and Ctermini are the primary sites of increased fast (ps-ns) timescale motions; interestingly, intermediate (μsms) timescale motions were observed on the helix of the primary binding site with Gα. Unexpectedly, we also discovered that hRGS4 maintains a dimer structure, even at low concentrations, whose interface must still be characterized. We have also recorded preliminary experiments on isotopically labeled hRGS4 bound to unlabeled (i.e. NMR invisible) Gαi1. An overlay of HSQC NMR spectra from apo and Gαi1-bound hRGS4 confirm evidence (from NMR and crystal structures) for a conformational change when hRGS4 binds its target. However, it was a surprise to see how extensive the chemical shift changes were: almost every backbone resonance was shifted to some extent. Further investigation of these changes should provide increased insight into the role of protein motions in RGS-Gα signaling interactions. We gratefully acknowledge support from NSF grants MCB-1158177 (RUI) and CHE-1040134 (MRI).

Poster Session: Protein Engineering & Synthetic Biology POST 12-143 Engineering Picomolar Affinity into a Rationally Identified 5 kDa Scaffold for Tumor Targeting

Max Kruziki, Patrick Holec, Benjamin Hackel Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, US

Small protein scaffolds, relative to antibodies, can provide superior stability, production, conjugation, and distribution. Yet while many scaffolds have been tested to generate synthetic ligands, the causes of the varying degrees of success or failure have not been systematically explored. We merged computational and experimental tools to elucidate the biophysical factors that dictate evolutionary potential to empower rational identification or design of protein scaffolds. We have identified the 45-amino acid Gp2 domain as an effective protein scaffold to engineer stable, picomolar affinity ligands. We developed and implemented a computational algorithm to efficiently evaluate naturally occurring protein domains on numerous metrics with potential impact on evolution of binding function. In one implementation, we identified all domains in the Protein Data Bank with two accessible loops (diversified paratope) and scored them on the size, shape, orientation, and target accessibility of their paratopes as well as protein size,


POSTER ABSTRACTS stability upon mutation, and dependence on disulfide bonds and/or cofactors. Machine learning was used to preliminarily weigh each metric based on historical scaffold performance. The top scaffold was the 45residue T7 phage gene protein 2 with a single alpha helix opposite a beta sheet with two adjacent loops amenable to mutation. We diversified the twelve loop amino acids using complementarity bias and length 8 diversity. The library of 10 mutants was displayed on the yeast surface and screened for binders to several protein targets. Novel ligand discovery and directed evolution yielded binders with affinities as strong as 200±100 pM and no observable nonspecific binding. Circular dichroism showed secondary structure comparable to the wild-type protein and Tm values from 70±4ºC to 80±1ºC for evolved ligands relative to 67±4º for wild-type. We evolved binders to epidermal growth factor receptor that effectively label human tumor cells with nanomolar affinity. More broadly, libraries from the top 30 scaffolds were created for comparative analysis of the ability to evolve specific, high affinity binding to numerous targets. We will discuss the relative efficacy of each scaffold as well as the scaffold design metrics that correlate most strongly with performance. Implications for future scaffold design, including synthetic protein topologies, will be addressed as well as other designs. POST 12-144 Utilizing Metal-Ligand Interactions to Promote Assembly of Collagen-Based Peptides into Functional Nanostructures

Jeremy Gleaton, David Przybyla, Charles M. Rubert-Perez, Jean Chmielewski Purdue University, Lafayette, Indiana, US

Collagen is a highly abundant protein that comprises up to one third of the total protein content within humans. Collagen serves as an essential scaffold and structural support throughout nature. Because of collagen’s striking structural strength and importance, efficient methods for mimicking natural collagen and the development of collagen biomaterials are actively being pursued. Here we present a collagen mimetic peptide, modified with bipyridines, that undergoes a hierarchical assembly into triple helices, followed by the formation of curved disks via hydrophobic interactions. Metal-ion mediated assembly of the curved disks is demonstrated to form hollow microspheres. These hollow microspheres encapsulate various molecular weight fluorescently labeled dextrans, and demonstrate a time dependent release of contents under a variety of thermal conditions. By gaining an understanding of the higher order assembly of collagen-mimetic peptide triple helices at a molecular level, we hope to obtain unique collagen-based materials on demand for a range of applications. POST 12-145 Live Cell Imaging of Molecular Conformations and Actions

Peter Yingxiao Wang Bioengineering, UC San Diego, La Jolla, California, US

Signaling molecules and their activities are well coordinated in space and time to regulate cellular functions in response to mechanical and chemical microenvironment. Based on fluorescent resonance energy transfer (FRET), we have developed genetically encoded biosensors to monitor the dynamic molecular activities (such as Src and FAK activities) in live cells at subcellular compartments when cells interact with their neighbors or mechanical/chemical microenvironment. In a recent study, we show that a ubiquitous signaling protein, Src Homology 2 (SH2) Domain-containing Protein Tyrosine Phosphatase 2 (Shp2), displayed unexpected plasticity of conformational changes via intramolecular interactions within Shp2 (cis-interaction). Utilizing Shp2 biosensors based on fluorescence resonance energy transfer (FRET), we found that two phosphorylated regulatory tyrosines upon stimulated phosphorylation can compete for the cis-interaction of the same SH2 domain within Shp2 to achieve plasticity. The antagonistic combination of contextual amino acid sequence and position (e.g. favorable position combined with


POSTER ABSTRACTS adverse sequence) can create a relatively small difference between the two phosphorylated tyrosines in their overall competitiveness for cis-interaction. Enlarging this difference by swapping the sequences at the two tyrosine positions resulted in loss of conformational plasticity and reprogrammed downstream ERK signaling dynamics. Thus, while the combinatorial effect of specific sequence and position of DNA cisregulatory elements on tuning gene expression has been well studied, our results unraveled a new and simple strategy to achieve cis-regulatory plasticity of protein conformation by coordinating the combination of sequence and position. We suggest that this strategy can serve as a general and basic design principle for natural and synthetic proteins, with their conformations and functions tunable by cisinteraction to regulate downstream physiological consequences. These proteins with plasticity can serve as programmable building blocks or nodes for higher order molecular machines and networks. POST 12-146 Protein Design: Preventing Protein Aggregation In Recombinant Erythropoietin

Manuel A. Carballo-Amador, Jim Warwicker, Alan J. Dickson Faculty of Life Sciences, University of Manchester, Manchester, UK

Protein design is a powerful approach for improving protein physicochemical properties with, potential consequences for stability, activity, and solubility of proteins. Protein solubility characteristics are important determinants of the success for recombinant therapeutic proteins in relation to expression, purification, storage and administration. Based on protein structure, an algorithm has been developed to predict protein solubility, defining polar and non-polar patches on protein surfaces. Using this algorithm, we predicted amino acid changes that would facilitate expression of forms of human erythropoietin (rHuEPO) of directed solubility in E. coli. We found that single point mutations (changing a single amino acid from positive to negative charge) verified the predicted effect on rHuEPO solubility (experimentally defined as the distribution between soluble and inclusion body fractions) in E. coli. Further application of this algorithm could provide a valuable tool in the design and engineering of proteins, with enhanced solubility and stability. POST 12-147 Intracellular Regulation of the NFE2L3 Transcription Factor

Meenakshi B. Kannan, Volker Blank Experimental Medicine, McGill University, Montreal, Quebec, Canada

Cap ‘n’ Collar (CNC) basic leucine zipper transcription factors play crucial roles in mammalian gene expression, stress response and cancer. NFE2L3 (NF-E2 related factor 3), also called Nrf3, was identified as a member of the CNC family. NFE2L3 forms heterodimers with small MAF proteins and binds to MARE (Maf recognition element) or ARE (Antioxidant response element) consensus sequences. Our laboratory has generated mice lacking the Nfe2l3 gene and showed that the knockout animals are highly sensitive to treatment with the carcinogen benzo[a]pyrene, leading to increased lymphoma formation, suggesting a tumor suppressor-like function in this context. Contrastingly, in breast cancer cells, we observe increased invasiveness in the absence of NFE2L3, which illustrates oncogene-like properties of NFE2L3. At the cellular level, we found that NFE2L3 exists in three different forms (A, B and C). The ‘A’ form is localized in the endoplasmic reticulum and is N-glycosylated, whereas the ‘B’ form, the full form of NFE2L3, is predominantly present in the cytoplasm and is also found in the nucleus, upon over-expression. The 'C' form of NFE2L3 is cleaved at the N-terminus and is localized in the nucleus. I have identified a functional bipartite nuclear localization sequence (NLS) in NFE2L3 that is required for its nuclear import. It is also necessary for it to transactivate its downstream targets. In contrast to wt NFE2L3, the NLS mutant fails to promote invasion in breast cancer cells. Cycloheximide studies revealed a rapid turnover of NFE2L3. Mass spectrometry and other biochemical analyses revealed ubiquitination and degradation of the NFE2L3


POSTER ABSTRACTS protein. My results also show the involvement of the ubiquitin ligase and tumour supressor, FBW7, in the degradation of NFE2L3. Together, my research will elucidate crucial regulatory mechanisms governing the NFE2L3 transcription factor and determine their significance in NFE2L3-mediated carcinogenesis.

POST 12-148 Phosphorylation and Binding Partners of the NFE2L3 Transcription Factor: Insights into its Role in Oncogenesis

Isadore Dodard-Friedman, Volker Blank Experimental Medicine, McGill University, Montreal, Quebec, Canada

Phosphorylation and Binding Partners of the NFE2L3 Transcription Factor: Insights into its Role in Oncogenesis Post-translational modifications play a major role in the regulation of protein function. Our laboratory has analyzed the NEF2L3 (NF-E2-like factor 3) protein, which is a member of the Cap 'n' Collar (CNC) transcription factor family. We found that NFE2L3 is a stringently regulated and post-translationally modified protein with a high turnover. Moreover, we discovered that NFE2L3 exists in the cell as three distinct forms located in different cellular compartments which we have called ‘A’, ‘B’ and ‘C’. Our cellular and mouse studies revealed that its function is linked to carcinogenesis and invasiveness, respectively. While strong evidence suggests it is glycosylated and ubiquitinated, the phosphorylation status of this protein is yet to be elucidated. Intriguingly, stringent in silico kinase screens have revealed a series of potential phosphorylation sites within this protein's transactivation domain. In order to better characterize NFE2L3 phosphorylation sites, a GST tagged NFE2L3 fusion protein comprising the transactivation domain was used as a substrate in a kinase screen developed by Screaton and colleagues. Out of the 420 kinases assessed, five were found to phosphorylate NFE2L3 including the cell cycle kinase PLK1. Phosphorylation of NFE2L3 by PLK1 was further confirmed by in vitro kinase assays. Furthermore, site directed mutagenesis of potential PLK1 phosphorylation sites converting respective serines to alanines was performed. In parallel, immunoprecipitation together with mass spectrometry analysis was used to identify potential NFE2L3 protein partners. These experiments uncovered a series of potential interactors. Of these, the golgi protein golgin-84 was confirmed to be a bona fide protein partner of NFE2L3 as confirmed by coimmunoprecipation and immunoblot experiments. Reverse immunoprecipitation revealed that only the ‘A’ form of NFE2L3 interacts with golgin-84. This data suggests the transcription factor may be processed in the golgi. Together, my research will lead to a better understanding of the pathways regulating NFE2L3 and linking it to important cellular functions. POST 12-149 An Orthogonal Genetic System for Rapid Evolution

Chang Liu Biomedical Engineering and Chemistry, University of California at Irvine, Irvine, California, US 1 We recently developed an orthogonal DNA replication system in the yeast Saccharomyces cerevisiae. This

system consists of an orthogonal DNA plasmid–DNA polymerase pair wherein the orthogonal DNA polymerase stably and specifically replicates the orthogonal plasmid in the cytoplasm of yeast. Engineered error-prone DNA polymerases show complete mutational targeting in vivo: per-base mutation rates on the plasmid can be increased substantially with no increase in genomic rates. Orthogonal replication therefore serves as a platform for in vivo continuous evolution of user-selected genes and as a system 1 whose replicative properties can be manipulated independently of the host's. A Ravikumar, A Arrieta, CC Liu. An Orthogonal DNA Replication System in Yeast. Nature Chemical Biology, 10, 175-177 (2014).


POSTER ABSTRACTS POST 12-150 A Recognition Model Of ACP-HCS Interaction For Programmed Beta-Branching In Type I Polyketide Synthases

Rohit Farmer1, Anthony S. Haines1, Matthew . Crump2, Christopher . Thomas1, Peter J. Winn1 1 School of Biosciences, University of Birmingham, Birmingham, United Kingdom, 2School of Chemistry, University of Bristol, Bristol, United Kingdom

Polyketide synthases (PKSs) are enzyme complexes that synthesise a wide range of natural products of medicinal interest, notably a large number of antibiotics. Type I polyketide synthases can introduce betacarbon branches into a growing polyketide chain via enzymes encoded by the “HMG-CoA synthase (HCS) cassette”. One of the first polyketide biosynthesis cluster in which the HCS cassette was discovered is responsible for the synthesis of the antibiotic mupirocin byPseudomonas fluorescens. MupH is the HMGCoA synthase homologue responsible for β-branching in the mupirocin synthesis pathway. To understand better what allows the HCS cassette to recognise β-branch-associated acyl carrier proteins (ACPs) of the mupirocin synthesis pathway, we have computationally docked the modelled MupH with the NMR structure of ACPs. The docking results were also supported by the evolutionary trace data and the physical properties of the interface residues. Hidden Markov models (HMM) were used to classify ACPs as branching and non-branching. HMM analysis highlighted essential features for an ACP to behave like a branching ACP. Through modelling and mutagenesis we identified helix III of the ACP as a probable anchor point of the ACP–HCS complex. The position of this helix is determined by the core of the ACP and substituting the interface residues modulates the interaction specificity. Our method for predicting βcarbon branching lays a basis for determining the rules for ACP-HCS specificity and expands the potential for engineering new polyketides. POST 12-151 Engineering Photo-Control Of Translation Initiation Using Photoactive Yellow Protein Hybrids

Anil Kumar1, Anna S. Jaikaran1, Alaji Bah2, 3, Julie Forman-Kay2, 3, G. Andrew Woolley1 1 Chemistry, University of Toronto, Toronto, Ontario, Canada, 2Biochemistry, University of Toronto, Toronto, Ontario, Canada, 3Hospital for Sick Children Research Institute, Toronto, Ontario, Canada

Photo-control of translation initiation could be a powerful tool for probing the role of translational processes in cellular biology. Translation initiation in eukaryotic cells is regulated by 4EBP (eukaryotic initiation factor 4E-binding protein). If 4EBP activity can be disrupted by light-induced conformational changes propagated from a linked photoactive yellow protein (PYP) domain, this may enable photocontrol of cap-dependent translation initiation. Here, we report designs and initial characterization of 4EBP-circularly permuted photoactive yellow protein (cPYP) chimeras. Three constructs were prepared, 54 60 one with only the primary binding site ( YXXXXLΦ : cPYP-4EBP-C0) and two longer constructs also 78 82 containing the secondary binding site ( IPGVT : cPYP-4EBP-C1, cPYP-4EBP-C1N1). All designed chimeras were expressed in E.coli and are highly soluble. UV-Vis spectra of the dark adapted and irradiated state confirm the photoswitchable properties of all designed constructs. Recovery rates were increased in the presence of 4E, consistent with coupling of conformational changes associated with the photocycle and 4E binding. Initial activity using an in vitro translation system based on HeLa cell extracts shows differential inhibition of translation by the constructs. POST 12-152 Gold Decorated Peptide Amphiphile Templates for Directed Silver Nanorods Growth

Shlomo Zarzhitsky1, 2, Hanna Rapaport2, 1



ILSE Katz Center for Meso-and Nanoscale Science and Technology, Ben-Gurion University, Beer-Sheva, Israel, 2Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University, Israel

Peptides are versatile building blocks that can be designed to assume particular structure with respect to predetermined function and biological activity. Tailoring the exact sequence of amino acids in peptides allows fine tuning of mechanical and chemical properties, along with self-assembly kinetic. Amphiphilic and anionic β-sheet peptides may form hydrogels at near physiological pH values. Our group has previously studied peptides with the amphiphilic motif (Phe-X) in peptides such as Pro-Glu-(Phe-Glu)5Pro. In the current study we were intrigued to explore whether an amphiphilic tripeptide, exhibiting one βpleated motif, i.e. Phe-X-Phe with X = hydrophilic amino acid can be triggered to self-assemble into bsheet fibrils. Relying on the crucial role of aromatic stacking interactions as the driving force in molecular self-assembly, we explored the self-assembly of this family of peptides with X = Glu\Lys\Cys\Thr. All these peptides were found to constitute self-supporting hydrogels under various conditions. During the assembly process FEF peptides were found to spontaneously stack in elongated fibrils composed of βpleated bilayers with preferred angular orientations between intersecting and stemming ones. These arrangements point to the influence of the phenylalanine aromatic moieties on the intermolecular interactions of neighboring fibrils. To explore the electrostatic potential activity of Glu residues, fibrils were exploited as templates for anchoring gold nanoparticles serving as nucleation seeds for directed mineralization of silver nanorods.

POST 12-153 Engineered Oligosaccharyltransferases With Greatly Relaxed Acceptor Site Specificity

Anne A. Ollis, Sheng Zhang, Matthew P. DeLisa Cornell University, Ithaca, New York, US The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (Nlinked) glycosylation and serves as the archetype for bacterial N-glycosylation. This machinery has been functionally transferred intoEscherichia coli, thereby enabling convenient mechanistic dissection of the Nglycosylation process in this genetically tractable host. For instance, glycosylation-competent E. coli were

used to demonstrate that the primary consensus site for glycan attachment recognized by the bacterial oligosaccharyltransferase (OST) PglB is (D/E-X-1-N-X+1-S/T; X-1, X+1 ≠ P). In contrast, N-glycosylation site selection by eukaryotic OST is less specific, requiring only a short N-X-S/T acceptor sequence. Here, we sought to identify the sequence determinants in PglB that govern its requirement for a negatively charged residue at the -2 position and effectively restrict bacterial glycosylation to a narrow set of polypeptides. This involved creation of a reliable genetic reporter for N-glycosylation in E. coli named glycoSNAP (glycosylation of secreted N-linked acceptor proteins) that was subsequently used to discover C. jejuni PglB variants with significantly relaxed substrate specificities. Several PglB variants were identified that no longer obeyed the “minus two rule”, glycosylating an array of noncanonical acceptor sequences including one in a eukaryotic N-glycoprotein. Taken together, these results underscore the


POSTER ABSTRACTS utility of the glycoSNAP assay for shedding light on poorly understood aspects of N-glycosylation and for engineering designer N-glycosylation biocatalysts. POST 12-154 The Dynamic Peptide Recognition And Stabilization Mechanism Of Human Leukocyte Antigen B*35:01

Saeko Yanaka1, Takamasa Ueno3, Kouhei Tsumoto2, Kenji Sugase1 1 Suntory Foundation for Life Sciences Bioorganic Research Institute, Osaka, Japan, 2Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan, 3Center for AIDS Research, Kumamoto University, Chuuoo, Japan

In an immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various antigenic peptides to cytotoxic T lymphocyte (CTL). The stability and long-lived presentation of the peptide-HLA complex (pHLA), important for efficient antigen-specific CTL activation, highly depends on bound peptides. Crystal structures of pHLA, however, are very similar to each other irrespective of the peptides. Thus, the inherent determinant for pHLA stabilization and peptide recognition remains elusive. In this study, we have examined the mechanism by which HLA-B*35:01 recognizes various peptides and stabilizes the complex by elucidating its conformational dynamics using relaxation dispersion NMR spectroscopy. HLA-B*35:01 is one of the most investigated HLAs in the light of the relationship between antigenic peptide and CTL activity. The NMR experiments of three pHLAs with different peptides showed that the peptide binding-domain in pHLAs fluctuates between the major and minor conformation in solution, and that the transition to the minor conformation is accompanied with a negative change of heat capacity ΔCp, indicating the more dehydrated well-packed conformation of the minor state. Interestingly, the minor populations detected by the relaxation dispersion experiments correlated well with the previously reported CTL activity duration. Taken all results together, we revealed that pHLA loosely recognizes various peptides with a highly identical conformation, and transiently forms a more dehydrated minor conformation in which the peptide is more tightly bound, resulting in circumvention of pHLA disintegration. We propose the dynamic recognition-stabilization mechanism as “the transient induced fit model”. POST 12-155 Engineering Ordered Protein Assemblies by Helix-Fusion Strategy

Yen-Ting Lai, Todd O. Yeates Chemistry and Biochemistry, UCLA, Los Angeles, California, US

Many natural proteins exhibit self-assembling properties. Large and ordered architectures, such as viral capsids, ferritins, clathrin coats, microtubules and bacterial S-layer can arise by repeated interactions between identical protein building blocks. Designing novel proteins that are capable of self-assembling into large and ordered architectures similar to those observed in Nature is a challenging task for protein engineers. Successfully designed protein assemblies with novel properties can lead to important applications in nanotechnology, material science and medicine. To design novel protein assemblies, the most critical factor is to fulfill the underlining symmetries of the target assemblies. For example, if one would like to engineer novel assemblies similar to viral capsids, then the icosahedral symmetry (point group 532) must be satisfied. Several strategies have been developed in the past decade to engineer protein building blocks that can self-assemble into highly ordered architectures; the most successful of these have been centered on the principles of symmetry. One strategy proposed by our group is to fuse natural protein homo-oligomers with a helix linker in a pre-determined orientation. Through careful in silicoselection, fusion protein molecules capable of forming structures with higher-order symmetries can be generated. In my presentation, I will discuss practical considerations that are essential for success in


POSTER ABSTRACTS the helix-fusion strategy. Successfully engineered tetrahedrally-shaped protein cages (16 nm in diameter) were verified by x-ray crystallography, small-angle x-ray scattering and electron microscopy. We have also created an even larger, highly-porous protein framework in the shape of a cube (20 nm in diameter) with underlying octahedral symmetry, and verified its structure by x-ray crystallography. These structural studies, however, also highlight that protein cages and frameworks engineered in this way tend to be flexible and can deviate considerably from perfect symmetry. Modifications to the original strategy, such as introduction of secondary contacts, can potentially improve the success rate of this method. 1. J. E. Padilla, C. Colovos, T. O. Yeates, Proc Natl Acad Sci U S A (2001) 98, 2217 2. Y.-T. Lai, D. Cascio, T. O. Yeates, Science (2012) 336(6085), 1129 3. Y.-T. Lai, K.-L. Tsai, M. Sawaya, F. Asturias and T. O. Yeates, JACS (2013) 135(20), 7738 POST 12-156 Total Synthesis And Chaperone-Mediated Folding Of A 312-Residue Mirror-Image Enzyme

Michael T. Jacobsen1, Matthew T. Weinstock1, 2, Michael S. Kay1 1 University of Utah, Salt Lake City, Utah, US, 2Synthetic Genomics, La Jolla, California, US

Non-natural D-proteins (composed of D-amino acids) are intriguing macromolecules. First, they are insensitive to natural proteases, making them appealing drug candidates. Second, they can facilitate structural analysis via racemic protein crystallography. Third, they possess identical physicochemical properties to their natural counterparts, making it possible to study otherwise toxic proteins in mirror image. Unfortunately, D-proteins can only be produced synthetically, hindering production. Furthermore, folding large and complex D-proteins will be challenging given the absence of D-chaperones. We hypothesized that a natural chaperone could fold a D-protein, which we addressed by synthesizing a model client protein in both L- and D-chirality and evaluating its folding by a natural chaperone. We selected dapA as a model client of the GroEL/ES chaperone. dapA is an essential enzyme in lysine production and bacterial cell wall synthesis, and its in vivo folding strictly depends on GroEL/ES. The syntheses of 312-residue dapA in both L- and D- (the longest reported synthetic proteins) were achieved using solid-phase peptide synthesis with convergent assembly via peptide hydrazides ligations. Rational mutagenesis and highly optimized synthesis and chromatography methods were necessary to complete this challenging synthesis. The fully assembled synthetic dapA proteins possess the correct molecular weight and structure (measured by CD), and they have similar activity to the recombinant enzyme. Importantly, both L- and D-dapA could be successfully refolded to a similar degree by GroEL/ES. These results offer a fundamental insight into chaperone mechanism: GroEL/ES recognizes and folds client proteins through non-specific hydrophobic interactions—activity that even extends to D-proteins. Because of this ambidextrous folding activity, a new tool (natural GroEL/ES) is now available to assist in Dprotein production. Ultimately, we aim to use the tools and strategies described here to produce a synthetic D-ribosome for routine production of D-proteins. Highly optimized synthesis strategies are needed to produce ribosomal D-proteins. This work suggests that their complex folding and assembly may be facilitated by natural chaperones. The D-ribosome will also serve as a critical springboard to the creation of mirror-image organisms. POST 12-157 Creating Self-Assembling Stimulus-Responsive Hydrogels From Protein Components

Danielle Williams1, Ashley Schloss1, Lynne Regan1, 2, 3 1 Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, US, 2Chemistry, Yale University, New Haven, Connecticut, US, 3Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, US


POSTER ABSTRACTS Stimulus-responsive hydrogels are promising vehicles for the controlled delivery of small molecules, cells, and other molecular cargo into the body. We have previously demonstrated the formation of selfassembling hydrogels from tetratricopeptide repeat (TPR) protein arrays that form non-covalent crosslinks with corresponding peptides upon mixing. This binding interaction is both pH- and ionic strengthdependent, allowing for encapsulation and subsequent release of cargo in response to a stimulus. The properties of this interaction directly translate to the macroscopic properties of the gel. Here we present the design of stimulus-responsive hydrogels made entirely of protein components. The modular nature of TPR domains allows for the engineering of interactions with different binding affinities/specificities that encode unique macroscopic properties. Original designs used functionalized star PEGs as the cognate peptide “cross-linkers” for the TPR arrays. Through the adaptation of SpyTag/SpyCatcher technology, we have developed a novel concatenation and branching scheme that eliminates the need for functionalized PEG. We can now create branched protein topologies with multivalent peptides that can be readily expressed in E. coli with the possibility of all proteins being monodisperse. We will discuss the properties of various different hydrogels created using this facile and robust production method and how they can be fine-tuned for biomedical applications. POST 12-158 Characterization Of A Novel Synthetic Biomaterial For Protein Immobilization

Carrie Marean-Reardon1, Patrick Reardon2, Thomas Squier3, Kathleen McAteer1 1 Washington State University, Richland, Washington, US, 2Pacific Northwest National Laboratory, Richland, Washington, US, 3Western University of Health Sciences, Lebanon, Oregon, US Enzymes catalyze a wide variety of processes that are important to environmental, industrial, and medical applications. However, many enzymes are inherently fragile, requiring specific conditions in which to operate. Polyethylene glycol diacrylate (PEGDA) is a thiol-reactive material that forms hydrogels and can be conjugated to cysteine-containing proteins to incorporate a usable binding site within the gel. In this case, the binding protein incorporated was calmodulin. The characterization of this method of protein immobilization was undertaken using a small protein, GB1, tagged with the M13 affinity peptide of calmodulin. Solution-state 15N-HSQC NMR spectra were collected on these gels and show the protein maintains its conformation. The spectra were consistent between free protein in solution and immobilized protein. The binding is reversible with the addition of EGTA, which chelates the calcium that the calmodulin needs to maintain binding. These results demonstrate that PEGDA hydrogels are a viable protein immobilization tool. POST 12-159 Protein Fragment Exchange: Converting an Arbitrary Binding Protein into a Robust FRET Biosensor

Huimei Zheng1, Jing Bi2, Mira Krendel2, Stewart N. Loh1 1 Biochemistry & Molecular Biology, State University of New York - Upstate Medical University, Syracuse, New York, US, 2Cell & Developmental Biology, State University of New York - Upstate Medical University, Syracuse, New York, US

Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial and temporal distribution of specific molecules in living cells. A major challenge facing biosensor development is how to functionally couple a biological recognition domain to an output module so that the binding event can be transduced to a visible and quantifiable signal (e.g. Förster resonance energy transfer, or FRET). Most designs achieve coupling by means of a binding protein that changes conformation upon interacting with its target. This approach is limited by the fact that few proteins possess such natural allosteric mechanisms, and for those that do, the conformational change is frequently not extensive enough to produce a large distance change between FRET donor and acceptor


POSTER ABSTRACTS groups. Here, we introduce protein FRagment EXchange (FREX) to address both problems. FREX employs two components: a folded binding protein and a fragment duplicated from it, the latter of which can be chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a conformational change in which the fragment exchanges positions with the corresponding segment of the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment reduces FRET of the unbound sensor to near zero, resulting in a ratiometric FRET response that is expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin III, a monobody binding scaffold that can be tailored to recognize multiple targets. Sensors labeled with Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and in serum. A genetically-encoded variant of this sensor is shown to be functional in mammalian cell cultures. This work highlights a novel approach, FREX, which converts an arbitrary binding protein into a biosensor with high-output, ratiometric FRET response.

POST 12-160 Design of an Albumin-Binding Human Protein by Mimicking the Contact Surface of a Bacterial Albumin-Binding Domain

Satoshi Oshiro1, Shinya Honda1, 2 1 Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan, 2Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

Attachment of a bacterial albumin-binding protein module is an attractive strategy to prolong the plasma residence time of protein therapeutics. However, an antigenicity of bacterial albumin-binding module in mice was previously reported. Thus, the induction of an unfavorable immune reaction by bacterial albumin binding module-fused protein or peptide is concerned. To address this issue, we designed an alternative albumin-binding protein by imparting albumin-binding affinity to a human protein, using molecular surface grafting technique. We conducted molecular surface grafting by transferring key residues of a bacterial albumin-module, Finegoldia magna protein G-related albumin binding domain (GA) module onto human derived 6 helix-bundle proteins. We utilized structural information (e.g. the calculation of Accessible surface area of mutation site and the estimation of folding energy change upon mutation) to avoid the deleterious mutation in molecular surface grafting. As a result, one of the designed proteins specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). Despite 13–15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural information. Competitive binding assay and thermodynamics analysis showed that the best binder have similar HSA-binding site of GA module. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another. Reference Oshiro, S. and Honda, S. Imparting Albumin-Binding Affinity to a Human Protein by Mimicking the Contact Surface of a Bacterial Binding Protein” ACS Chemical Biology, 9 (4), 1052–1060 (2014)

POST 12-161 Developing Soluble Co-Receptor Mimetics For The Study Of HIV Env/Receptor Interactions

Agnes Hajduczki, Virgilio Bundoc, Edward A. Berger National Institutes of Health, Bethesda, Maryland, US


POSTER ABSTRACTS The HIV envelope glycoprotein (Env) mediates virus entry by initiating fusion of the viral envelope with the cell membrane upon receptor engagement. The surface-exposed gp120 subunit is a dynamic molecule that undergoes a series of stepwise conformational changes in response to interactions with the primary receptor CD4, and co-receptors, CCR5 or CXCR4. Obtaining structural information on the various intermediates during viral entry is a key focus of antiviral and vaccine research and could open the doors for more effective treatment and prevention. The co-receptors are G-protein-coupled receptors, anchored with seven membrane-spanning helices, leaving the N-terminus and three extracellular loop regions exposed outside the plasma membrane and available for interactions. Due to the inherent insolubility of membrane proteins, working with the intact co-receptors outside the context of the membrane is not a viable option. This project aims to develop recombinant soluble co-receptor mimetics featuring critical determinants of CCR5 using two approaches: fusing the CCR5 moieties to soluble CD4 by flexible polypeptide linkers and using a globular, stable scaffold protein to orient the co-receptor determinants in an optimal conformation to recreate the binding interaction with gp120. We have successfully overexpressed and purified the recombinant proteins from mammalian cells. Characterization of the gp120-binding properties of the variants is underway using a vaccinia-based cell fusion assay where soluble CD4 has been shown to induce membrane fusion between Env-expressing effector cells and target cells bearing CCR5, but no CD4. When the soluble CD4 fused co-receptor mimetic is added to the fusion reaction, there is strongly impaired activation of membrane fusion compared with sCD4 alone, suggesting that the CCR5-derived portion of the protein competes with cell-surface CCR5 for binding to gp120. Conversely, the mimetic displays stronger neutralization activity than sCD4 in a standard fusion assay. The soluble variants of CCR5 will be used to elucidate the conformational changes in gp120 that immediately precede membrane fusion, and potentially for collaborative high-resolution structural analyses of the gp120-coreceptor complex. POST 12-162 Constructing Highly Detectable Fluorescence Reporter Protein Forin Vitrosingle Molecular Screening

Kotaro Nishiyama1, Norikazu Ichihashi1, 2, Yasuaki Kazuta1, 2, Tetsuya Yomo1, 2 1 Osaka Univeristy, Suita, Japan, 2Exploratory Reasearch for Advanced Technology Japan Science and Technology Agency, Suita, Osaka, Japan

Recently, as the improvement of micro-compartment technology, biochemical reactions are reconstructed inside micro-compartments such as lipid vesicles. To detect the gene expression inside microcompartments, reporter proteins that produce fluorescence are widely used. The fluorescence proteins such as GFP have problem in the strength of signal (Liu HS et al., 1999, Lang et al., 2006) comparing to enzymatic reporter proteins such as β-galactosidase(β-Gal) (Yu H et al., 2003), which keep degrading substrate and increasing signal. β-Gal is widely used because of its strong substrate degradation reactivity and variety of substrates (Zabin Z et al., 1975). β-Gal becomes active by forming tetramer. When it loses its amino terminus (e.g. residue position 23-31, called ω-protein), cannot form tetramer, and become inactive dimer. This dimer recovers its reactivity by binding with protein which complements lost residue (called α-protein). This complementation is called “a-complementation” and famous as blue-white screening. In in vitro, it has been used to detect RNA self-replication reaction in liposomes (Nishiyama et al., 2012) because short size of a-protein is advantageous for RNA replication reaction. However, enzymatic activity of a-complementation is weaker than that of b-galactosidase in in vitro. In this study, we attempted to improve the α-protein activity by optimizing the α-protein size and also by the liposomebased directed evolution (Nishikawa et al., 2012). [Result] We perfomed liposome-based directed evolution method for α-protein. In this method, we first performed random mutagenesis of the DNA encoding α-protein, and then encapsulate the DNA in liposome with cell-free translation system under the


POSTER ABSTRACTS condition of 1 DNA molecule per liposome. After incubation at 37°C, collected the liposomes which volume is 10 fL and show stronger fluorescence intensity than certain threshold by using a fluorescence activated cell sorter (FACS). We then amplified the collected DNA by PCR and encapsulated the DNA into liposome again. We repeated this cycle for 23 times and got mutant which show 1.7 times higher acomplementation activivty than that of wild type. To further improve the activity, we then searched the optimum size of α-protein . We constructed α-protein of various size ranging 81 bp to 687 bp, and found that 180 bp (alpha-180), shoter than the original one (570bp), exhibit the highest acitivy. POST 12-163 Model Building Of Antibody-Antigen Complex Structures Using GB/SA Scores

Narutoshi Kamiya1, Noriko Shimba2, Haruki Nakamura1 1 Osaka University, Suita, Japan, 2Panasonic Corporation, Seika-cho, Soraku-gun, Japan

A structure prediction method for antibody (Ab) and antigen (Ag) complexes, which consists of conformer generation of Ab and Ag, molecular docking to make a decoy set, and a ranking the set using a scoring function, is one of the most important technics to support the design of antibody drugs and biosensors. Since the scoring functions used in the conventional docking programs are not always suitable for Ab and Ag, the prediction result is not accurate. In addition, there is no evaluation method whether the predicted complex is real or not. In this work, we present a scoring function and an evaluation method using molecular dynamics (MD) simulation to improve the accuracy. We introduced a score using the Generalized-Born/Surface Area method, GB/SA score, where interaction energy between Ab and Ag including solvation free-energy is calculated. To test the GB/SA score, we executed the ranking of 82 AbAg decoy sets generated by the docking program, SurFit [1]. The area under the curve (AUC) obtained from the GB/SA score (= 0.985) was better than that using the SurFit score (= 0.882), suggesting that our score improved the docking accuracy. We also carried out the ranking of decoy sets in the ZDOCK benchmark, and obtained a better AUC than that using ZDOCK. We used our new MD program, psygeneG [2], which utilizes GPU for acceleration of the electrostatic treatment by our original zero-dipole summation method [3]. We have attained similar dynamics properties to the particle mesh Ewald method in membrane protein and DNA-water-ion systems [4, 5]. We executed short 10-ns MD simulations to evaluate the decoy structures, where the top 8 to 10 decoys from each of three decoy sets were chosen for trials. During MD, all native-like decoys kept each initial complex structure, and some (not all) nonnative decoys dissociated. We calculated an average GB/SA score over the last 6-ns MD trajectories. All of near-native decoys exhibited favorable scores, and most of non-native decoys did not. Thus, our approach combining SurFit and short MD with GB/SA scores is effective to build correct Ab-Ag models. References [1] Kanamori E. et al. 2013. In Biomolecular forums and functions pp. 160. World Scientific, Singapore. [2] Mashimo T. et al. J. Chem. Theory Comput. 9, 5599 (2013). [3] Fukuda I. et al. J. Chem. Phys. 134, 164107 (2011). [4] Kamiya N. et al. Chem. Phys. Lett. 568, 26 (2013). [5] Arakawa T. et al. PLoS ONE 8, e76606 (2013). POST 12-164 Engineering Novel Phosphopeptide Recognition Modules that Recognize Targets In Vitro and in E. coli

Nicholas Sawyer1, 2, Lynne Regan1, 2, Brandon Gassaway3, 4, Jesse Rinehart3, 4, Adrian Haimovich3, 5, Farren Isaacs3, 5 1 Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, US, 2Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, US, 3Systems Biology Institute, Yale University, West Haven, Connecticut, US, 4Cellular and Molecular 131

POSTER ABSTRACTS Physiology, Yale University, New Haven, Connecticut, US, 5Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, US

Protein phosphorylation is a widespread biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signals is implicated in a wide variety of diseases. Thus, detection and quantification of protein phosphorylation is highly desirable for diagnostic as well as academic applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in E. coli. We first re-design a model tetratricopeptide repeat (TPR) protein and characterize its interaction with a target phosphopeptide in vitro. We then use this phosphopeptide-protein interaction to benchmark a split mCherry assembly assay in Escherichia coli. We demonstrate that split mCherry assembly coupled to fluorescence-activated cell sorting (FACS) can be used to separate highly fluorescent clones from a mixture of split mCherry peptide-protein pairs. This high-throughput strategy can thus be used to isolate proteins that specifically recognize a variety of different phosphopeptides. POST 12-165 N-linked Glycosylation of HIV-1 core gp120 is Not Required for Native Trimer Formation or Viral Infectivity

Ujjwal Rathore1, Piyali Saha1, Sannula Kesavardhana1, Aditya A. Kumar1, John R. Mascola2, Raghavan Varadarajan1, 3 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India, 2Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, Maryland, US, 3Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

Human immunodeficiency virus 1 (HIV-1) is the causative agent for Acquired Immunodeficiency Syndrome (AIDS). The envelope (env) glycoprotein of HIV-1 is a trimer of the receptor binding soluble subunit gp120 and the transmembrane gp41 heterodimers. gp120 is the primary viral component which is exposed on the virion surface and is the main target for neutralizing antibodies. Glycans constitute approximately 50% of the molecular mass of gp120 and shield env from immune recognition. The structure of gp120 can be subdivided into three parts, the inner domain, the outer domain (OD) and the bridging sheet. OD is heavily glycosylated and contains important neutralization epitopes. Glycans are believed to be indispensable for proper folding of gp120 and viral infectivity. However, the role of glycosylation in the folding of gp120 has not been rigorously tested. Using rationally designed mutations and yeast surface display, we show that glycosylation is not essential for in vivofolding of OD alone or OD in the context of core gp120. Two mutants of core gp120 were isolated, which retained a single inner domain glycan, and broadly neutralizing antibody (bNAb) binding. In previous studies, whenever multiple glycosylation sites were mutated in combination, it resulted in loss of viral infectivity which led to the hypothesis that a certain level of glycan coverage is essential for maintaining infectivity of the virion. In contrast, the present study shows that virions retain infectivity even in the absence of all glycans from core gp120. bNAbs against HIV-1 show a very high level of affinity maturation. Germline (GL) reverted variants of these bNAbs fail to recognize mature env. Thus immunogens based on mature env will likely not be able to activate the target GL-B cells. Immunogens that bind well to precursors of bNAbs as well as the corresponding mature antibodies may help to elicit such bNAbs upon vaccination. In the current study, we show that recognition of a GL-bNAb increases substantially with the progressive loss of glycans from JRFL E168K pseudoviruses. Glycan free OD domain immunogens bind to mature as well as a GL reverted variant of bNAb VRC01 with nM affinity. The present results inform immunogen design, targeting mature as well as GL bNAbs, clarify the role of glycosylation in gp120 folding and illustrate general methodology for design of glycan free, folded protein derivatives. POST 12-166


POSTER ABSTRACTS Silicon Transporters: From Membrane Proteins To Nanotechnology

Laura Senior, Sarah Ratcliffe, Michael Knight, Adam Perriman, Stephen Mann, Paul Curnow University of Bristol, Bristol, United Kingdom

Diatoms are abundant and diverse single-celled algae that are sheathed in a silica-coated outer cell wall. This structure, known as the frustule, forms hierarchical silica structures which are patterned with nanoscale precision at ambient pH and temperature. In order to form these frustules diatoms must source silicon from their environment. It is thought that this is done through membrane proteins that specifically interact with silicon and are known as silicon transporters (SITs). When the SITs are energised by an electrochemical sodium gradient they transport silicic acid (soluble silicon) into the diatom cell, where it undergoes polymerisation to form silica. This work has focused on the reconstitution of a recombinant SIT from the diatom Thalassiosira pseudonana into synthetic liposomes in order to replicate this biomineralization process. The SIT is recombinantly expressed and purified before being reconstituted into liposomes that contain biomineralizing peptides. The transporter is then energised by applying a transmembrane sodium gradient so that it actively pumps silicic acid from the outside to the inside of the proteoliposome. The small internal volume of the proteoliposome should cause the transported silicic acid to rapidly approach saturation; under these conditions the biomineralizing peptides should catalyze and template the formation of nanostructured silica. This novel method incorporates many of the principles of diatom biomineralization such as encapsulation and active transport, that are absent in bulk silicification experiments. Further work will attempt to synthesise novel silica structures by adjusting reaction conditions or functionalising the silica products by trapping fluorophores, enzymes and other reactive species within the silica matrix.

POST 12-167 Characterization of Thermotoga maritima Maltotriose Binding Protein

Laura Masson, Jonathan Dattelbaum University of Richmond, Richmond, Virginia, US Thermotoga martima is a thermophilic, Gram-negative bacteria found primarily in hot springs and

hydrothermal vents. Its thermophilic characteristics mean that proteins found in this bacterium should be extremely stable, even at very high temperatures, making them ideal for manipulation and biosensor design. The protein of interest for our project is the T. maritima maltose binding protein (TmMBP), which is a member of the periplasmic binding protein superfamily. Using site-directed mutagenesis of residues in and around the binding pocket, we constructed eight single cysteine mutants of TmMBP. The individual Cys residues will be covalently modified with environmentally-sensitive fluorophores to interrogate maltose binding to the protein. The altered fluorescence characteristics will be used to determine a dissociation constant for maltose. We will then use circular dichroism to evaluate the stability of each mutant. Ultimately, we hope to design a TmMBP biosensor that is stable, can bind molecules other than maltose, and will fluoresce when the target molecule is bound.


POSTER ABSTRACTS POST 12-168 Zinc Induces Self-Assembly Of Bacterial Thermoalkalophilic Lipases: A Strategy For Thermostability Emel Timucin, Osman U. Sezerman

Sabanci University, Istanbul, Turkey

Bacterial thermoalkalophilic lipases are both thermostable and aggregation-prone. Moreover, these lipases possess a unique zinc site. Zinc ions have been shown to be a prevalent cause of protein aggregation; especially in human diseases affecting the central nervous system. Yet any mechanism linking zinc and aggregation in the context of thermostability has not been characterized. In this study we investigated the effects of zinc ion on the aggregation mechanism of the thermoalkalophilic lipase from Bacillus thermocatenulatus (BTL2). Dynamic light scattering analysis showed that the recombinant BTL2 was dispersed in dimeric form with a hydrodynamic radius (RH) of 7 nm and when treated with 2+ Zn chelator, it adopted monomeric form (RH=3.6 nm). Although the zinc free BTL2 restored 90% activity at 25°C, it showed only 50% activity at 50°C, suggesting that zinc is essential for self-assembly which confers thermostability to BTL2. After 12 hours incubation of BTL2 with 50 µM ZnCl2, it formed ordered aggregates (RH>27 nm) able to bind congo red. These aggregates showed significant loss in activity (80%) at 25°C, while they displayed relatively marginal decrease in activity (30%) at 50°C. Although the zinc induced aggregates showed very low activity at 25°C, most likely due to hindrance of the catalytic site by aggregation, 50% more activity could be restored from these aggregates at 50°C. This particular observation implies that increased kinetic energy dissociates aggregates into functional forms. Using CD spectroscopy we further investigated the impact of temperature on the kinetics of zinc induced aggregation and found that the rate of aggregation was much slower at 50°C compared with the rate at 25°C (Fig. 1), confirming that increased kinetic energy curtails the zinc induced intermolecular interactions and impedes with the self-assembly of BTL2. To this end, we suggest that thermoalkalophilic lipases are prone to self-assemble through interactions mediated by zinc, which can be largely reversed by temperature switches. Essentially the reversibility of the zinc induced aggregation by high temperatures has a biological relevance, in which aggregation becomes a concrete preference of thermoalkalophilic lipases for thermostability. In line with the above findings, we will also discuss the roles of hydrophobic amino acids on the mechanism of zinc induced aggregation through a mutation (W211A) located in close proximity (~5 Å) to zinc. POST 12-169 The SasG E-G5 Protein Fold Forms A Stable Contiguous Rigid Nanorod Of Tunable Length

Fiona Whelan1, Dominika T. Gruszka2, Jane Clarke2, Jennifer R. Potts1 1 Department of Biology, The University of York, Heslington, North Yorkshire, United Kingdom, 2Department of Chemistry, Cambridge University, Cambridge, United Kingdom

Every year, infections associated with implanted medical devices affect millions of patients worldwide, resulting in significant morbidity, mortality and high costs to health services. Central to the antibiotic resistance and persistence of these infections is the formation and repeated dissemination of bacterial communities termed biofilms. Protein-dependent biofilm formation is mediated by SasG in S. aureus. SasG comprises an N-terminal functional domain implicated in bacterial adherence that is projected from the cell surface by a series of near identical sequence repeats, terminating in a covalent cell-wall tether. Negative stain EM studies have shown that SasG forms a uniform fringe of fibres projecting up to 54 nm from the cell surface [1]. We have previously determined the structure of the minimal repeat sequence, defining a novel G5 fold with an N-terminal extension domain 'E' that form a highly anisotropic rod-like fold [2]. Models of a repetitive E-G5 fold suggest the protein should form a stable extended nanorod. We have produced a truncation series of repetitive sequences on biologically


POSTER ABSTRACTS relevant scales and performed a range of biophysical and structural analyses including AUC, SEC-MALLS, QELS and SAXS. These analyses indicate that stepwise addition of E-G5 repeats generates stable, soluble, contiguous rigid nanorods up to 63 nm in length and only 2 nm in width of tunable length in repeat increments. 1. Corrigan, R.M., et al., The role of Staphylococcus aureus surface protein SasG in adherence and biofilm formation. Microbiology, 2007. 153 (Pt 8): p. 2435-46. 2. Gruszka, D.T., et al., Staphylococcal biofilm-forming protein has a contiguous rod-like structure. Proc Natl Acad Sci U S A, 2012. 109(17): p. E1011-8. POST 12-170 Computational Design Of Allosteric Antibody

Olga Khersonsky, Sarel Fleishman Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Allosteric regulation is central to the control of many metabolic and signal-transduction pathways, and thus it was named “the second secret of life”. The mechanisms of allostery, however, are largely remaining enigmatic. De novo design of allosteric proteins is particularly challenging due to the need to model the backbone degrees of freedom, and to define the multiple protein states. Recently, AbDesign algorithm was developed in our lab for de novo design of antibodies. It is guided by natural conformations and sequences, and exploits the modular nature of antibodies to generate an immense space of conformations, which can be used as scaffolds for design of stable high-affinity binders. We have used 2+ AbDesign to design an allosteric antibody, in which Zn is an allosteric effector of fluorescein binding. We 2+ introduced Zn binding site into the H3 CDR of high-affinity fluorescein-binding antibody, with the 2+ assumption that without Zn chelation, the floppiness of H3 backbone would prevent fluorescein from binding. We used as a template the fluorescein-binding antibody (pdb ID 1x9q), and spliced into it various 2+ H3 segments to obtain a variety of scaffodls. Zn binding site was introduced into these scaffolds with RosettaMatch, and the sequence was subsequently optimized by design. ~100 designs were 2+ experimentally tested by yeast display for fluorescein binding in the presence or absence of Zn . We have found that fluorescein binding by 1x9q is very sensitive to any changes and requires atomic accuracy. One 2+ of the designs demonstrated significantly higher fluorescein binding in the presence of Zn , and this allosteric behaviour is currently being further investigated. POST 12-171 Development And Optimization Of An e.Coli-based Display Platform For Selection Of Affinity Proteins

Filippa Fleetwood, Ken Andersson, Stefan Ståhl, John Löfblom School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden

Combinatorial protein engineering using surface display is commonly used for the generation of specific affinity proteins. An advantage of cell surface display over other systems such as phage display is the possibility to use flow-cytometric cell sorting. Autotransporters is a class of surface proteins in Gramnegative bacteria that enable efficient translocation of passenger proteins to the outer membrane. The aim of this project is to investigate and further develop an autotransporter-based display system in Escherichia coli for combinatorial protein engineering applications. Potential advantages of this system include a high surface expression level of the autotransporter AIDA-I, which is beneficial for flowcytometric purposes, as well as a high transformation frequency due to the use of Gram-negative bacteria, which will allow for large library sizes. Optimization data including evaluation of different promoters, and selection from a mock library of Affibody molecules will be presented.


POSTER ABSTRACTS POST 12-172 Computational Redesign of Metagenomic Enzymes

Justin B. Siegel Genome Center, University of California at Davis, Davis, California, US

The development of new computational methods to enable the efficient exploration of protein sequencestructure-function space is critical for the design of enzymes with novel functions. We have developed a new method to explore the structurally relevant sequence-space of an enzyme family, and identify protein sequences that can be used as novel starting points for enzyme engineering. This method takes advantage of the recent explosion of available protein sequences derived from genomic data. Within an enzyme family there are now commonly thousands of available protein sequences that are diverse in sequence and broadly cover the structurally relevant sequence-space for the enzyme family. By modeling the three-dimensional structure of each “metagenomic enzyme” we can evaluate the active site, and predict if it can be readily engineered for the specific reaction of interest. We have applied this method to engineer alpha-keto acid decarboxylases, which resulted in a novel enzyme experimentally validated to be four orders of magnitude more specific for long chain keto-acids than any enzyme reported to date. POST 12-173 Accurate Prediction of Protein Stability by Explicit Negative Multistate Design

James A. Davey, Christian K. Euler, Roberto A. Chica Chemistry, University of Ottawa, Ottawa, Ontario, Canada

Current computational protein design (CPD) methodologies allow for the in silico screening of amino acid sequences on a scale that is experimentally impossible to achieve. While CPD can reliably enrich predictions of stable protein sequences, the methodology is currently unable to accurately correlate predicted and experimentally-validated stabilities of protein sequences. Traditionally, CPD is performed using a positive design approach whereby sequences are scored and ranked on a model of the folded protein structure, approximated by either a single fixed protein backbone or a backbone ensemble. In an attempt to reconcile calculation and experiment, we present the first negative design approach that utilizes a backbone ensemble as an explicit negative state model. Our negative state model, intended to approximate the unfolded protein, was created from an off-target backbone ensemble shown to poorly predict and enrich stable protein sequences. Conversely, our model for the folded protein was created from an on-target ensemble shown to correctly predict and enrich stable protein sequences. A correlative 2 (R = 0.82) quantitative structure-activity relationship (QSAR) was generated using a training set of 18 known stable Streptococcal protein G domain β1 (Gβ1) sequences. Our QSAR was then applied to the prediction of stability for 10 new Gβ1 sequences. Experimental validation of the stabilities of these 10 new 2 sequences shows excellent agreement between prediction and experiment (R = 0.77) providing a proofof-concept for the application of off-target backbone ensembles in explicit negative multistate design for the prediction of protein stability. POST 12-174 Plastic Protein Design: A Novel Design Algorithm Using Backbone And Side-Chain Ensembles To Model Protein Flexibility

Christian D. Schenkelberg1, Derek J. Pitman3, Yao-ming Huang2, Christopher Bystroff1 1Department of Biology, Rensselaer Polytechnic Institute, Troy, New York, US, 2Department of Bioengineering and Therapeutics, University of California, San Francisco, San Francisco, California, US, 3Department of Biological Chemistry, University of California, Los Angeles, US

Developing and implementing an effective and realistic three dimensional protein model for computational protein design remains a major challenge in structural biology. It has long been


POSTER ABSTRACTS understood that protein design based on a static backbone conformation is not adequate to predict probable amino acid sequences (Kuhlman and Baker, 2000). Previous attempts at modeling backbone flexibility have relied on performing design on multiple static conformations of the backbone (Allen and Mayo, 2010) or introducing backbone movements as part of the protein design move-set, such as in the backrub algorithm (Smith and Kortemme, 2008). In order to efficiently model protein backbone flexibility during computational design, we have implemented a design scheme termed “plastic” design which incorporates elements of these existing backbone flexibility models. Plastic design involves designing a protein primary sequence on an ensemble of related structures of a single protein where each member of the ensemble is designed in parallel with the other members, computing a Boltzmann-like weighted average energy as the score of the design. Ensembles are generated primarily from the backrub modeling application (Friedland et al., 2009) but can be generated via other existing methods, such as molecular dynamics. Rotamers are selected in the context of an ensemble generated by rotating all combinations of the sidechain χ angles by ±10° from a “parent” rotamer derived from the Dunbrack rotamer library (Shapovalov and Dunbrack, 2011). An appropriate “child” rotamer for each backbone is selected from this ensemble by finding the lowest energy rotamer from among all the child rotamers in the rotamer ensemble. We have found that plastic protein design yields predictions that more realistically simulate protein behavior as modeled by molecular dynamics trajectories. POST 12-175 Repeat-Protein Directed Synthesis Of Gold Nanoparticles With Tunable Morphology And Optical Properties

Tijana Grove, Xi Geng Chemistry, Virginia Tech, Blacksburg, Virginia, US Fundamental advances in biotechnology, medicine, electronics, and energy require methods for control of structure and function on nanometer scales. Extensive research has focused on the bio-enabled synthesis of Au nanoparticles (AuNP) as fundamental building blocks for the construction of catalytic, optoelectronic, and biosensing devices. However, the mechanism responsible for biomolecule-directed nanoparticle and nanostructure formation remains unclear. Here we will discuss morphology and optical properties of AuNPs synthesized in the presence of modular repeat-protein arrays. Rod shaped, modular consensus sequence tetratricopeptide repeat proteins, CTPRs, containing 3, 6, or 18 tandem repeats were used in-situ for synthesis of Au nanoparticles in the presence of zwitterionic buffers MOPS, HEPES, and PIPES. Transmission electron microscopy and optical spectroscopy data indicate that the protein concentration, shape, and choice of buffer all have implications on Au nanoparticle morphology, ranging from flowers to spheres. Circular dichroism and fluorescent spectroscopy provide further information on the molecular interaction between CTPR proteins and both Au precursor and AuNP. POST 12-176 Designed Affinity Reagents Directed to Heat Shock Protein C-terminal Motifs

Robert Wells, Akiko Koide, Shohei Koide University of Chicago, Chicago, Illinois, US

Affinity reagents that bind with high affinity and specificity to target proteins are indispensable for biological research. There is an increasing need for affinity reagents that bind to pre-defined linear protein motifs, especially those targeted by modular peptide-binding domains, such as SH2, PDZ and TPR. However, it is generally difficult to recognize such short, flexible peptides with high specificity. To address this challenge, our group has established a new class of designed binding proteins called ‘affinity clamps’. An affinity clamp is a two-domain fusion protein, consisting of a ‘primary domain’, chosen from nature that binds to the desired target, but may have low affinity and/or low sequence specificity. This


POSTER ABSTRACTS primary domain is fused via a flexible linker to an ‘enhancer domain’, which is engineered to bind to the target-primary domain complex. Together, these domains ‘clamp’ to the target peptide to increase the interaction surface area, and are able to achieve a dramatic increase in affinity and specificity. Affinity clamps have been successfully created using PDZ and SH2 primary domains with the FN3 "monobody" as the evolved enhancer domain. Here, we aim to expand the sequence space of affinity clamp recognition to charged C-termini. C-termini are ideal targets for selective protein capture, because they are unique chemical moieties present in every protein, can be easily predicted from genomic data and can be mimicked by short peptides. As proof-of-principle, we chose the C-terminal sequences of the human heat shock proteins as affinity clamp targets. They are important for co-chaperone binding and protein function. Therefore, we envision that these affinity clamps will be unique inhibitors of heat shock protein function, as well as useful affinity reagents. Using a TPR primary domain, we have generated affinity clamps with greatly enhanced affinity to the human HSP90 C-terminus, while retaining high specificity. Further in vitro and in cell characterization are underway. Our results strongly suggest the feasibility of developing affinity clamps using TPR domains and the generality of the affinity clamp concept. Supported by the NIH grant R01-GM090324.

POST 12-177 Consensus Design Of A NOD Receptor Leucine Rich Repeat Domain With Binding Affinity For A Muramyl Dipeptide (MDP), A Bacterial Cell Wall Fragment

Rachael Parker, Ana Mercedes-Camacho, Tijana Z. Grove Chemistry, Virginia Tech, Blacksburg, Virginia, US

Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N- and C- capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand. CLRR2 will be used as a scaffold to incorporate binding affinity for other nonproteinaceous ligands.


POSTER ABSTRACTS POST 12-178 Solvent Engineering To Improve Enzymatic Synthesis Of A Sugar-Based Surfactant

Rodrigo A. Arreola-Barroso, Gloria Saab-Rincón Ingenieria Celular y Biocatalisis, Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico

Surfactants are important molecules to maintain pharmaceutic, cosmetic and food products in one phase. Some alpha-amylases can produce the non-toxic surfactant alkyl-glucoside from the abundant substrate starch though an alcoholysis reaction, in a single step, when in the presence of alcohols.Thermotoga maritima enzyme AmyA has been the best alpha-amylse to carry out alcoholysis reactions. This ability has been improved through amino acid substitution on its sequence. However, further improvement in this synthetic reaction through media engineering has not been explored. In the present work we explore reaction media engineering to further improve the yield of the synthetic reaction. Here we show that a decrease in water activity by a cosolvent is enough to increase the production of alkyl glycosides. We found that the addition of 2-methyl-2-butanol increases three-fold butyl glycosides synthesis compared to the condition where only a water-butanol mixture is used. Additionally, a proper cosolvent allows the alcoholysis reaction to run in one phase when using long chain alcohols, which potentiates the use of higher alcohol concentrations in the reaction. Our work shows that solvent engineering can be used to increase the production of alkyl glycosides using alpha-amylases, opening new opportunities for the improvement of this reaction. POST 12-179 Delivery Of Natural And Non-Natural Chemical Entities Into Cells Using Anthrax Toxin

Amy Rabideau, Xiaoli Liao, Bradley Pentelute Chemistry, MIT, Cambridge, Massachusetts, US

Delivery of polypeptides, especially those containing non-natural functionalities, into the cytosol of cells is a major challenge in the biotechnology field. We explored the capability of protective antigen (PA), the pore-forming protein of anthrax toxin in the delivery of these entities. To do this, we conjugated various cargos covalently to the C-terminus of a 263-residue domain (LFN) recognized by PA. We found that PA can efficiently transport a wide range of natural and non-natural chemical entities including single domain antibody mimics, small molecules, and peptides with backbone, side chain, and stereochemical modifications. This platform also enabled the delivery of a functional peptide and protein cargo into the cytosol of cells that can be used to disrupt specific protein-protein interactions in cancer cells. POST 12-180 Thermostabilization of Aspergillus oryzae Cutinase

Danielle A. Basore1, 4, Abhijit Shirke2, 4, Glen Butterfoss3, Richard Gross2, 4, Christopher Bystroff1, 5, 4 1 Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 2Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, US, 3Center for Genomics and Systems Biology, New York University, Abu Dhabi, United Arab Emirates, 4Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, US, 5Computer Science, Rensselaer Polytechnic Institute, Troy, New York, US

Cutinases are esterases produced by various phytopathogenic fungi to break down cutin, the biopolyester coating found on leaves and fruit. They also are powerful hydrolases that can cleave ester bonds of rigid polyesters such as poly(ethyleneterephthalate), PET, opening up new options for polymer recycling and surface modification reactions. To increase the rate at which cutinases can hydrolyze PET, it is useful for o the enzyme to function at temperatures above the glass transition which, for PET, is 75 C. To function at


POSTER ABSTRACTS such temperatures there is a need to increase the thermal stability of cutinases. Consequently, work is in progress to improve the thermal stability of the cutinase from Aspergillus oryzae (AoC) using Rosetta and other rational design approaches focusing on both surface interactions and core packing to reduce o the void space. Wild-type AoC has a Tm of 61 C, an additional disulfide bond not present in other cutinases and an extended groove near the active site. Of particular interest were two sets variants with changes in amino acids at the surface of AoC: i) A102D, Q105R, G106E and ii) N133A, S140P, E161T, o A166P. These variants increased the Tm by 6 and 4 C, respectively. By combining these 7 mutations the o Tm was further increased by 2 C. However, the relative activity was approximately half that of the wild type AoC. The reasons for this increase in stability and loss in activity are currently under study. POST 12-181 Beta-Hairpin Tags For Increased Expression Of Helical Peptides

Melissa E. Lokensgard, John J. Love Chemistry and Biochemistry, San Diego State University, San Diego, California, US Many small helical domains can only be expressed at high levels in E. coli when fused to larger amino-

terminal tags (such as Ubiquitin, SUMO, GST, MBP, thioredoxin, etc.) or when cloned in periplasmic expression vectors. However, StreptococcalProtein G (Gβ1), a 56aa domain, can be easily expressed in high yield using simple pET cytosolic overexpression systems with IPTG-inducible promoters. Gβ1 has also been successfully used as an amino-terminal fusion partner for poorly-expressed proteins. Gβ1 and other tags can improve not only yield, but also solubility and folding of the target protein, and some can double as handles for chromatographic purification and crystallization. These fusion tags are thought to act by facilitating or accelerating folding of the fused protein and either stabilizing the folded state or destabilizing the unfolded state. We have discovered a twelve amino acid fusion tag derived from a betahairpin in Gβ1 that dramatically increases the overexpression yields of three evolutionarily unrelated helical proteins when fused to either terminus. Deconvoluted circular dichroism spectra show that the fusion proteins contain beta-character proportional to the added hairpin, suggesting the tag sequence may adopt a native-like conformation without disrupting global folding of the helical target. Additionally, the proteins have similar or marginally increased thermal stabilities and demonstrate cooperative, reversible unfolding. Expanding on this, we show that a similar 17-amino acid hairpin sequence can also serve as an expression-enhancing tag when fused to either terminus of the same poorly-expressed helical proteins, and we are currently investigating possible mechanisms for this finding. POST 12-182 Gradient Diversity Enriches Combinatorial Protein Library Design

Daniel R. Woldring, Benjamin J. Hackel Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, US

Discovering new protein functionality via combinatorial library currently lacks efficiency, particularly in small ligand scaffolds. This study hypothesizes that combinatorial protein libraries would benefit from optimizing site specific amino acid distribution rather than the traditional heavily diversified paratope and fully conserved framework. The latter methodology is vulnerable to overly destabilizing the paratope as well as missing beneficial intramolecular contacts with the new paratope. We examined the hypothesis that the most efficient evolution is achieved with a combinatorial library exhibiting a gradient of diversity from extensive diversity in the paratope hot spot to full conservation in the framework. Importantly, this gradient includes moderate diversity, with structural bias, within the paratope interfacing with target yet peripheral to the hot spot. Moreover, more mild diversity is included adjacent to the interfacial residues to yield optimal intramolecular contacts with the newly identified paratope. To test the hypotheses, we investigate the evolvability of small proteins in the context of binding affinity using the beta-sheet


POSTER ABSTRACTS fibronectin(III) and the three-helix affibody domain. Naïve combinatorial libraries were constructed to incorporate gradient diversities across individual sites that were hypothesized to frequently be involved with protein-protein interactions or of structural importance. Yeast surface display and magnetic- and fluorescence-activated cell sorting were used to isolate clones demonstrating high affinity, specific binding to six protein targets. Thousands of unique and highly functional clones were isolated, sequenced via Illumina MiSeq and analyzed to elucidate the most evolutionarily favorable amino acid distributions within 25 fibronectin sites and 15 affibody sites. We found that a subset of fibronectin sites demonstrate improved evolvability by implementing only mild diversity, with a wild-type bias, such as T76, a semiburied position in the peripheral paratope. Additionally, restricting diversity at structurally relevant positions – both within (V29 and G79) and distal to (D23 and S85) the paratope – is evolutionarily favorable. Collectively, our results provide an efficient strategy for quantifying the balance between interand intra-molecular interaction potential at each scaffold position to optimize amino acid distributions for enhanced combinatorial library designs. POST 12-183 Expanding The GFP Toolbox Through A Better Understanding Of Its Folding Pathway

Keith Fraser1, 2, Colleen Lamberson1, 2, Victoria Jones1, 2, Erin Gilbert1, 2, David Rosenman1, 2, Yao-ming Huang3, Derek Pitman4, Shounak Banerjee1, 2, Yan Xia5, Angela Choi1, 2, Rachel Altshuler1, 2, Luis Garreta6, John Karonicolas5, Donna E. Crone1, Jonathan S. Dordick1, 2, 7, Christopher Bystroff1, 2, 8 1 Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 2Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, US, 3UCSF, San Francisco, California, US, 4Institute of Genomics and Proteomics, UCLA, Los Angeles, California, US, 5Molecular Biosciences, University of Kansas, Lawrence, Kansas, US, 6Universidad del Valle, Cali, Colombia, 7Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, US, 8Computer Science, Rensselaer Polytechnic Institute, Troy, New York, US

During the biotech explosion over the last 20 years we have seen an ever expanding role for the green fluorescent protein (GFP) in vivo and in vitro assays. One of the primary drives of this has been the work done to broaden the spectroscopic range of this protein. These successes have highlighted how amenable GFP is to design. With this in mind we attempted to take advantage of the designability of GFP in an attempt to improve its folding efficiency and better understand its folding pathway. Wt GFP has been reported to have a tendency to misfold in vitro; in an attempt to fix this problem we targeted the cis proline at P89 in GFP and redesigned this position and some surrounding residues. The engineered mutant (all trans GFP or AT-GFP) showed faster folding kinetics and no misfolding. We have observed that the introduction of a cis peptide bond (G57P58) significantly impedes folding. We were able to improve the folding efficiency of this mutant on the order of the wt via a G57A point mutation. We have previously reported that GFP tolerates the omission of one of its secondary structural elements (LOO-GFPs). Depending on the structural element that is removed the ability of the rest of the molecule to fold and form a soluble protein varies. Co-expression with the missing element results in reconstitution of fluorescence to varying degrees. We interpret preservation of native like structure in LOO-GFPs as an indicator of the order of folding of secondary structural elements. CD data along with equilibrium and stop-flow fluorescence data have allowed us to further refine our folding pathway model for GFP. We have also supplemented these experimental data with a computational model of the intermediates present along the GFP unfolding pathway. POST 12-184 Are Peptide Nucleic Acids (PNAs) Recognized by Aminoacyl-tRNA Synthetases (aaRSs)?

Crystal Serrano1, Long Nguyen1, Anthony Bell1, Filbert Totsingan2



University of Southern Mississippi, Hattiesburg, Mississippi, US, 2New York University, New York, New York, US

PNAs (peptide nucleic acids) are synthetic hybrids of nucleic acids and peptides. PNA was originally discovered during the development of gene targeting drugs. Like DNA and RNA, PNA possess standard purine and pyrimidine bases. As do peptides and proteins, PNA contains a polyamide backbone – typically aminoethyl glycine (AEG). The neutral PNA backbone provides greater stability than the natural ribose-phosphate in hybridization to complimentary strands of DNA and RNA. Hence, it is not surprising that PNA has been investigated extensively as an antigene reagent. The seminal report by Peter Nielsen and Orgel clearly showed that PNA is capable of transferring genetic information to RNA via templatedirected syntheses. These data indicate that PNA might have played a key role in the establishment of primordial protein/peptide translation systems. The objective of this study was to determine if PNAs are recognized by extant translational machinery such as aminoacyl-tRNA synthetases (aaRSs). Here, we 32 show, using a P based aminoacylation assay, that PNA-RNA duplexes are efficiently recognized and aminoacylated by AlaRS. The relative charging efficiency of PNA-RNA duplexes are comparable to RNAAla RNA duplexes based on tRNA mini- and micro-helices. The biophysical characteristics of PNA-RNA and RNA-RNA duplexes were evaluated using: electrophoretic mobility shift assays (EMSA), circular dichroism (CD) and UV spectroscopy. The CD investigations revealed that, as expected, RNA-PNA duplexes were o more stable (D Tm ≈ 10 C) than the corresponding RNA-RNA duplex. EMSA and UV analysis showed that PNA-RNA duplexes form more readily at lower pH values. This is the first investigation that shows aaRSs charge synthetic nucleic acids (PNA). Future investigations will focus on screening PNAs against aaRSs to develop antibiotics. POST 12-185 Protein Rings and Tubes as Versatile Templates for Self-Assembled Bionano Structures

Ali A. Malay2, Zuben Brown1, Kenji Iwasaki1, Jonathan G. Heddle2 1 Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, and CREST, Japan Science and Technology Agency, Osaka, Osaka, Japan, 2Heddle IRU, RIKEN, Wako, Saitama, Japan

The natural world demonstrates the power of proteins to act as self-assembled nanometric structures able to carry out a diverse range of interactions with matter including as nanostructural components, catalysts and nanomachines. It would therefore be very useful to build our own proteins to carry out reactions and functions that we desire. Unfortunately current technology does not allow us to design proteins of significant size ab initio. An approach to overcome this limitation is to use naturally occurring proteins and alter their properties such that they assemble into the desired structures with desired properties (a semi-synthetic approach). In this work we will summarize our progress with two proteins, the nanotubular tomato mosaic virus (ToMV) and the toroidal protein TRAP (trp-RNA binding attenuation protein). These proteins have proved very useful as templates for the production of non-biological nanotubes and nanowires with potential applications in electronics and sensors and as building blocks for self-assembly of virus-like particles via unique catalytic chemistries.

POST 12-186 Bio-Inspired Tectons: The Architecture & Engineering Of Synthetic Ring-Forming Proteins

Francesca Manea, Bridget C. Mabbutt Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, New South Wales, Australia 142

POSTER ABSTRACTS The vast structural and chemical diversity of proteins makes them highly attractive as bio-building blocks or ‘tectons’ for fabrication of materials by self-assembly. Through supramolecular engineering, quaternary structure and function can be manipulated to fabricate materials with new or improved properties. I am exploiting self-assembling Lsm proteins to construct novel nanostructures of ring morphology. In vivo, the Lsm proteins form the core of the ribonucleoprotein (RNP) complexes crucial to RNA metabolism. They assemble as oligomeric rings via the association of seven distinct Lsm protomers to generate an RNA-binding scaffold. I have engineered synthetic Lsm rings composed of fused protein components, so creating simplified structures with reduced symmetry. In solution, recombinant Lsm selforganise into overlaid ring pairs through electrostatic interactions mediated by salt concentration. I have further driven oligomerisation of Lsm ring tectons into coherent architectures by engineering Cys residues into ring faces for covalent stacking. These disulfide-bonded Lsm clusters are readily disengaged by a reducing step. The cluster architecture of high MW Lsm complexes has been visualised by electron microscopy (EM) to augment solution-state studies. Crystallisation of Lsm forms offers potential to understand the quaternary forms, from clusters to tubules. Other avenues of conjugation between Lsm rings are also being explored, including self-assembling peptides and metal-binding via engineered Histags. Ultimately, these novel nanomaterials will have applications in RNA housing and delivery capsules or as next-generation bio-inspired nanosensors. POST 12-187 A Designed Small Protein For Controlling Site-Specific Mineralization Of Silica And Calcium On Dnas

Kenji Usui2, Kazuma Nagai2, Hiroto Nishiyama2, Aoi Yamada2, Makoto Ozaki2, Takaaki Tsuruoka2, Kin-ya Tomizaki1 1Department of Materials Chemistry, Ryukoku University, Otsu, Japan, 2Faculty of Frontiers of Innovative Research in Science and Technology, Konan University, Kobe, Japan

Biomineralization, (mineralization in a biological system), precipitation of inorganic compounds, is controlled by some natural proteins. These proteins can control spatiotemporally both the size and the shape of the inorganic deposits with high reproducibility and accuracy during the biomineralization processes. Short peptides derived from sequences of these biomineralization proteins can also precipitate inorganic compounds [1]. Because these peptides are easy to design and synthesize compared to the natural proteins, they are promising molecules for the construction of organic-inorganic nanostructures with mineralization. There have been few studies to date using these peptides for well-controlled sitespecific mineralization yielding homogeneously-shaped inorganic precipitates. In this study, we focused on silica and calcium mineralization and demonstrated a site-specific mineralization using DNA and designed peptides with peptide nucleic acids (PNA) [2]. After design and synthesis of the peptides, we checked its ability of silica mineralization using atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX). Then we attempted more complicated mineralization. Calcium as the second mineralized inorganic compound, was precipitated on the silica-peptide-DNA structure. Preliminarily, TEM-EDX images showed dumbbell shape nanostructures, long chains for calcium-DNA and nanometer-sized spheres for silica and calcium precipitates. Our results will provide good examples of the controlled distribution of inorganic compounds and organic compounds. Such system would be one of the most powerful tools for bottom-up methods in the nanobiotechnology and material fields. [1] Knecht, M.R.; Wright, D.W. Chem. Commun. 2003, 3038. [2] Sano, S.; Tomizaki, K.-Y.; Usui, K.; Mihara, H. Bioorg. Med. Chem. Lett. 2006, 16, 503. POST 12-188 Enhancing Predictions Of Surface Entropy Reduction For Improved Crystallizability Of Proteins


POSTER ABSTRACTS Derek J. Pitman1, Thomas Holton1, Luki Goldschmidt2, 1, Zygmunt Derewenda3, David Eisenberg1 1 University of California, Los Angeles, Los Angeles, California, US, 2University of Washington, Seattle, Washington, US, 3University of Virginia, Charlottesville, Virginia, US

The resistance of certain proteins to forming well-ordered crystals impedes the determination of highresolution structures and expends considerable resources on unsuccessful crystallization trials. A successful strategy for improving the crystallizability of these problematic proteins is surface entropy reduction (SER), the mutation of high-entropy residues such as lysine or glutamate on the protein surface to low-entropy residues such as alanine, facilitating the formation of crystal contacts. We have applied this concept to develop a prediction server, SERp, which takes a primary sequence as input and uses expected surface entropy, secondary structure prediction and evolutionary conservation to determine probable sites for SER mutations (Goldschmidt et al., Protein Science 16(8):1569-76). The SERp server has allowed for the determination of numerous new crystal structures. SERp can also interface with the XtalPred server for prediction of protein crystallizability (Slabinski et al., Bioinformatics 23(24):3403-5). Here we review the SERp server in its current form, outline a future revision of the server infrastructure, and discuss potential extensions for the methodology, including improved predictions for membrane proteins and an increased emphasis on the use of known structural data and previous SER successes in refining the entropy reduction theory. Entropy reduction can thus be viewed as a mechanism to explore the more general design of crystal contacts. The SERp server can be accessed at:

POST 12-189 Repeat Protein Scaffolds For Assembly Of Functional Nanostructures

Sara H. Mejias1, 2, Pierre Couleaud1, 2, Javier Lopez3, 4, Begoña Sot1, 2, Carmen Atienza3, Teresa Gonzalez4, Aitziber L. Cortajarena1, 2 1 Nanobiosystems, IMDEA Nanociencia, Madrid, Spain, 2CNB-CSIC-IMDEA Nanociencia Associated Unit, Madrid, Spain, 3Química Orgánica, Universidad Complutense, Madrid, Spain, 4IMDEA Nanociencia, Madrid, Spain

Self-assembly of biological molecules into defined functional structures has a tremendous potential in nanopatterning, and the design of novel materials and functional devices. Molecular self-assembly is a process by which complex three-dimensional structures with specified functions are constructed from simple molecular building blocks. We present the study and characterization of the assembly properties of modular repeat proteins, in particular designed consensus tetratricopeptide repeats (CTPRs) which 1 stability and function can be tuned, and their appplication as building blocks in order to generate 2 functional nanostructures and materials. 1. We show the assembly of repeat proteins into thin nanometric fibers. We have characterized the polymerization kinetics and thermodynamics and developed a quantitative simple model of the polymerization for rational control of features of the final 3 nanostructures (Fig. 1a). 2. We show data on the design and conjugation of CTPR building blocks with gold nanoparticles (Fig 1b) and how these protein scaffolds are used for nanometer-precise arrangement of the nanoparticles, toward the generation of nanoscale systems with numerous appplications in nanotechnology. 3. We show preliminary data on the use of CTPR proteins to template donor-acceptor pairs for electroactive materials. In order to achieve an efficient electron transfer the arrays of molecules need to be ordered with defined inter-molecular distances. We show the conjugation of CTPRs scaffolds with electroactive molecules and the characterization of the hybrid structures (Fig. 1c). References 1. (a) Cortajarena, A. L.; Regan, L., Calorimetric study of a series of designed repeat proteins: modular structure and modular folding. Protein Sci 2011, 20, 341-352; (b) Cortajarena, A. L.; Liu, T. Y.; Hochstrasser, M.; Regan, L., Designed proteins to modulate cellular networks. ACS Chem. Biol. 2010, 5 (6), 545-552. 2. Mejias, S. H.; Sot, B.; Guantes, R.; Cortajarena, A. L., Controlled nanometric fibers of self-assembled


POSTER ABSTRACTS designed protein scaffolds. Nanoscale 2014. 3. Grove, T. Z.; Regan, L.; Cortajarena, A. L., Nanostructured functional films from engineered repeat proteins. J. R. Soc. Interface. 2013, 10(83), 20130051. POST 12-190 Generation of Novel Amino Acid Dehydrogenase Activity through Evolution of a Highly Specific Alanine Dehydrogenase

Emily Mundorff Chemistry, Hofstra University, Hempstead, New York, US Mutants of alanine dehydrogenase (AlaDH) from the M. tuberculosis have been identified with activity

towards leucine, norleucine and norvaline. This enzyme shares no significant structural or sequence similarity with the Phe/Glu/Val/Leu Dehydrogenase superfamily and has been demonstrated to have high specificity towards alanine. Position F94 was identified through molecular modeling as being essential for substrate specificity. Modeling studies predicted that mutating this residue to either alanine or serine would create room in the binding pocket for bulkier substrates. Indeed both mutants facilitated the generation of activity on leucine, norleucine and norvaline while decreasing the activity for its natural substrate. Saturation mutagenesis of the active site identified improvements upon these activities. This result indicates that even though this enzyme bears no significant relationship to other amino acid dehydrogenases, it still has the potential to evolve these functions.

POST 12-191 Rational Protein Design, SPR Binding Analysis, And Iterative Optimization Of Student-Designed Single-Chain Immunoreceptors

Benjamin J. McFarland, Andrew Daman Chemistry and Biochemistry, Seattle Pacific University, Seattle, Washington, US The success of rational protein design can be enhanced by synthesizing and testing multiple designed proteins in the lab. We previously optimized binding of the MICA protein surface to its protein ligand, the homodimeric NKG2D immunoreceptor, with an iterative process resulting in seven MICA mutations that successfully enhanced the interaction. Here we apply these techniques to designing a single-chain form of NKG2D with an optimized homodimer interface. To effectively assess the binding of multiple designs, we assign multiple undergraduates to make the proteins in a biochemistry teaching lab course and to test them for binding in a survey of physical chemistry lab course, guiding and recording their progress with a set of GENI online tools we have developed. Surface Plasmon Resonance (SPR) binding analysis was made possible in this context with a BIAcore X instrument, which gave similar results as a BIAcore T100 instrument at 60 ng/ml) shows cross-reactivity with mouse IgG. GAH profiles show slower association (ka) and faster dissociation (kd) rates for binding to mouse IgG, compared to the specific human IgG surface. Overlay of comparative sensograms provides an estimate of 0.4% (GAH to mouse IgG) cross-reactivity. Longer association times (10-16 min) can detect cross-species binding in the nd 0.1% - 0.5% range. ProteOn Manager Concentration Analysis uses initial binding slopes of 2 Ab nd standards to estimate cross-reactive 2 Ab concentrations in unknown samples. Quantitation (at < 5% nd error) for 2 Abs below 50 ng/ml is demonstrated.

Poster Session: Cellular Structures POST 02-224 Dissecting the repetitive C-terminal domain of RNA polymerase II

Stephen Fuchs, Mohammad Mosaheb, Summer M. Morrill Biology, Tufts University, Medford, Massachusetts, US

Tandemly repeating amino acid sequences play diverse, functionally important roles in the cell. One in particular, the C-terminal domain (CTD) of RNA polymerase II (RNAPII) plays an essential role in coordinating processes such as mRNA processing with ongoing transcription. Protein factors recognize specific phosphorylated forms of a tandemly-repeating seven amino acid consensus sequence (YSPTSPS). This sequence is conserved across most eukaryotes and varies in length from 26 repeats (yeast) to 52 repeats (humans). This work attempts to explore a very long-standing question regarding CTD function – do repeats have overlapping function or do they show some level of specificity based on either sequence or proximity to the rest of the RNA polymerase II complex. Here we present evidence that repeats indeed show independent function. POST 02-225 Bacterial Microcompartments: Substrate Transport through Shell Pores

Sunny Chun2, 1, Chiranjit Chowdhury3, Jiyong Park1, Kendall N. Houk1, Thomas A. Bobik3, Todd O. Yeates2, 1 1

Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, US, 2Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, US, 3Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa, US

Bacterial Microcompartments (BMCs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency and prevent the loss of volatile or toxic intermediates. Operons encoding at least seven apparently metabolically distinct BMCs have been identified in 20% of bacteria, including BMCs involved in utilizing inorganic carbon (i.e. bicarbonate), 1,2-propanediol, and ethanolamine. Ranging from 100 to 150 nm in diameter, BMCs are encapsulated in a protein shell consisting of hexagonal tiles of the conserved BMC-fold proteins. The BMC-fold proteins homo-oligomerize into hexagonal or pseudo-hexagonal assemblies with unique functions; most possess central pores presumed to be involved in transport of substrates and products. The mechanisms of small molecule transport through, and retention of toxic intermediates by, BMCs remain poorly understood. We hypothesize that


POSTER ABSTRACTS the BMC shell pores serve a role for molecular transport by passive selective diffusion. One such poreforming BMC-fold protein is PduA of the Propanediol utilization (Pdu) BMC. Here, we present the high resolution crystal structures of PduA pore mutants S40Q and S40L, which support their observed phenotypes of increased and decreased BMC activity, respectively (unpublished). Initial findings using Molecular Dynamics to investigate the free energy of small molecule penetrance through the PduA pore (r=2.8Å) also indicates a higher free energy barrier for the toxic intermediate propionaldehyde than for substrate 1,2-propanediol (unpublished). Using both biochemical and computational approaches, we identify the importance of the key residue S40 and hydrogen-bonding of the small molecule metabolites. Our experiments shed insight on BMC shell pore dynamics and function, furthering our understanding of BMC systems. POST 02-226 Sequence Signature for Recognition of Histone H3 Arginine (R2)

Francisca Essel, Suvobrata Chakravarty, Tao Lin Chemistry and Biochemistry, South Dakota State University, Brookings, South Dakota, US Recognition of covalently modified as well as unmodified N-terminal histone residues by specific factors play a crucial role in regulating chromatin structure and assembly. Hence, a large effort in the past decade has gone into discovering protein factors that read (recognize) site-specific histone peptide segments. However, majority of these have focused on the recognition of covalently modified histone amino acids (e.g. methylated, and acetylated Lysine) while there are fewer reports on the recognition of unmodified histone Lysine and Arginine. Here we focus on identifying protein factors capable of recognizing unmodified histone H3R2. Sequences of human PHD (plant homeodomain) family having a distinct pattern of negatively charged residues that are predicted to form salt-bridges with H3R2 are tested for site-specific binding using a fluorescent protein based colorimetric assay. We observed that the first PHD finger domain of Lysine-specific Demethylase 5B (KDM5B-PHD1) strongly interacts with H3R2 with 8.85 mM while H3K4 contributes negligibly to the interaction of unmodified histone H3-1-11 residues i.e. KDM5B-PHD1 belongs to a PHD family subgroup that is distinct from the subfamily specific for the recognition of unmodified H3K4. Consistency of the binding result with that of ITC suggests a simple assay to probe protein-protein interaction. POST 02-227 Microfluidic Control And In Situ Monitoring Of Microtubule Maturation Kinetics Provide Evidence For A New Stabilising Cap Model Of Dynamic Instability

Christian Duellberg1, Nicholas Cade1, David Holmes2, Thomas Surrey1 1 Microtubule Cytoskeleton Lab, Cancer Research UK, London Research Institute, London, United Kingdom, 2London Centre for Nanotechnology, University College London, London, Greater London, United Kingdom

Switching between growth and shrinkage is an important property of microtubule ends. The molecular mechanism underlying this switch is still not well understood. Using a novel microfluidics-assisted total internal reflection fluorescence microscopy assay, we directly tested predictions of the most prominent current models of dynamic instability. We manipulated in a controlled manner GTPase cycle-associated transition kinetics and directly linked these kinetics to microtubule stability. With these new tools in hand and in combination with rapid tubulin concentration changes, we found that the mechanism of catastrophe induction cannot be explained satisfactorily by recently proposed models. We further demonstrate that the density and the turnover (but not the length) of a conformational cap recognized by EB1 family proteins determines filament stability and that fluctuations of the cap density are directly linked to momentary filament stability. Our model for catastrophe induction effectively combines


POSTER ABSTRACTS properties of previous coupled and stochastic GTP cap models and takes structural changes at microtubule ends into account. It explains microtubule lifetime distributions under steady state and after rapid tubulin wash-out. Our results provide a unified conceptual framework that explains the molecular mechanism of catastrophe induction. POST 02-228 Investigating The Function Of Suppressor Of IKK-epsilon

Sean W. McKinley2, Kenneth F. Lawrence2, Jessica K. Bell1 1 Biochemistry, Virginia Commonwealth University, Richmond, Virginia, US, 2Microbiology & Immunology, Virginia Commonwealth University, Richmond, Virginia, US

Pathogen challenge elicits an immediate response by the innate immune system. Several innate immune signaling pathways converge to activate TANK binding kinase 1 (TBK1), a ubiquitously expressed kinase. TBK1 phosphorylates substrates that mediate pro-inflammatory cytokine and type I interferon production or sequestering of intracellular bacteria. We have defined a new TBK1 substrate, Suppressor of IKKepsilon (SIKE). With respect to other TBK1 substrates, SIKE acted as a nanomolar, mixed-type inhibitor of TBK1. Although SIKE could influence TBK1 activity as a high affinity substrate, the primary function of SIKE remains unknown. Preliminary co-immunoprecipitation assays of SIKE suggested that the SIKE interaction network impinged upon the cytoskeleton and RNA transport. We hypothesize that SIKE links innate immune signaling to mRNA transport. To further characterize SIKE’s function within the antiviral response, we used immunofluorescence assays to identify colocalization between SIKE and cellular markers in epithelial and myeloid cell types. Colocalization was quantified at the level of whole cell or region of interest. SIKE colocalized with cytoskeleton components (actin and tubulin), endosomal and plasma membrane markers (Rab11, LAMP-1, and LC3), and ribosomes (S6). In parallel experiments to identify SIKE function, recombinant SIKE protein purification strategies were developed to produce material for structural analysis. Purification schemes utilizing a 6XHis tagged construct under denaturing and native conditions with Ni-NTA or TALON resin and incorporation of ion exchange have been tested. For each scheme, size exclusion chromatography and SDS-PAGE/Coomassie/silver stain were used to assess purity. The optimal purification scheme targeted purification of SIKE from the insoluble pellet under denaturing conditions using TALON resin to minimize co-purification with E. coil protein, SlyD. Funds to support these studies provided by CCTR Endowment Fund and NIH R21AI107447. POST 02-229 Structure Of BDBT Reveals A Role For Noncanonical FK506 Binding Protein In Regulation Of The Fly Circadian Clock

Boadi Agyekum, Jin-Yuan Fan, Anandakrishnan Venkatesan, Jeffrey Price, Samuel Bouyain UMKC, Kansas City, Missouri, US

In the fly circadian cycle, DOUBLETIME (DBT) is a principal kinase that phosphorylates PERIOD (PER) and targets it for degradation. Bride of DOUBLETIME (BDBT) is a novel DBT-binding protein identified in a proteomic screen. RNAi-mediated knock-down of BDBT produced behavioral arrhythmicity, high levels of hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME, suggesting that BDBT is a novel clock regulatory protein. To gain insight into the function of BDBT in the Drosophila circadian clock, we determined the crystal structure of the first 211 amino acid residues of BDBT. Our analysis revealed that the closest structural homologue of the BDBT N-terminus (residues 1-120) is FK506 binding protein 1A (FKBP1A) even though they share only limited sequence identity. FKBP1A is a peptidyl-prolyl cis-trans isomerase, but only 2 of the 13 residues involved in isomerase activity are conserved in BDBT, suggesting that BDBT is not enzymatically active. Instead, our biochemical experiments indicate that the N-terminus of BDBT binds to DBT. Although there is no BDBT homologue in vertebrates, sequence analyses indicate


POSTER ABSTRACTS that the C-terminus of BDBT resembles the tetratricopeptide repeat region of the non-canonical FK506binding proteins FKBP51, FKBP52, and FKBPL, which bind the vertebrate DBT homologue CK1δ and CK1ε. Thus we propose that these proteins may function as functional homologues of BDBT in vertebrates. POST 02-230 The PKD-Related Proteins ANKS6, BICC1, And ANKS3 Form A SAM Domain Interaction Network

Catherine N. Leettola1, Mary J. Knight1, Duilio Cascio2, James U. Bowie1 1 Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, US, 2Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, US

Polycystic kidney disease (PKD) is the most common genetic disorder leading to end-stage renal failure in humans. Mutations in the proteins ANKS6 and BICC1 are responsible for disease in rat and mouse models of PKD, respectively, yet the exact mechanism of disease causation remains unknown. Both ANKS6 and BICC1 contain SAM domains, which are known protein-protein interaction domains that are capable of binding each other to form heterodimers, helical polymers, or sheets of polymers that can act as scaffolds. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. Using a negGFP native gel binding assay, we have identified the SAM domain of ANKS3 as a direct binding partner of both the ANKS6 SAM domain and the BICC1 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. Crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex reveal the molecular details of these SAM domain associations. An R823W mutation in the SAM domain of ANKS6 is causative of disease in the PKD/Mhm(cy/+) rat model of autosomal dominant PKD. Using circular dichroism, we determined that this point mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. ANKS6, ANKS3, and BICC1 each contain other functional protein domains in addition to their SAM domains. We propose that a network of interactions between the SAM domains of ANKS3, ANKS6, and BICC1 defines a variety of scaffolds responsible for correctly organizing a variety of protein complexes and that this network of interactions is responsible for proper kidney development and function.

Poster Session: Chemical Biology & Enzymology POST 03-231 The Variations Of Protein Splicing: Regulation And Mechanism Of Non-Canonical Inteins

Kenneth Mills, Julie N. Reitter, Michael Nicastri, Jennie Williams, Kathryn Colelli, Michelle Marieni Holy Cross, Worcester, Massachusetts, US

Protein splicing is the post-translational excision of an intervening polypeptide, or intein, concomitant with the ligation of the flanking domains, or exteins. The inteins that interrupt the PolII of Methanoculleus marisnigri (Mma) and Pyrococcus abyssi (Pab) each facilitate protein splicing with C-terminal Gln in place of the highly conserved Asn. Thus, the inteins splice by a non-canonical first step. The Mma PolII intein is regulated by the redox state of an internal disulfide bond, and the third step of splicing is inefficient with C-terminal Gln. The Pab intein is regulated by both temperature and redox state. The thermal stability of the unspliced Pab intein precursor allows for structural study and analysis of the kinetics of each step of splicing in vitro. NMR solution structures of the Pab PolII intein reveal that the structure is rigid, with a


POSTER ABSTRACTS disordered loop absent in an otherwise homologous P. horikoshii intein, and a b-hairpin specific to inteins from archaebacteria. This material is based upon work supported by the National Science Foundation under grant MCB-1244089 and the Camille and Henry Dreyfus Foundation. POST 03-232 Directed Evolution Of A Highly Sensitive Peroxidase Reporter And Application To Electron Microscopic Visualization Of MICU1.

Stephanie S. Lam1, Jeffrey D. Martell1, Kimberli J. Kamer2, Vamsi K. Mootha2, Alice Y. Ting1 1 Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, US, 2Systems Biology, Harvard University, Cambridge, Massachusetts, US

Peroxidases are exceptionally versatile enzymes that have been widely utilized for biomarker detection, fluorescence and electron microscopies, and proteomic mapping. Horseradish peroxidase (HRP) and engineered ascorbate peroxidase (APEX) are two peroxidase reporters now available but HRP is inactive when expressed in the cytosol of cells, while APEX lacks the exceptional sensitivity of HRP. In an effort to close this gap, we performed yeast display evolution to engineer a novel reporter, APEX2, that is vastly more sensitive than APEX in both living and fixed mammalian cells. The improved properties of APEX2 permitted us to examine the precise localization of MICU1, a regulator of calcium import into mitochondria, by electron microscopy. We observed clear localization in the intermembrane space, in contrast to previous reports of localization in the matrix, necessitating revision of a previously proposed mechanistic model.

POST 03-233 Characterization Of The Alpha-Proteobacteria Wolbachia Pipientis Protein Disulphide Machinery Reveals A Regulatory Mechanism Absent In Gamma-Proteobacteria

Patricia M. Walden1, Maria A. Halili1, Julia K. Archbold1, Fredrik Lindahl1, David P. Fairlie1, Kenji Inaba2, Jennifer L. Martin1 1 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia, 2Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Aoba-ku, Japan

Dengue fever, caused by the dengue virus, is a severe and deadly disease that is still vastly expanding in tropical and subtropical regions [1]. One way to eliminate the dengue virus is to reduce the spread of its major vector, the mosquito Aedes aegypti. The endosymbiotic bacteria Wolbachia pipientis can infect Aedes aegypti and inhibit dengue virus replication and transmission [2]. Because of this unique ability of Wolbachia to interfere with the dengue virus, the bacterium has been exploited as a novel biocontrol tool against dengue fever in tropical countries. However, the interaction between Wolbachia and its mosquito host at a molecular level still remains poorly understood. We hypothesize that Wolbachia secretes proteins into its host that alter physiological processes within the mosquito. Like many other bacteria, Wolbachia possesses disulfide bond forming (Dsb) proteins that introduce stabilizing disulfide bonds into these secreted proteins. The genome of theWolbachia strain wMel encodes three Dsb-like proteins that could be important for stabilizing secreted proteins: alpha-DsbA1, alpha-DsbA2, and an integral membrane protein, alpha-DsbB. Alpha-DsbA1 and


POSTER ABSTRACTS alpha-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA proteins, would need to be oxidized to the disulfide form to serve as a disulfide bond donor towards substrate proteins. Here we show that the integral membrane protein alpha-DsbB oxidizes alpha-DsbA1, but not alpha-DsbA2. The interaction between alpha-DsbA1 and alpha-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of alpha-DsbB [3]. This is the first extensive investigation of the Dsb folding machinery in Wolbachia. Exploiting Wolbachia for halting the spread of Dengue fever is an ongoing strategy in tropical climates. References: [1] Kyle, J. L. & Harris, E. Global spread and persistence of dengue. Annu. Rev. Microbiol. 62, 71–92 (2008) [2] Walker T, et al. (2011) The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature 476:450–453. [3] Walden PM, Halili M, Archbold JK, Lindahl F, Fairlie D, Inaba K, Martin JL. (2013) The alpha-proteobacteria Wolbachia pipientis protein disulfide machinery has a regulatory mechanism absent in gamma-proteobacteria. PloS One, 8 11: e81440.1-e81440.9. POST 03-234 The Metal-Dependent FAD Pyrophosphatase/FMN Transferase Activity Of Periplasmic FlavinTrafficking Protein (Ftp): A Potential Role In Flavoprotein Biogenesis

Ranjit K. Deka1, Chad A. Brautigam2, Wei Z. Liu1, Diana R. Tomchick2, Michael V. Norgard1 1 Microbiology, The University of Texas Southwestern Medical Center, Dallas, Texas, US, 2Biophysics, UT Southwestern Medical Center, Dallas, Texas, US The syphilis spirochete Treponema pallidum (Tp) is a highly enigmatic bacterium that still cannot be cultivated in vitro, and a syphilis vaccine remains elusive. Tp lacks many biosynthetic pathways and has

evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoires. We recently described in Tp an ABC-type riboflavin transporter (RfuABCD) that likely is essential for the organism's survival within its human host. We also discovered treponemal Ftp (Ftp_Tp) as the first bacterial metal-dependent FAD pyrophosphatase/FMN transferase that traffics/processes FAD into AMP and FMN in the periplasm to generate flavoproteins for use in the redox reactions. In previous studies it was shown thatSalmonella enterica Ftp (Ftp_Se) binds FAD, but that it lacked pyrophosphatase activity. Although the crystal structures of these Ftps are quite similar and the FAD binding residues are well conserved, a bi-metal catalytic center is present only in Ftp_Tp. It would thus be instructive to discern what molecular features allow some Ftp proteins to recognize and hydrolyze FAD whereas others are restricted to FAD binding only. Structure-based mutagenesis revealed that a single amino acid change 2+ in E. coli Ftp (Ftp_Ec) converted it from an FAD-binder to an Mg -dependent FAD pyrophosphatase (similar to Ftp_Tp). Further, we have shown in vitro and in vivo that both types of Ftps are capable of flavinylating proteins via the covalent attachment of FMN to the threonine sidechain of an appropriate sequence motif. These discoveries are of widespread relevance because Ftps are essential for flavoprotein biogenesis in the bacterial periplasm. We also propose that Tp likely possesses a flavin-based redox system that underlies a "flavin-centric" lifestyle for Tp's survival in the human host. POST 03-235 Characterization Of Hyperthermophilic Tyraenzymes Involved In Aromatic Amino Acid Biosynthesis

Irina Shlaifer, Joanne L. Turnbull Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada

TyrA enzymes belong to a family of prephenate dehydrogenases that are dedicated to the biosynthesis of the aromatic amino acid L-tyrosine. L-tyrosine is a precursor of valuable compounds such as aromatic metabolites, pigments and hormones. Designing catalytically efficient and stable enzymes for tyrosine overproduction in microorganisms serve as an environmentally friendly ans sustainable alternative to the traditional methods of obtaining this amino acid from animal sources. Enzymes from hyperthermophilic


POSTER ABSTRACTS organisms are of particular interest in industrial and green chemistry applications as they are stable at neutral pH and over a wide range of temperatures. In this study, the genes encoding putative TyrA enzymes from two hyperthermophilic archaea were cloned and the proteins were recombinantly expressed in Escherichia coli and characterized. One is a trifunctional enzyme from Nanoarchaeum equitans which has a prephenate dehydrogenase (PD) domain fused to chorismate mutase (CM- the preceding enzyme in the pathway) and to the prephenate dehydratase (PDT) that is dedicated to the biosynthesis of L-phenylalanine). The second enzyme is the bifunctional CM-PD from ChrenarchaeonIgnicoccus hospitalis. Unlike the TyrAs from most organisms which are mainly inhibited by the end product L-tyrosine, we have predicted by amino acid sequence alignment that these two enzymes will be insensitive to regulation by L-tyrosine. Accordingly, kinetic analysis of the enzymes confirms that the PDs are insensitive to L-tyrosine regulation implying that the enzymes are good candidates for the production of industrial aromatic compounds from renewable resources. These are the first studies exploring the aromatic amino acid biosynthetic pathway from the two archaeal organisms which may also provide insight into the strategy adopted for the efficient processing of labile metabolites such as chorismate and prephenate in hyperthermophilies. POST 03-236 Probing Novel Antibiotic Targets Within Sialic Acid Catabolism

Rachel A. North2, Sarah A. Kessans2, Hironori Suzuki2, Michael D. Griffin1, Renwick C. Dobson2, 1 1 Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, Australia, 2Biomolecular Interaction Centre & School of Biological Sciences, University of Canterbury, Christchurch, New Zealand Staphylococcus aureus is a Gram-positive bacterial pathogen, notorious for its ability to evolve resistance

mechanisms against novel antibiotics. The ever-increasing prevalence of this superbug and the continual progression of antibiotic resistance highlight the urgency to characterize novel drug targets and develop new drugs against this pathogen. The enzymes involved in the catabolism of sialic acids are promising targets, yet to be exploited. Sialic acids constitute a large family of amino sugars and are undoubtedly one of the most important carbohydrate classes in biology. Found at the terminus of glycan molecules attached to the surface of eukaryotic cells, they are involved in a diverse range of cellular functions. Interestingly, several bacterial pathogens capable of colonizing heavily sialylated niches can scavenge sialic acid from their surrounding environment and degrade it as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid requires a cluster of genes known as the ‘NanNag’ cluster. The Nan-Nag cluster of genes encodes five catabolic enzymes (lyase, kinase, epimerase, deacetylase and deaminase) that degrade sialic acid via a series of reactions into fructose-6-phosphate (see figure). This pathway has been well documented in several bacterial pathogens that colonize mucousrich niches, such as methicillin-resistant Staphylococcus aureus, suggesting that the ability to utilise sialic acid as a ubiquitous carbon and nitrogen source is important for colonization and persistence. More significantly, the Nan-Nag genes have proven essential for Staphylococcus aureus growth on sialic acids, making the pathway a viable target for drug design against this pathogen. A biophysical and structural characterization of the catabolic enzymes involved in the breakdown of sialic acid from methicillinresistant Staphylococcus aureus will be presented. Data obtained from analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography will be included. Crystal structures have been obtained for N-acetylneuraminate lyase (1.7 Å), and N-acetylmannosamine-6-phosphate 2-epimerase (1.8 Å). Understanding the structural nature of these enzymes will provide us with the preliminary information necessary for antibiotic development. POST 03-237


POSTER ABSTRACTS The Role of Protein Glycosylation in Laccases from Lentinus sp.

Wei-Chun Liu1, Manuel Maestre-Reyna2, Wen-Yih Jeng3, Cheng-Chung Lee2, Chih-An Hsu1, Tuan-Nan Wen4, Andrew H.-J. Wang2, Lie-Fen Shyur1 1 Agricultural Biotechnology Research Center , Academia Sinica, Taipei, Taiwan, 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 3Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan,4Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan

Laccases are blue multicopper oxidases catalyzed various organic and inorganic compounds by reducing O2 to water. Despite several decades’ research, the roles of protein glycosylation in laccases are still obscure. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. The nLcc4 is composed of three cupredoxin-like domains D1-D3, and each domain folds into a Greek key β-barrel topology. The T1 and T2/T3 copper binding sites 75 238 458 and three N-glycosylated sites at Asn , Asn , and Asn were elucidated. Initial rate kinetic analysis -1 -1 revealed that the specific activity, kcat, Km, and kcat/Km of nLcc4 with ABTS were 2,996 Umg , 3,412 s , 65.0 -1 -1 ± 6.5 μM, and 52 s μM , respectively, and the values for lignosulfonic acid determined using isothermal -1 -1 -1 -1 titration calorimetry was 39 Umg , 44 s , 42.0 ± 2.1 mM, and 1.05 s mM , respectively. Endo Hdeglycosylated nLcc4 (dLcc4), leaving one GlcNAc residue at the three N-glycosylated sites, exhibited similar kinetic efficiency and thermal stability to that of native nLcc4. However, the N75D, N238D and N458D mutant enzymes secreted in the cultural media of Pichia pastoris cells maintained only 4-50% activity of the wild-type laccase. Molecular dynamics simulations analysis of various states of (de)glycosylation in nLcc suggest that the local H-bonds networks between domain connecting loop D2-D3 and glycan moiety play a crucial role in laccase activity. Together, this study provides new insights in the structural and functional roles of glycosylation in Basidiomycetes fungal laccase. POST 03-238 FBP17 Plays A Role In The Morphological Control By Regulating The Activity Of Rho Subfamily GTPase CDC42

Jun Zhang, Lin Ming-ming, Zhang Qian-ying, Wang Yun-hong, Li Xin Institute of Molecular Medicine and Oncology, Chongqing Medical University, Chongqing, China

The formin-binding protein 17 (FBP17) is widely expressed in eukaryotic cells and found to be involved with endocytosis and migration in macrophage. In these cellular processes, the N-terminal EFC (extended FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was previously shown to have membrane binding and deformation activities. In hepatocyte, it was interestingly found that knockdown of the endogenous FBP17 by RNAi didn’t affect the cell endocytosis and migration, but leaded to the cell morphological transition from the epithelioid to fibroid. Further investigation demonstrates that the activity of CDC42, which is governed by the exchange of GTP and GDP, significantly reduces with the down-regulation of FBP17 and withdrawal of the silence could simultaneously recover the FBP17 expression, the activity of CDC42 and the cell morphology. Polypeptide specifically targeted to the RBD domain of FBP17, a putative Rho family protein binding site, can also attenuate the CDC42 activity with a similar morphological response, which strongly suggests that FBP17 might play an important role in morphological control to keep moderate CDC42 activity possibly by the physical interaction of its RBD domain with CDC42. Co-localization of RBD domain of FBP17 and CDC42 has been directly observed in the cell cortex. Furthermore, F-actin stabilizing reagent phalloidine can abrogate the morphological responses mediated by both the FBP17-siRNA and target polypeptide, which implies that the inhibition of CDC42 activity by knockdown of FBP17 initially results in disrupting the equilibrium of F-actin dynamics, followed by rearrangement of actin cytoskeleton in cortex and subsequently forces the morphological remodeling. It is indicated that FBP17 could be responsible for the


POSTER ABSTRACTS versatile functions in different cellular milieus via distinct molecular pathways. Key words: FBP17; morphological remodeling; CDC42; cell cortex; actin dynamics This work is funded by the grants from Chongqing Education Commission (KJ080301), Chongqing Science&Technology Commission (CSTC, 2010BB5366) and National Natural Science Foundation of China (NO. 20803098). POST 03-239 Semi-Synthesis and Applications of Fluorophore/Thioamide pairs Containing Proteins

Solongo Batjargal, E. James Petersson University of Pennsylvania, Philadelphia, Pennsylvania, US

Our laboratory is interested in elucidating the structures of misfolded proteins on the single residue level using fluorescence spectroscopy. We have shown that a thioamide, a substitution of the carbonyl oxygen with a sulfur atom in the peptide backbone, is capable of quenching a variety of fluorophores. We have developed methods for incorporating fluorophore/thioamide pairs into full-length proteins through a combination of unnatural amino acid (Uaa) mutagenesis and native chemical ligation (NCL). We have applied our small thioamide probes to studies of α-Synuclein (αS), an intrinsically disordered protein implicated in Parkinson’s disease. The conformational changes of the αS monomers labeled with fluorescein/thioamide pairs have been examined in the presence of osmolytes as well as in aggregation prone states through fluorescence spectroscopy. In addition, we have developed an easy and efficient method of isolating the full-length proteins containing Uaa insertions by exploiting the unique selfsplicing properties of inteins. A C-terminal His-tagged intein provides a convenient handle to pull out only the full-length protein, and the subsequent removal of the intein in a traceless manner yields the Uaa-labeled protein in pure form. This technique should be of general use to the Uaa mutagenesis community as it provides a simple solution to the “truncation problem.” POST 03-240 Computational Design of an Unnatural Amino Acid Dependent Metalloprotein with Atomic Level Accuracy

Jeremy Mills1, Sagar D. Khare1, Jill M. Bolduc2, Farhad Forouhar3, Vikram K. Mulligan1, Scott Lew3, Jayaraman Seetharaman3, Liang Tong3, Barry L. Stoddard2, David Baker1 1 Biochemistry, University of Washington, Seattle, Washington, US, 2Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, US, 3Biological Sciences, Columbia University, New York, New York, US

Genetically encoded unnatural amino acids (UAAs) could provide a foundation for the development of proteins with novel chemistries or functions. However, as success in this endeavor requires precise placement of the UAA and surrounding residues within a protein scaffold, it represents a difficult challenge. Computational protein design methods could potentially be employed to address this problem, but had not been used in concert with UAAs prior to this work. We report the use of the Rosetta computational protein design suite to engineer a novel metalloprotein in which the UAA (2,2′bipyridin5yl)alanine (Bpy-ala) serves as a primary ligand of the bound metal. In a first design attempt, important considerations were identified for design approaches that utilize Bpy-ala as a metal ligand. A second round of design identified a series of candidate proteins in which octahedral binding sites were formed from Bpy-ala, two scaffold based metal liganding residues, and two water molecules. Experimental characterization of these candidates identified MB_07 as a protein possessing 2+ 2+ 2+ 2+ the ability to bind with high affinity a host of divalent cations including Co , Zn , Fe , and Ni . X-ray 2+ 2+ crystallographic characterization of MB_07 bound to Co and Ni showed excellent agreement between the computational models and the designed metalloprotein; RMSDs over all atoms in the binding site are 2+ 0.9, and 1.0 Å respectively. The dissociation constant of MB_07 for Zn was measured to be ~40 pM,



representing a ~2x10 increase in affinity of the designed protein for this metal relative to the UAA alone. The methodologies developed in the course of this study should facilitate the design of novel proteins with properties that would be difficult to achieve exclusively using naturally occurring amino acids. POST 03-241 Acrolein-modified High Density Lipoproteins Promote Atherogenesis

Alexandra Chadwick1, Rebecca L. Holme1, Yiliang Chen2, Kirkwood A. Pritchard1, Daisy Sahoo1 1 Medical College of Wisconsin, Milwaukee, Wisconsin, US, 2Blood Research Insititute, Blood Center of Wisconsin, Milwaukee, Wisconsin, US

High density lipoprotein (HDL) prevents atherosclerosis through its key roles in the reverse cholesterol transport pathway (RCT), where HDL’s main function is to transport excess cholesterol from the periphery to the liver for excretion. Cholesterol delivery into hepatic cells is achieved by interaction of HDL with its receptor, scavenger receptor BI (SR-BI). Mounting evidence shows that modification to HDL by environmental factors may reduce/eliminate its anti-atherogenic functions. Acrolein, a highly reactive aldehyde found in tobacco smoke, has recently been identified as an HDL modifier in vivo, although its affects on HDL function in RCT have not been fully characterized. We hypothesized that acrolein modifications to HDL (acro-HDL) generate a dysfunctional particle that: (i) is unable to mediate SR-BImediated selective uptake of HDL-CE and efflux of free cholesterol (FC) and (ii) upregulates inflammatory pathways in macrophages that promote atherogenesis. To test this hypothesis, human HDL was first modified with 250uM acrolein. Immunoblot analysis confirmed the presence of protein crosslinking after one hour of acrolein modification, as well as the presence of acrolein adducts on apoA-I and apoA-II, the two major HDL proteins. Mass spectrometry analyses also verified the expected acrolein adduct shifts of 56, 76, and 94 Da. Next, we assessed acro-HDL’s functionality during the beginning of RCT. Our data revealed that acro-HDL displayed a 30% decrease in accepting FC effluxed out of SR-BI-transfected COS7 cells, as compared to native HDL. Oil Red O staining of murine macrophages supported these findings. We then evaluated acro-HDL’s functionality at the end of RCT and demonstrated that acro-HDL (050mg/mL) was 10-25% less efficient at delivering HDL-CE to COS7 cells transiently expressing SR-BI. Finally, we used qRT-PCR to demonstrate that acro-HDL, but not native HDL, stimulated atherogenic pathways by increasing mRNA expression of scavenger receptors (CD36 and LOX-1) and inflammatory markers (MCP-1, IL-8, ICAM-1, and TNF-a) in human THP-1 differentiated macrophages, paralleling the effects of oxidized LDL . Altogether, our findings reveal that acrolein modification of HDL produces a dysfunctional particle that is proatherogenic. More detailed investigations into how oxidative modification of HDL contributes to atherosclerosis will be extremely important in combating cardiovascular disease. POST 03-242 Control Of Protein Production And Virus Replication By Pharmacological Blockade Of Degron Detachment

Hokyung K. Chung1, Conor Jacobs1, Yunwen Huo6, Jin Yang5, Stefanie A. Krumm4, Richard K. Plemper4, 7, Rodger Y. Tsien5, 2, Michael Z. Lin6, 3 1 Department of Biology, Stanford University, Stanford, California, US, 2Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California, US, 3Department of Pediatrics, Stanford University, Stanford, California, US, 4Department of Pediatrics, Emory University, Atlanta, Georgia, US, 5Department of Pharmacology, University of California, San Diego, La Jolla, California, US,6Department of Bioengineering, Stanford University, Stanford, California, US, 7Institution for Biomedical Science, Georgia State University, Atlanta, Georgia, US


POSTER ABSTRACTS A generalizable method to rapidly control protein production while minimizing protein modification is desirable for many biological applications. Here, we describe a novel method for pharmacological shut-off of protein production using an essentially traceless protein tag. This tag is genetically encoded to the protein of interest (POI) and consists of the self-cleaving HCV protease and degron. By default, this tag is separated from the POI and leaves it structurally unmodified; however, administration of a clinically approved HCV protease inhibitor blocks degron detachment on the subsequently synthesized POI. The POI fused to the degron will then be proteolytically removed. We call this system, Small-Molecule Assisted Shutoff (SMASh). The traceless characteristic of SMASh is especially beneficial for controlling a wide range of proteins whose structures need to be intact for their functions such as virus replication factors. We illustrated the usefulness and versatility of SMASh by showing reversible control of various proteins’ production in multiple mammalian cell lines. Importantly, SMASh enables us to engineer drugsusceptibility into the measles virus, an oncolytic RNA virus that is in clinical trials but does not have a specific inhibitor. This drug-regulable SMASh-incorporated virus would hold potential as a safe platform virus in translational applications such as cancer therapy or new multivalent live vaccine. Together with the advantages of the commercial HCV inhibitor–non-toxicity, high selectivity and cell-permeability– we envision that the rapid, reversible, uniquely traceless and compact nature of SMASh will allow a variety of applications in biomedical research and technology. POST 03-243 Understanding The Metabolism Of Enteric Pathogen Campylobacter jejuni

Adnan Ayna, Peter Moody Leicester University, Leicester, United Kingdom

Fructose bisphosphatase (EC is the enzyme that converts fructose-1,6-bisphosphate to fructose 6-phosphate in gluconeogenesis. In mammals the enzyme is subject to metabolic regulation but regulatory enzymes of bacterial FBPases are relatively less understood. Our study aimed to shed some lights on the metabolism of enteric pathogen Campylobacter jejuni. Homology modeling and sequence alignments show that the FBPase from C. jejuni (CJFBPase) is expected to have the same overall fold as the other type 1 FBPases for which structural information is available. However, alignments of the sequences show different trends in AMP, Glc-6-P and PEP binding sites. Some of the critical residues of AMP binding sites may be missing in C. jejuni and some seem not to be conserved. The regulatory properties of the enzymes have been investigated and surprisingly it is found that physiological level of ATP has an inhibitory effect on the activity of CJFBPase suggesting that there might be a novel ATP binding site on the enzyme. PEP also has an inhibitory effect on CJFBPase. It is not clear how PEP inhibits the activity as it doesn’t have the conserved PEP binding site. Both molecules are uncompetitive inhibitors of the enzyme as they decrease Km and Vmax. To test the effect of these molecules on the thermal stability of the enzymes the effect of thermal variation on the stability was investigated with CD using 1 mM PEP and 2.5 mM ATP. Both molecules increased the stability of the enzyme supporting the suggestion that there might a novel ATP binding site of the enzyme. In order to fully understand the mechanism of inhibition, crystal structure of the enzyme needs to be solved. POST 03-244 Chitosan-Binding Modules (CBM32) of a chitosanse From Paenbacillus sp. IK-5 ---Amino Acid Residues Responsible For Chitosan Binding---

Shoko Shinya3, Takayuki Ohnuma3, Hisashi Kimoto2, Hideo Kusaoke1, Tamo Fukamizo3 1 Fukui University of Technology, Fukui, Japan, 2Fukui Prefecture University, Fukui, Japan, 3Kinki university, Nara-shi, Japan


POSTER ABSTRACTS A chitosanase isolated from Paenibacillus sp. IK-5 has two carbohydrate binding modules (DD1 and DD2) at its C-terminus (Kimoto et al., J. Biol. Chem. 2002, 277, 14695-14702). DD1 and DD2 are highly specific to chitosan oligosaccharides, (GlcN)n. Although the amino acid sequence of DD1 is highly homologous (72 %) to that of DD2, the affinity of DD1 to (GlcN)n is much higher than that of DD2. NMR titration experiments and docking simulations showed that in both DD1 and DD2, (GlcN)nbind to the shallow binding cleft formed by several loops extruded from the b-sandwich structure (Shinya et al., J. Biol. Chem. 2013, 288, 30042-30053). However, we found that Glu36 located in the (GlcN)n-binding site of DD1 is substituted with tyrosine in DD2, as shown in Fig. 1. To identify the structural factors resulting in the different affinities between DD1 and DD2, Glu36 of DD1 was mutated to tyrosine (DD1-E36Y), and the reverse mutation (Tyr→Glu) was also conducted at the 36thposition in DD2 (DD2-Y36E). For the NMR titration experiments, 15N- and 13C-labeled DD1, DD1-E36Y, DD2, and DD2-Y36E were obtained using E. coli expression system in an M9 minimal medium containing 15N-NH4Cl and 13C-glucose. Based on the chemical shift perturbations of the 1H-15N HSQC resonances, (GlcN)4 binding ability was evaluated for individual module proteins. The resonances of amino acids located at the (GlcN)n binding site of DD1 were affected strongly, whereas the resonances of DD1-E36Y were almost unaffected. In contrast, the resonances of DD2-Y36E were more strongly affected than those of DD2. ITC experiments were also conducted to determine the association constants (Ka) of (GlcN)4 for individual proteins. The Ka values for (GlcN)4 were 4.7x 105 M-1 for DD1 and 5.6 x 103 M-1 for DD1-E36Y. The mutation of Glu36 of DD1 significantly reduced the binding affinity. The Ka values were 5.8 x 103 M-1 for DD2 and 3.1 x 104 M-1 for DD2-Y36E. The mutation of Tyr36 of DD2 enhanced the binding affinity. Thus, we concluded that amino acid substitutions at the 36th position significantly control the binding ability of DD1 and DD2. POST 03-245 Crystal Structure Of A Family GH18 (Class V) Chitinase From Cycad, Cycas revolta---- Structural Factors Controling The Transglycosylation Activity Of The Enzyme

Tamo Fukamizo1, Naoyuki Umemoto1, Takayuki Ohnuma1, Toki Taira2, Tomoyuki Numata3 1 Department of Advanced Bioscience, Kinki University, Nara, Japan, 2Department of Bioscience and Biotechnology, University of Ryukyus, Nishihara, Okinawa, Japan, 3Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan A family GH18 chitinase from cycad, Cycas revolta, (CrChiA) is the first plant chitinase that has been found

to possess transglycosylation (TG) activity. To identify the structural factors controlling the TG activity, we solved the crystal structure of the chitinase, and compared the structure with those of GH18 chitinases from Arabidopsis thaliana (AtChiC) and Nicotiana tabaccum (NtChiV), which do not exhibit TG activity, All three chitinases have an (α/β)8 barrel fold with an insertion domain (green region), as shown in Fig. 1. In the acceptor-binding site (+1, +2, and +3) of CrChiA, several aromatic side chains form a cluster, stabilizing the platform for the oligosaccharide binding. Among the aromatic side chains in the cluster, Trp168 was found to make a face-to-face stacking interaction with the +3 sugar. However, the cluster in the identical region of AtChiC is smaller than that of CrChiA. Tryptophan residue is not conserved in this cluster of AtChiC. Such a cluster was not found in the identical region of NtChiV. This situation may bring about the lower affinity for acceptor binding to AtChiC and NtChiV. In the catalytic DxDxE motif (the bottom of Fig. 1), the side chain of the middle Asp (Asp117) is always oriented toward Glu119 in CrChiA, whereas in NtChiV and AtChiC, the side chain of the middle Asp (Asp113 and Asp114, respectively) is oriented toward the first Asp (Asp111 and Asp112, respectively). The orientation of the middle Asp toward Glu in the DxDxE catalytic motif was also found in the Serratia chitinase (SmChiA) mutant (W167A), and suggested to be one of the molecular signs for efficient TG activity in GH18 enzymes. CrChiA, NtChiV, and AtChiC were also employed for allosamidin binding experiments using isothermal titration calorimetry. No significant difference in the free energy changes of allosamidin binding (ΔG○) to subsites -3, -2, and -1


POSTER ABSTRACTS was found for these chitinases; -9.7, -9.8, and -9.5 kcal/mol for CrChiA, NtChiV, and AtChiC, respectively. However, the solvation and conformational entropy changes (ΔSsolv and ΔSconf) for CrChiA were considerably different from those for NtChiV and AtChiC but simillar to those for SmChiA, which catalyzes TG reaction. Taken together, structural factors controlling the TG activity were found to lie on the entire region of the binding cleft; the acceptor-binding site, the catalytic center, and the allosamidin binding site. POST 03-246 Split Intein Mediated Peptide Cyclization

Shubhendu Palei2, 1, 3, Henning D. Mootz2, 1, 3 1 Department of Chemistry and Pharmacy, Muenster, North Rhine Westphalia, Germany, 2Institute of Biochemistry, Muenster, Germany, 3International NRW Graduate School of Chemistry, Muenster, Germany

Cyclic peptides are in the focus for therapeutic applications because of their unique properties. Peptide backbone cyclization is one of the major strategies to stabilize peptides and proteins. Cyclic peptides are more stable towards pH change, heat denaturation, protease degradation than their linear counterparts. The cyclic backbone also has less conformational flexibility and small entropy which increase their binding affinity towards the peptide or protein interface. Therefore cyclic peptides are also used as inhibitors for protein-protein interactions or for different enzymes. There are different methods for achieving cyclization of peptide backbone either by chemical or biological means. Protein trans-splicing (PTS) is one of the robust ways of making cyclic backbone in vitro as well as in vivo. In PTS, the extein sequences from two different protein and/or peptide partners are joined by a new peptide bond without the use of any external energy or cofactor. It is purely an autocatalytic process. Here we develop a novel method of peptide backbone cyclization using PTS to make a range of cyclic peptides of different sizes. We make the use of SspDnaB split intein to make cyclic peptides. This technique can be used as an excellent tool to make cyclic peptides even in complex environments in vitro (cell lysates) or in vivo (inside a living cell) with a great selectivity.

POST 03-247 Exploring the Morpheein Forms of B. cenocepacia HMG-CoA Reductase

Riley Peacock, Michelle Brajcich, Courtney Boyd, Jeffrey Watson Chemistry and Biochemistry, Gonzaga University, Spokane, Washington, US

Isoprenoids are a key class of biological molecules required by all three kingdoms of life, and are synthesized by one of two metabolic pathways. The mevalonate pathway is found in eukaryotes, archaea and some eubacteria, and proceeds through the enzyme HMG-CoA reductase (HMGR), which catalyzes the rate-limiting and first committed step. The DXP pathway is found in most eubacteria, and does not use HMGR at any step. The opportunistic lung pathogen Burkholderia cenocepaciacodes for HMGR, yet biosynthesizes isoprenoids using the DXP pathway. B. cenocepacia is a significant cause of fatality in cystic fibrosis patients, in part due to its natural resistance to most available antibiotics. Therefore, understanding the function and regulation of HMGR from B. cenocepacia (BcHMGR) may aid in combating these difficult-to-treat infections. Our evidence from various kinetic, spectroscopic and chromatographic techniques suggests that BcHMGR exists in at least three active quaternary forms, each with distinct size and charge properties. In addition, our data suggests that the equilibrium between these states depends on pH, enzyme concentration, and substrate concentration, as changing these elements independently yields a shift in the relative concentration of these forms, and an associated change in the kinetic activity of the enzyme. This data suggests that BcHMGR follows the morpheein


POSTER ABSTRACTS model of allostery, and further suggests that the enzyme may be an important asset to B. cenocepacia and a potential antibiotic target. POST 03-248 Dynamic Functional Switch in Poliovirus 3C Protease

Yan M. Chan, David D. Boehr Chemistry, Pennsylvania State University, University Park, Pennsylvania, US

Viral genomes are very efficient; they are typically compact but nevertheless encodes numerous elements that are essential for regulation of both its own replication and packaging, and of the host cell's machinery. Viruses have developed intelligent strategies to overcome their biological information storage pro problem. For example, the 3C protein from the picornavirus family of positive-strand RNA viruses is responsible for binding of RNA control sequences to regulate translation and replication, interacting with phosphoinositide lipids (PI) to regulate the maturation of virus replication organelles, and acting as the pro main protease to cleave host and virus proteins to further regulate host and virus processes. 3C can also pro pro pro be found as a domain in the 3CD polyprotein. 3C by itself and 3CD have different protease pro specificities, and likely different RNA and PI binding capabilities. The domains in 3CD are tethered by a pro pol flexible linker and do not make specific 3C -3D interactions. Surprisingly, we have found that by extending the C-terminal tail of 3C with just a few amino acid residues, the RNA and PI binding properties alter dramatically. These functional changes are accompanied by changes in the structural dynamics of 3C, as measured by NMR relaxation methods. We propose that this finding has critical bearing on 3C pro pro function; proteolytic processing of the C-terminus is the switch from 3CD to 3C (by itself) activities. Such a simple, but elegant, mechanism does not require any additional domain-domain interactions in the pro pro 3CD polyprotein to regulate 3C function, and can help explain functional differences between pro pro 3C and 3CD that have confounded virologists and structural biologists for years.

POST 03-249 Lighting the Cellular Fuel Gauge with Fluorescent Sensors for Imaging Single-Cell Metabolism

Mathew Tantama, Juan Ramón Martínez-François, Rebecca Mongeon, Gary Yellen Neurobiology, Harvard Medical School, Boston, Massachusetts, US

How do neurons support their metabolically-demanding signaling activities? Despite our growing knowledge of brain circuitry, we still do not understand how neurons and glia tune their cellular metabolism to support different types of signaling. In order to interrogate this linkage between metabolism and brain activity, genetically-encoded biosensors have been developed to visualize cellular energy status using live-cell optical microscopy. In particular, a fluorescent biosensor, PercevalHR, has now been optimized to sense the range of intracellular ATP:ADP ratios expected in mammalian cells and to respond to changes within seconds. Additionally, this newly engineered biosensor can be used with either one- or two-photon fluorescence excitation microscopy with live samples. To demonstrate that it is a sensitive reporter of physiological changes in energy consumption and production, PercevalHR has been used to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli. PercevalHR has also been used to visualize intracellular ATP:ADP while simultaneously recording electrical currents from ATP-sensitive potassium (KATP) channels, enabling the direct correlation between cytosolic ATP:ADP and KATP channel open probability in intact single cells. Thus, PercevalHR should be a versatile tool that will provide access to a new level of molecular and cellular detail in the study of energy metabolism. Furthermore, this combination of genetically-encoded biosensors, live-cell imaging, and


POSTER ABSTRACTS electrophysiology will be used to study the metabolic components of neurodegeneration in aging and in diseases such as epilepsy and Parkinson's. POST 03-250 Structural Analysis And Molecular Dynamics Of The Self-Sufficient P450 CYP102A5 And CYP102A1: A Combined Computational/Experimental Approach To Increase The Efficiency Of Biocatalyst Engineering

Maximilian Ebert4, 1, Brahm Yachnin2, Guillaume Lamoureux3, 1, Albert Berghuis2, 1, Joelle Pelletier4, 1 1 PROTEO, Montreal, Quebec, Canada, 2McGill University, Montreal, Quebec, Canada, 3Concordia University, Montreal, Quebec, Canada, 4Université de Montréal, Montreal, Quebec, Canada

P450s catalyze the oxidation of non-activated carbon atoms, which is chemically demanding. Members of the CYP102 family are termed “self-sufficient P450s”, meaning that they contain all the machinery necessary to ensure the electron transfer and active site regeneration in one single protein. However, the macromolecular assembly remains unknown. Here we report results of SAXS analysis that bring new insights into the formation of the active complex. The recently reported new member CYP102A5 is highly interesting due to its sequence similarity to the intensively studied member CYP102A1, accompanied by a significant increase in electron transfer rate and improved regioselectivity. Homology models were generated, compared and used to identify the structural basis for these differences in catalytic activity. Based on this result we identified residues that may be involved in the gating and substrate capturing mechanism in CYP102A5. Predictions of differences in substrate incorporation and product release from the active site were computed using the adaptive biasing force (ABF) method. With this pioneering application of ABF in enzyme engineering, we were able to predict all known important residues for fatty acid substrate binding in CYP102A1, as well as two additional residues which were identified and analyzed in vitro to support the in silico finding. This newly developed computational biology approach, in addition to conformational studies, will help to guide directed evolution efforts towards the oxidation of non-native substrates. POST 03-251 Isolation And Characterization Of Proline Specific Dipeptidyl Peptidase IV From The Tenebrio Molitor Larval Midgut

Valeriya F. Sharikova1, Irina Goptar1, Yulia Smirnova2, Brenda Oppert3, Irina Filippova1, Elena Elpidina2 1 Chemistry, Lomonosov Moscow State University, Moscow, Moscow, Russian Federation, 2A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation, 3Center for Grain and Animal Health Research, Stored Product Insect Research Unit, Manhattan, Kansas, US The yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) is a stored product pest and a biochemical model organism of the family Tenebrionidae. The main food proteins of this insect are storage seed proteins - prolamins, which are also present in the diet of most people. Prolamins contain 10 - 30% proline and 30 - 50% glutamine residues. Several proline- and glutamine-rich prolamin peptides, which are resistant to proteolysis by human digestive enzymes, cause autoimmune Celiac Disease in 1% of the susceptible human population. In this regard, proline specific peptidases (PSP) are urgently needed for therapeutics, capable of hydrolyzing peptide bonds formed by proline residues that are resistant to proteolysis by peptidases of broad substrate specificity. We have carried out a bioinformatic search for PSP in the T. molitor larvae gut transcriptome and found 12 sequences similar to human PSP. Two predicted dipeptidyl peptidase IV (DPP IV) sequences were found, one with the highest mRNA expression level among all PSPs in the larval transcriptome. These enzymes are serine exopeptidases from the S9 family, which cleave Xaa-Pro dipeptides from the N-terminus of polypeptides. The major DPP IV-like


POSTER ABSTRACTS enzyme was purified from the T. molitor larvae midgut and analyzed by mass-spectrometry analysis. The amino acid sequence of the enzyme coincided with that of the highly-expressed DPP IV found in T. molitor gut transcriptome. The isolated enzyme was characterized by substrate specificity, pHdependence, pH stability, and inhibitor sensitivity. The importance of DPP IV in insect digestion as well as the potential for new treatments of Celiac Disease will be discussed. This work was supported by ISTC grant 3455 and RFBR grants 12-03-01057-a, 14-04-91167-NSFC_a. POST 03-252 Characterization Of Hydrolytic Enzyme-Producing Bacteria Isolated From Paper Mill

Manel Ghribi5, 2, Fatma Meddeb-Mouelhi2, 3, Marc Beauregard1, 2, 4 1 biologie médicale, Université de Québec à Trois Rivieres, Trois-Rivieres, Quebec, Canada, 2CRML, TroisRivieres, Quebec, Canada, 3Buckman North America, Vaudreuil-Dorion, Quebec, Canada, 4PROTÉO, Laval, Quebec, Canada, 5physique,chimie et biochimie, Université de Québec à Trois-Rivières, TroisRivieres, Quebec, Canada

Enzymes act as biocatalyst in many industries, such as textiles, detergent, food, animal feed, bio-fuel, paper and pulp, pharmaceutical, to name a few. Cellulases and hemicellulases are efficient hydrolytic enzymes used in the pulp and paper industry to reduce the cost of production. In addition, industrial enzymes reduce the environmental impact by replacing harmful chemicals. The leading industrial enzyme suppliers (Novozymes, Genencor) offer a limited library of enzymes. Thoses enzymes are poorly adapted to the need of the paper industry. Therefore, our partner, Buckman North America, wants to expand his enzymes portfolio by characterizing new enzymes-producing bacteria. Pulp and paper mills offer untapped biodiversity for microorganisms that use cellulose-based substrates as nutriments. In this project, we will isolate and characterize cellulose and hemicellulose degrading microorganisms from paper mill sludges. To conserve microorganism’s biodiversity, we used two temperatures (37 ᵒC and 50 ᵒC) for the isolation step. Detection of extracellular enzymatic activities was carried out on minimum agar plate medium supplemented with either cellulose (carboxylmethylcellulose or Avicel) or beechwood xylans. Bacteria strains showing extracellular cellulase and/or xylanase activities were isolated from various sludges (primary, secondary, presses and machines) found in a paper mill. These bacteria were identified based on their morphology, biochemical characterization and DNA 16s sequencing. The biorefining potential of these enzymes will be evaluated. POST 03-253 Isolation of NRPS and PKS Gene Clusters from Soil Microbes

Danielle N. O'Hara, Connor P. Craig University of Richmond, Richmond, Virginia, US

Greater than 90% of microbes living in soil are unculturable due to their complex nutrient and temperature requirements for growth. These microorganisms present a potential source of natural products that could be developed for biotechnological and pharmaceutical uses. Microorganisms with phosphopantetheinyl transferase (PPTase) activity are of high interest due to the role PPTase plays in activating non-ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene cluster products. The proteins expressed by these gene clusters synthesize complex natural products that are utilized by the microorganism or its host for selective advantages. We screened for NRPS and PKS gene clusters in microbes isolated from soil on the University of Richmond campus. Genomic DNA was isolated from each of these samples and was used to construct metagenomic libraries. We will continue to screen the libraries for PPTase activity in order to identify positive clones. We extended this study to include a library of pigmented microbes previously isolated from Chesapeake Bay sponges, Clathria


POSTER ABSTRACTS prolifera and Halichondria bowerbanki. The library was screened for antibacterial activity using gram positive and Gram negative co-cultures.

POST 03-254 Treatment Of Kraft Pulp With Enzymes For Improving Beatability And Physical Properties

Li Cui1, 2, Fatma Meddeb1, 3, Marc Beauregard1, 2 1 CRML, Université du Québec à Trois-Rivières, Trois-Rivieres, Quebec, Canada, 2PROTEO, Université Laval, Québec, Quebec, Canada, 3Buckman North America, Vaudreuil-Dorion, Quebec, Canada Over the last few years, the importance of enzymes as biotechnological catalysts for the pulp and paper industry has been demonstrated. Among the enzymes mostly studied, hydrolases such as cellulases and hemicellulases have been investigated for their potential impact on refining energy costs. Unfortunately, energy reduction by prior enzyme treatments often had a negative impact on certain paper properties. In this study, five different commercial cellulase formulations were used for the modification of fiber properties. PFI refining was employed at 3000 and 4500 revolutions to mimic the impact of various levels of refining, particularly on fiber size and morphology. With the five enzyme preparations, it was possible to decrease the number of PFI revolutions by 50% and achieve the same target freeness value (decrease in pulp CSF by approximately 200 mL) afforded by more intense refining without enzyme. The fiber morphology changed to different extent according to various enzymatic treatments. Subsequently, the carbohydrates in the filtrate released during enzymatic treatment was studied by ion chromatography (IC). IC results showed good agreement with the enzyme activity measured independently. The impact of enzymatic treatment on physical properties of handsheet was also investigated. The enzyme impact on tear index was exceptional compared to most properties measured in this study. Enzymes had a deleterious impact on tear even without any mechanical refining. A slight decrease in tear strength was observed with enzyme C1 and C4 at pH 7 after mechanical refining (less than 10%) while the most important decrease in tear was observed after C2, C3, C5 treatments. The reason for this phenomenon appears to be that C1 and C4 had xylanase activity. We conclude that xylanase activity could preserve and/or improve the properties of handsheets made from enzymatic treated pulp, and that the balance between cellulolytic and hemicellulolytic activities is the key to optimization of biorefining, leading to energy reduction and improving handsheet properties.

POST 03-255 Structural And Biochemical Investigation Of The Intramolecular Interactions Of Ceramide Transfer Protein

Jennifer Prashek, Seungkyung Kim, Xiaolan Yao University of Missouri-Kansas City, Kansas City, Kansas, US

Ceramide transfer (CERT) protein transfers ceramide, a key intermediate in sphingolipid biosynthesis, from the endoplasmic reticulum to the Golgi where it is converted to sphingomyelin. CERT contains several domains and motifs. The amino terminal pleckstrin homology (PH) domain targets CERT to the Golgi by specifically recognizing phosphatidylinositol-4-phosphate (PtdIns(4)P). The carboxyl terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain catalyzes the transfer of ceramide. Following the PH domain is a short stretch rich in serine and threonine residues called the Serine Rich (SR) motif. Phosphorylation of the SR motif reduces the ceramide transfer activity of CERT. This is achieved through both inhibition of the PH domain binding to PtdIns(4)P and START domain transfer of ceramide. It has been shown that this inhibition requires both the PH and START domains suggesting an 1,2 auto-inhibitory intramolecular interaction . We set out to investigate the structural basis of phosphorylation inhibition of CERT function. We generated mutations of ten serine and threonine


POSTER ABSTRACTS residues of the SR motif to glutamate to mimic the phosphorylated form of CERT. We also generated isolated PH and START domains to further investigate the domain-domain interaction within CERT using a combination of structural and biochemical methods. Our results show that the interaction of the PH and START domains may contribute to the inhibition of PH binding to PtdIns(4)P that occurs upon SR motif phosphorylation. We are currently investigating the effect of disrupting the PH-START interaction on the activity of the phosphorylation mimic of full length CERT. POST 03-256 Evaluating Interpretation Of B-factors For Collective Motion Modeling

Edvin Fuglebakk1, Nathalie Reuter1, Konrad Hinsen2 1 University of Bergen, Bergen, Norway, 2Centre de Biophysique Molećulaire, Centre Nationalde la Recherche Scientifique, Orleáns, France The interpretation of crystallographic B-factors in terms of thermal motion is prevalent in many branches of protein science. It is therefore important to understand its inherent limitations. One common criticism against interpreting functional motion from B-factors is that they are influenced by many non-thermal factors. Another concern is that the thermal component of B-factors arises from motion in a highly restrictive crystalline environment. This environment is expected to dampen collective motion, the kind of motion often involved in functional rearrangments like conformational change. I will present results that reveal potential problems with the use of B-factors as a model of thermal motion of solvated proteins. We have compared the collective motions of several elastic network models, a kind of protein model commonly validated and parameterized against B-factors. We obtained collective motion predictions from elastic network models and compared them with molecular dynamics simulations for seven solvated protein structures. We find that models that give good reproduction of B-factors are severely compromised in their ability to recapitulate collective motions. Moreover, we compare the elastic network models with a null model of restricted collective motion, and find that models parameterized to reproduce B-factors are in close agreement with this null model. We therefore find it important to consider the effect of the crystalline environment when interpreting Bfactors, and avoid inference about slow collective motion from this data.

POST 03-257 Structure/Function Relationships in Carboxylesterase EstGtA2 from Geobacillus thermodenitrificans

Jessica K. Moisan1, 2, Fatma Meddeb-Mouelhi1, 3, Marc Beauregard1, 4 1 3351 Boulevard des Forges, Université du Québec à Trois-Rivières, Trois-Rivières, Quebec, Canada, 2PROTEO, Québec, Quebec, Canada, 3Buckman, Vaudreuil-Dorion , Quebec, Canada, 4PROTEO, Québec, Quebec, Canada

Carboxylesterases from thermophiles have become objects of special interest for structural investigation and for a broad range of biotechnological applications. Their range of physicochemical properties makes them enzymes of great interest for textile, food and pulp and paper industries. Carboxylesterases (EC belong to a class of hydrolases adopting the α/β hydrolase fold. These enzymes are active at alkaline pH and high temperature. Understanding the relationship between structure and enzymatic properties is essential in order to produce novel biocatalysts mutants with desirable properties for a given application. Five mutants of the recombinant Geobacillus thermodenitrificans carboxylesterase (EstGtA2) were generated by site-directed mutagenesis in order to study the contribution of specific salt bridges in the stabilization and refolding of EstGtA2. One particular mutant (M1a), in which a salt bridge has been disrupted by a point mutation (R37A), was found to have an increased melting temperature. This is an


POSTER ABSTRACTS unexpected change in enzymatic properties considering the fact that salt bridges often contribute to thermal stability. In order to better understand the impact of this point mutation (R37A), yield production has been optimized for both the wild type protein EstGtA2 and the mutant M1a (R37A). Biophysical and biochemical studies were performed to enable better understanding the structure-function relationships. The enzymes were characterized by circular dichroism for conformational analysis, by enzymatic assays for activity and specificity and by dynamic light scattering to determine solution conditions which are optimal for the sample to be monodisperse. In the near future our goal is to determine the three dimensional structures by X-ray crystallography, allowing for a detailed investigation of structural key elements which control the properties of the carboxylesterase EstGtA2. POST 03-258 Up-Regulation Of Rich1 Causes S-Phase Arrest And Reduces Cell Adhesion In Epithelial Cells

Lin Ming-ming, Zhang Qian-ying, Wang Yun-hong, Li Xin, Zhang Jun Institute of Molecular Medicine and Oncology, Chongqing, China

Rich1, a previously identified Rho GTPase-activating protein (GAP) domain-containing protein, was found to have close relationship with Rho GTPase family members in an array of cellular processes. In addition to GAP domain, the protein also contains a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain and plays a role in stimulating GTP hydrolysis to terminate Rho signaling. The regulation functions of Rich1 in epithelial cells are still not completely clear. In the present investigation, it was shown that up-regulation of Rich1 expression to over 3-fold by transient transfection of full-length Rich cDNA vector caused a slight S-phase arrest with corresponding growth inhibition, but strongly reduced cell adhesion in each of the six types of epithelial cells including HL7702, Hela, 293T, A594, H1299 and T24 enrolled in the experiments. Further in-depth survey in HL7702 cells revealed that up-regulation of Rich1 could greatly increase the GTPase activities of both the CDC42 and RAC1, which could attenuate the Rho protein (CDC42 and RAC1) signaling, and subsequently lowered the phosphorylation level of ERK1 as well. A truncated Rich1 construct without GAP domain could not result in any of the biological effects. Co-localization of Rich1 with CDC42 and RAC1 could be observed in cytoplasm respectively. According to the discovery, it is indicated that Rich1, via its GAP domain, might act as a crucial upstream negative regulators of the Rho GTPases-ERK1 signaling triggered by CDC42 or/and RAC1 in the control of epithelial cell cycle, growth and adhesion. Key words: Rich1; CDC42; RAC1; S-phase arrest; cell adhesion This work is funded by the grants from Chongqing Education Commission (KJ080301), Chongqing Science&Technology Commission (CSTC, 2010BB5366) and National Natural Science Foundation of China (NO. 20803098). POST 03-259 Tracking Wood Fibers Decrystallization With Carbohydrate Binding Module

Yannick Hébert-Ouellet1, 2, Vinay Khatri1, 2, Fatma Meddeb-Mouelhi1, 3, Marc Beauregard1, 2 1 UQTR, Trois-Rivières, Quebec, Canada, 2PROTEO, Québec, Quebec, Canada, 3Buckman North America, Vaudreuil-Dorion, Quebec, Canada

There are many steps in the complex process of converting wood into paper. One of these steps is refining. Refining is essential for modifying the characteristics of wood fibers so that it may form paper sheet with a specific set of properties. One important modification of wood fibers is the fibrillation of the exposed S2 layers which promote the formation of hydrogen bonds and increase the available bonding surface. This modification also partially converts crystalline cellulose into amorphous cellulose. On the other hand, the majority of the energy devoted to paper manufacturing is consumed by the refining of wood fibers. Therefore, improving energy efficiency through the understanding of wood fibers refining at a molecular level is of paramount importance for the pulp and paper industry. In this study, we specifically


POSTER ABSTRACTS track the decrystallization of cellulose on the surface of refined paper sheets through the utilization of Clostridium thermocellum CipA carbohydrate binding module 3a fused to eGFP. Correlations between crystalline cellulose quantification, energy of refining and paper physical properties are shown. Advantages, limitations and applications of this assay to enzymatic prerefining are also discussed. POST 03-260 Evolution of Structure and Mechanistic Divergence in Di-Domain Methyltransferases from Nematode Phosphocholine Biosynthesis

Soon Goo Lee, Joseph M. Jez Biology, Washington University in St. Louis, St. Louis, Missouri, US

The phosphobase methylation pathway is the major route for supplying phosphocholine to phospholipid biosynthesis in plants, nematodes, and Plasmodium. In this pathway, phosphoethanolamine Nmethyltransferases (PMT) catalyzes the sequential methylation of phosphoethanolamine to phosphocholine. In the PMT, one domain (MT1) catalyzes methylation of phosphoethanolamine to phosphomonomethylethanolamine and a second domain (MT2) completes the synthesis of phosphocholine. The x-ray crystal structures of the di-domain PMT from the parasitic nematode Haemonchus contortus (HcPMT1 and HcPMT2) reveal that the catalytic domains of these proteins are structurally distinct and allow for selective methylation of phosphobase substrates using different active site architectures. These structures also reveal changes leading to loss of function in the vestigial domains of the nematode PMT. Divergence of function in the two nematode PMT provides two distinct anti-parasitic inhibitor targets within the same essential metabolic pathway. The PMT from nematodes, plants, and Plasmodium also highlight adaptable metabolic modularity in evolutionarily diverse organisms.

POST 03-261 ERK1 and 2 - Exploring Isoform Differences

Jen Liddle, Natalie Ahn University of Colorado Boulder , Boulder, Colorado, US

The MAP kinase pathway includes multiple isoforms of each member in the pathway for most vertebrates, raising the question of whether these isoforms are redundant or have evolved unique functional niches. The prototypical MAP kinases, Erk1 and Erk2, share ~ 85% sequence identity, are activated by the same stimuli, and phosphorylate the same set of substrates. However, mouse knockout models show strong phenotypic differences, and incomplete redundancy is shown by a strong preference for Erk2 in specific contexts, such as T-cell proliferation, mouse embryonic survival, platelet signaling, thymocyte maturation, and EMT capabilities. A significant body of work has established multiple methods that could provide distinct regulatory mechanisms in vivo, including tissue-specific differential expression, nuclear translocation rates, and the possibility of unique scaffolding interactions. However, biophysical data suggest that intramolecular mechanistic differences can provide an additional and novel layer of signaling regulation. We present our results characterizing the differences between Erk1 and Erk2 using hydrogenexchange mass spectrometry, mutagenesis, and kinetic assays of autoactivation as well as substrate phosphorylation.


POSTER ABSTRACTS POST 03-262 Chemical Modification of MitoNEET

Megan Laffoon1, Michael Menze2, Mary Konkle1 1 Chemistry, Eastern Illinois University, Charleston, Illinois, US, 2Biology, Eastern Illinois University, Charleston, Illinois, US

MitoNEET, a [2Fe-2S] cluster protein, has the capability to bind to the antidiabetic drug pioglitazone. Possible cellular functions of mitoNEET include as an electron-transport protein or an ironsulfur transfer protein. The ligation of the [2Fe-2S] cluster of mitoNEET is unique in using one histidine and three cysteine residues. Recombinant expression of mitoNEET was accomplished using e. Coli bacteria and was subsequently purified by using affinity fast protein liquid chromatography. After expression and purification, UV-visible spectroscopy is used to observe the local environment of the cluster through observation of the ligand to metal charge transfer (LMCT) bands. MitoNEET was reacted with the chemical modifier diethylpyrocarbonate (DEPC) using a range of pH values. DEPC reacts with histidine to form a chromophore at 250 nm. However, a change in the LMCT bands after DEPC addition indicates modification of His87, the ligating histidine of mitoNEET. Additionally, the reactivity of the cysteine residues of mitoNEET, all of which ligate the metal cluster, were evaluated against the biologically-relevant molecules glutathione and fumarate. The goal of chemical modification of mitoNEET is to investigate the reactivity of the ligating residues of mitoNEET and to understand how these modifications impact protein stability and chemical reactivity towards small molecules like pioglitazone and macromolecules like protein binding partners of mitoNEET. POST 03-263 Biochemical Basis For The Extended Spectrum Cephalosporinase Activity Of A Clinical AmpC βlactamase Variant

Jozlyn R. Clasman, Brianna M. Jackman, Cynthia M. June, Rachel A. Powers, David A. Leonard Grand Valley State University, Allendale, Michigan, US

Class C β-lactamases provide antibiotic resistance to a diverse array of microbial species through their hydrolytic action against a wide-range of penicillin and cepholosporin β-lactam drugs. These β-lactamases can also expand their spectrum of activity through mutations that evolve under the pressure of antibiotic treatment. One example of this phenomenon is the appearance of a serine → asparagine mutation (S287N) in the AmpC β-lactamase during an outbreak of E.coli in France. Subsequent analysis revealed that the S287N variant conferred a high level of ceftazidime resistance on E.coli compared to wild-type AmpC. Also, steady-state kinetic analysis revealed that oxyiminocephalosporins and imipenem have lower KMvalues for the variant compared to wild-type suggesting that these drugs might bind the active site with higher affinity in the presence of the mutation. In order to investigate the structural basis of these interesting phenotypes, we used X-ray crystallography to determine the acyl-intermediate structures of AmpC S287N with ceftazidime and cefotaxime bound (1.88 Å and 1.97 Å, respectively), and compared our results to the previously-published structure of the ceftazidime acyl-intermediate of wild-type AmpC. Our analysis indicates that the S287N mutation allows the loop formed by residues 286-296 to adopt a new conformation, greatly relieving the steric clashes between the exocyclic carbon of the ligand’s dihydrothiazine ring and the side-chain of residue N289. These results may lead to the design of new cephalosporins that are less susceptible to the hydrolytic action of class C β-lactamases.


POSTER ABSTRACTS POST 03-264 Common Substitutions Enhance The Carbapenemase Activity Of Oxa-51-Like Class D β-lactamases From Acinetobacter SPP

Joshua M. Mitchell, David A. Leonard Grand Valley State University, Allendale, Michigan, US Carbapenem resistance in Acinetobacter baumannii is a growing threat to effective treatment of

infections. Acquired carbapenemases, particularly OXA-23, OXA-24/40 and their variants are a major source of resistance, especially when combined with porin deletion. A.baumannii has a chromosomal carbapenemase (OXA-51), though its weak activity against most β-lactams has allowed it to escape the high degree of scrutiny given to other carbapen-hyrolyzing class D β-lactamases (CHDLs). With more than 50 clinical variants of OXA-51 documented, it appears that many mutations have arisen independently several times and are sometimes associated with an increase in the minimum inhibitory concentration values for carbapenems. Based on homology with other β-lactamases, many of these clinical mutations are predicted to be in the active site. Starting with an OXA-51 gene, we synthesized, expressed and purified the I129L and L169V variants of OXA-51. Steady-state kinetic analysis shows that OXA-51 displays high KM and low kcat values for both penicillins and cephalosporins. OXA-51 affinity for carbapenems appears to be tighter (KM 5-150 µM), although much weaker than that seen for OXA23/OXA-24/40 (doripenem KM10-30 nM). Binding and turnover of penicillins and cephalosporins were little improved for either of the variants, but carbapemem KM values (and KS values) were much lower for both mutants, suggesting greatly increased affinity that approaches or equals that seen with OXA-24/40 and OXA-23. The increased affinity for both mutants with respect to substrates containing α-hydroxyethyl groups can be explained by mutation-induced remodeling of the active to better accommodate that group.

POST 03-265 Exploring the Potential of Arylboronic Acids as Inhibitors of OXA-24 β-lactamase

Josephine P. Werner, Rachel A. Powers Chemistry, Grand Valley State University, Allendale, Michigan, US

β-lactam antibiotics, like penicillin, are crucial to the field of medicine. Yet due to over-prescription, many bacteria are now resistant to them. The most widespread resistance mechanism to β-lactams is expression of β-lactamase enzymes. β-lactamases hydrolyze the amide bond of the defining four membered β-lactam ring, rendering the antibiotic inactive. One way to overcome resistance is through inhibition of βlactamase enzymes. However, current inhibitors for β-lactamases also contain a β-lactam ring. Therefore, a critical need exists for a novel, non-β-lactam inhibitor. The carbapenem-hydrolyzing class D β-lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze the newer carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β-lactamase inhibitors. Boronic acids are known to be transition state analogue inhibitors of class A and C β-lactamases, but they have not been extensively characterized as inhibitors of the class D β-lactamases. Boronic acids do not contain a βlactam ring in their structure, providing a novel way to inhibit class D β-lactamases. We ordered and tested thirteen arylboronic acids for inhibition of the CHDL OXA-24. Several were identified as inhibitors of OXA-24, with KI values ranging from 33.7 mM to 6.23 μM. These arylboronic acids serve as a starting point in optimization efforts in the development of a novel series of inhibitors for class D β-lactamases.


POSTER ABSTRACTS POST 03-266 Structure-based Discovery of a Novel Inibitor of OXA-1 β-lactamase

Leslie A. Wyman, Neil V. Klinger, Rachel A. Powers Grand Valley State University, Allendale, Michigan, US

β-lactams, like penicillin, are the most clinically prescribed antibiotics. However, due to their overuse, resistance has developed. β-lactamase enzymes are the most common resistance mechanism used by bacteria to combat the effects of these drugs. These enzymes efficiently hydrolyze the β-lactam ring that defines this class of antibiotics. In response, β-lactamase inhibitors were created to disrupt this type of bacterial resistance. Alone, β-lactamase inhibitors have minimal antibiotic activity, but when given in combination with a partner β-lactam, they enable the antibiotic to work by inhibiting the β-lactamase enzymes produced by resistant bacteria. Unfortunately, the structures of the inhibitors also contain a βlactam ring. The chemical similarity has allowed for resistance to develop against the inhibitors as well. Additionally, these compounds do not inhibit members of the class D β-lactamases. Therefore, there is an urgent need for the discovery of a novel β-lactamase inhibitor that does not resemble a β-lactam. A structure-based approach was used to discover possibilities for potential novel β-lactamase inhibitors of the class D β-lactamase OXA-1, a key clinical target. The program DOCK was used to screen the ZINC database of commercially available compounds. Twenty-one compounds from the fragment subset were ordered and tested experimentally for inhibition of OXA-1. Of the twenty-one fragments tested, seven inhibited OXA-1 with a Ki < 5 mM. Subsequently, the structure of OXA-1 in complex with fragment 19 (0.469 mM) was determined to 1.98 Å resolution. Using this structure as a template, optimization of a novel series of OXA-1 inhibitor is currently underway. POST 03-267 Kinetic And Biochemical Investigations Of Thermostable Acid Phosphatase From Zea Mays And Glycine Max Cotyledons: A Therapeutically Important Enzyme

Nidhee Chaudhary1, Subhash Chand2, Nameet Kaur1 1 Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India, 2Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT), Delhi, New Delhi, India

Acid phosphatases (APs), a group of ubiquitous enzymes found in many plant, animals and microorganisms, catalyze and facilitate important physiological changes within cells. Human APs are normally found at low concentrations. However, pronounced changes in their synthesis occur in particular diseases, where unusually high or low enzyme expression is seen as part of the pathophysiological process. Deficiency of APs causes various diseases like; Osteomalacia, Rickets, Bone malformations. In the present investigation few kinetic and biochemical parameters of acid phosphatase (APs) from germinating seedlings (cotyledons) of Zea mays and Glycine max named as AP I and AP II, respectively, have been studied. The extracted APs from the two sources were partially purified into two fractions each; based on 0-40% and 40-80% saturation level of (NH4)2SO4. The 0-40% fraction (both cases) was found to have -1 -1 higher specific activity; the values being 88.86 units mg and 97.56 units mg in AP I and AP II, 0 0 respectively. AP I and AP II were found to be thermally stable up to 75 C and 60 C, respectively. Values of Km, temperature coefficients (Q10) and activation energy (Ea) were found to be 1.42, 1.25 mM; 1.7, 1.5 and -1 44.7, 40.6 kJmol for AP I and AP II, respectively. Out of the tested detergents viz.; Sodium Lauryl Sulphate (SLS), Cween-60, Tween-80 and Tween-20; SLS acted as strong inhibitor of both; AP I and AP II, with -4 -4 inhibition constants (Ki) 7.6 x 10 M and 2.2 x 10 M, respectively. Various metal ions (salts) inhibited AP I 2+ 2+ 2+ 2+ + 2+ and AP II activity in the order of Mg >Cu >Zn >Ca >Na >Ba and 2+ 2+ 2+ 2+ + 2+ 2+ Cu >Zn >Mg =Mn >Na >Ba >Ca , respectively. The values of inhibition constant (Ki) for first two 2+ 2+ 2+ 2+ -5 -5 potential inhibitors of AP I (Mg , Cu ) and AP II (Cu , Zn ) were found to be 1.7 x 10 , 2.5 x 10 M and




3.7 x 10 , 8.5 x 10 M, respectively. These results suggest that the APs which play an important role in energy transfer and release of inorganic phosphate are strongly affected by certain metal ions and detergents. This study may provide direction in knowing various aspects of APs from very commonly consumed crop plants, their potential in future development of food supplements, lead to a further understanding of their function and may serve to extend the therapeutic application of these enzymes. Above all, the high thermo stability enhances their importance making them industry friendly with good economics. POST 03-268 Investigation Of The Dynamic Amino Acid Networks in a (β/α)8 Barrel Enzyme

Jennifer M. Axe, Kathleen F. O'Rourke, Eric M. Yezdimer, Nicole E. Kerstetter, Xianrui Yuan, David D. Boehr Chemistry, Penn State University, University Park, Pennsylvania, US

Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. We used nuclear magnetic resonance chemical shift correlation analyses to identify longrange amino acid networks in the alpha subunit of tryptophan synthase, both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). Additionally, we compared the networks present in the wild-type enzyme to that of the T183V variant (resulting in the severing of an essential hydrogen bond between the β2α2 and β6α6 active-site loops). The amino acid networks that stretch from the surface of the protein all the way into the active-site are different between the resting andworking states and the loss of the hydrogen bond between the dynamic β2α2 and β6α6 loops results in substantial changes to the network surrounding catalytic residue Glu49. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. The amino acid networks are likely important for coordinating structural changes necessary for enzyme function and regulation. POST 03-269 An Uncommon Phytochelatin Synthase Gives Hints On How To Improve Their Catalytic Efficiency On Heavy Metal Hyperaccumulator Organisms

Jorge D. Garcia1, David G. Mendoza-Cozatl2, Rafael Moreno-Sánchez1 1 Biochemistry, Instituto Nacional de Cardiologia "Ignacio Chavez", Mexico City, Distrito Federal, Mexico, 2Plant Sciences, University of Missouri, Columbia, Missouri, US

Phytochelatin synthase (PCS) produce GSH polymers (phytochelatins, PCs) that bind and inactivate heavy metals. Despite being a very slow enzyme, PCS is essential for cadmium detoxification and 2+ accumulation. Euglena gracilis is an aquatic protist that synthesizes PCs to hyper-accumulate Cd . We have cloned and characterized the recombinant EgPCS, finding significant differences compared to plant PCSases. In contrast to all previous characterized PCSases, EgPCS functions as a monomeric enzyme able 2+ 2+ to synthesize PC2 to PC4, in the presence of Zn , Cd or S-methyl-glutathione (S-methyl-GS). Kinetic analysis of EgPCS suggests a two-substrate reaction mechanism for PC2 synthesis with Km values of 18 mM for GSH and 2 mM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and catalytic efficiency with Zn-GS2 (instead of Cd-GS2). Moreover, its heterologous expression in Cd-sensitive 2+ yeast conferred resistance to Cd . Phylogenetic analysis showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. The EgPCS N-terminal domain contains a typical catalytic core (Cys-70, His-179 and Asp-197), suggesting that the acyl-enzyme intermediary is formed using a GSH molecule, as reported for other PCSases. Our results indicated that the intermediary was exclusively formed with GSH because no activity was observed using g-EC or PC2 as substrate. An EgPCS mutant comprising only the N-terminal 235 amino acid residues


POSTER ABSTRACTS (EgPCS_Nter235aa) was inactive, while the chimera AtPCS_Nter218aa::EgPCS_Cter241aa showed activity with CdGS2 but not with Zn-GS2, suggesting that the Zn-GS2 specificity is conferred by the EgPCS_Nter domain. Additionally, this chimeric enzyme showed an optimal pH (7.5) closer to physiological pH versus optimal pH for AtPCS (8) and EgPCS (≥8). We conclude that EgPCS showed typical features of PCSases (conserved N-terminal domain and kinetic mechanism), but also exhibited distinct characteristics such as preference for Zn-GS2 over Cd-GS2 as co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs. Our results also suggested that the C-ter domain is essential for activity/stability in a monomeric PCS and that biochemical engineering on this domain may lead us to obtain an enzyme with higher catalytic efficiencies under physiological conditions

POST 03-270 Nonlocal Effects Of Metal Ion Binding At The Catalytic Site Of A Protein-DNA Complex.

Kaustubh Sinha1, 2, Michael R. Kurpiewski2, Sahil Sangani1, Andrew D. Kehr1, Gordon S. Rule1, Linda JenJacobson2 1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, US, 2Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, US

Protein- DNA interactions play a critical role in a variety of biological processes. These interactions are important for chromatin structure, gene regulation, DNA repair and modification and also recombination. 2+ Divalent metal ions, especially Mg , are an important cofactor in a large number of nucleic acid processing enzymes (polymerases, nucleases, restriction enzymes, recombinases, transposases etc.). They are not only involved in catalysis (activation of a nucleophile and stabilization of the leaving group) but also make the formation of specific protein-DNA complexes energetically more favorable. Even though the crystal structures of a number of nucleic-acid processing enzymes have been solved, there is a void in our understanding of the effect of metal ion binding on the structure (and dynamics) of protein-DNA complexes. Does metal ion binding affect only the residues close to the metal ion binding site or does it trigger structural and dynamic changes at sites far removed from the binding site? We have used EcoRV, a well characterized restriction endonuclease, as a model to study the effect of metal ion binding on protein-DNA complexes. Binding studies and NMR have been used to characterize the effect of metal binding to the EcoRV-DNA complex. We have shown that lanthanide ions competitively inhibit the cleavage of DNA and occupy sites similar to the divalent ions. Lanthanides are thus excellent candidates for replacing the divalent metal ions to form inactive complexes. We show that each active site can 3+ 3+ 2+ 3+ accommodate 2 smaller Lu or Gd ions (size similar to Mg ) but only one larger La ion (size similar to 2+ Ca ). Chemical shift perturbation studies show that the metal binding induces structural changes at distal regions of the protein. Deuterium exchange and proton exchange studies show that the metal ion binding also causes long range dynamic changes in the complex. POST 03-271 Recognition and Conversion of Flunitrazepam by Cyp3A4 is Altered by Caffeine

Mark Volker, Lauren Sparks, Larry R. Masterson Chemistry, Hamline University, Cambridge, Minnesota, US Flunitrazepam (FNTZ, also referred to as ‘roofies’) is prescribed to treat insomnia due to its strong sedative effects. However these effects can be abused in social settings when FNTZ is administered with predatory intent in alcoholic beverages, resulting in enhanced durations of sedation and amnesia. These beverages often contain a combination of ethanol and the stimulant caffeine, both of which have been shown to interact with the enzyme involved in the metabolism of FNTZ, Cyp3A4. In this study, the steady-state kinetics of Cyp3A4 conversion of FNTZ was measured in the presence of ethanol, ethanol 186

POSTER ABSTRACTS and caffeine, and caffeine alone. In addition, the binding thermodynamics of FNTZ with human Cyp3A4 was measured under these conditions. The rate of formation for the two products of Cyp3A4-mediated catalysis, desmethyflunitrazepam (DM FNTZ) and 3-hydroxyflunitrazepam (3-OH FNTZ), was shown to decrease in the presence of caffeine. Specifically, a 4-fold decrease in the rate of DM FNTZ formation was observed (kcat changed from 1.15 min-1 to 0.27 min-1 in the presence of caffeine), while a small but significant change in the rate of 3-OH FNTZ formation was observed (kcat changed from 3.90 min-1 to 2.87 min-1 in the presence of caffeine). Ethanol, and the combination of ethanol and caffeine, was also shown to diminish Cyp3A4 activity. Changes in the absorbance spectrum for the heme cofactor at the active site of Cyp3A4 were used to probe the binding affinity of FNTZ. The recognition of FNTZ by Cyp3A4 was characterized by a Kd of 22.0 µM. In the presence of caffeine, the absorbance spectra revealed a change in the binding mode of FNTZ to Cyp3A4 and a concomitant increase in Kd to 185 µM. The increase in Kd in the presence of caffeine indicates a decrease in the stability of the complex, possibly due to a reorientation of FNTZ at the enzyme active site, as has been shown with the substrate acetaminophen. Importantly, these factors indicate that product formation occurs at a diminished rate in the presence of caffeine, providing a possible biochemical mechanism for the prolonged effects observed during the misuse of FNTZ. POST 03-272 Fluorogenic Probes for Mycobacterial Esterase Profiling

Katie Tallman, Kimberly Beatty Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon, US

Fluorogenic probes go from a dark to a bright state following enzyme-mediated hydrolysis and provide a rapid readout of enzyme activity. We have developed three novel fluorogenic esterase probes: two derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), and one from 2’,7’-dichlorofluorescein (DCF). Notably, DDAO excites above 600 nm but does not require near-infrared detection capabilities. We spectrally characterized and validated our probes with a panel of commercially available esterases and lipases. All three probes were efficiently hydrolyzed in the presence of enzyme, but resisted spontaneous cleavage in aqueous solution. We used these probes to identify differences in enzyme activity patterns produced by a variety of mycobacterial species, including members of the Mycobacterium tuberculosis (M. tb.) complex. We anticipate that these probes will enable us to annotate the M. tb. proteome under diverse conditions and to identify new diagnostic targets. POST 03-273 Visualizing Cell Interactions With Genetically Encoded Bioluminescent Tools

Krysten A. Jones1, David Li2, Elliot E. Hui2, Mark A. Sellmyer5, Jennifer A. Prescher3, 1, 4 1 Molecular Biology and Biochemistry, Univeristy of California Irvine, Irvine, California, US, 2Biomedical Engineering, University of California Irvine, Irvine, California, US, 3Chemistry, University of California Irvine, Irvine, California, US, 4Pharmaceutical Sciences, University of California Irvine, Irvine, California, US, 5Radiology, University of Pennsylvania, Philadelphia, Pennsylvania, US

Cell-cell interactions underlie diverse physiological processes and disease mechanisms, including immune function and cancer metastases. Despite their critical importance, cell-cell contacts are difficult to examine in live organisms without invasive procedures. Bioluminescence imaging provides adequate sensitivity to examine small numbers of cells in vivo, but lacks sufficient spatial resolution to image microscopic cell contacts. To re-tool this technology for visualizing cellular interactions, we engineered bioluminescent proteins to produce light only when two distinct cell populations are nearby. These probes comprise “split” fragments of Gaussia luciferase (Gluc) fused to the leucine zipper domains of Fos and Jun. When cells expressing the Gluc fragments are in close proximity, Fos and Jun facilitate rapid


POSTER ABSTRACTS assembly of functional Gluc, and thus light emission. The split Gluc reporters were used to repot on cellcell interactions in vitro and in models of metastatic disease. Such studies are enhancing our understanding of cell-cell interactions relevant to human health and disease. POST 03-274 Specific Inhibition of Enolase From Entamoeba histolytica

Normande Carrillo-Ibarra2, 1, Cesar Augusto Sandino Reyes-Lopez2, Jose Correa-Basurto1, Elibeth Mirasol Melendez2, Claudia Guadalupe Benitez Cardoza2 1 Medicine, Instituto Politecnico Nacional, Mexico City, Mexico, 2Biochemistry, Instituto Politecnico Nacional, Mexico City, Mexico City, Mexico Entamoeba histolytica is the enteric protozoan that infects approximately 500 million people worldwide,

of which nearly 50 million become ill, resulting in almost 100,000 deaths annually. Clinical manifestations include diarrhea, dysentery and in some cases, liver, brain or lung abscesses. Metronidazole is an effective therapy, yet some parasites do persist in 40-60% of treated patients. Also, there are some reports indicating the generation in vitro of strains resistant to Metronidazole. These reasons make new therapeutic strategies an urgent need. Enolase is the enzyme that catalyzes the reversible dehydration of 2-phosphoglycerate to phosphoenol-pyruvate, using magnesium as a cofactor. It also participates in different physiopathological processes, revealing enolase as a virulence factor in amoeba, since it has been described as a plasminogen receptor, allowing the cellular invasion of the pathogen. Since E. histolytica depends on glycolysis, enolase is an attractive therapeutic target for rationally designed drugs. Therefore, we sought to determine structural differences between both amoeba and human enolases, using molecular dynamics simulations. Using such differences, we are screening in silico many compounds. Those with the best scores will be probed in vitro to find the best inhibitors. POST 03-275 Effects of Intrauterine and Extrauterine Exposure to 1800 MHz GSM-Like Radiofrequency Radiation on Liver Regulatory Enzymes Activities in one-month-old male New Zealand Rabbits

Nuray N. Ulusu Koç University, Istanbul, Turkey In recent years, people are more exposed to Radiofrequency Radiation (RFR) from raising wireless communication technologies. Public concerns about the possible bio-effects of RFR have risen dramatically over the past two decades and this largely depends on the widespread use of advanced RF devices in people's everyday life. However; intense exposure to RF radiation may negatively affect the vulnerable people of population. Firstly; children have developing and growing tissues and organs, they are more sensitive to non-ionizing radiation impacts than adults. Children absorb more the Radio Frequency (RF)/Microwave (MW) energy in their tissues than adults at Wi-Fi frequencies. Children’s skulls are thinner, their brains are smaller, and their brain tissue is more conductive. Children especially are vulnerable to RF exposure, because of the susceptibility of their developing nervous systems. Radio Frequency (RF)/Microwave (MW) radiation may penetrate children’s heads. Secondly, children have been using mobile phones much earlier and longer than adults. Actually, their exposure has begun as fetus, due to the widely used telecommunication systems during the pregnancy of their mother. With the increment in the daily life usage of telecommunication systems, concerns about the adverse effects of RFR on children in growing age have also been increased. In this study, investigation of the possible bio-effects of RFR on the developing male infants was aimed. A totally thirty-six New Zealand White male rabbits, one- month old, were randomly divided into four groups which are composed of 9 rabbits. 1800 MHz GSM-like RF whole- body exposure for 15 min/day during two weeks was applied to offspring Group II and Group IV in accordance with the experimental protocol that was reviewed and approved by 188

POSTER ABSTRACTS the Laboratory Animal Care Committee of Gazi University. Offspring Group I and Group III were also kept in plexiglas cage under experimental setup for 15 min/day during two weeks but device was switchedoff. The activities of enzymes related with pentose phosphate pathway; glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione-dependent metabolism enzymes; glutathione peroxidase, glutathione reductase, glutathione S-transferase, were measured in liver tissues of young male rabbits. POST 03-276 Imaging Protein-Protein Interactions, Post-Translational Modifications, and Non-Protein Biomolecules by CorrelativeLight and Electron Microscopy

John T. Ngo1, Daniela Boassa2, Stephen R. Adams1, Thomas J. Deerinck2, Sakina F. Palida1, Varda LevRam1, Mark H. Ellisman2, Roger Y. Tsien1, 3 1 Department of Pharmacology, University of California, San Diego, La Jolla, California, US, 2National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California, US, 3Howard Hughes Medical Institute, Chevy Chase, Maryland, US

Electron microscopy (EM) achieves the highest spatial resolution in protein localization and has long been the main technique to image cell structures with nm resolution, but making specific molecules standout for EM has long been challenging. Recently, powerful genetically encoded tags have been introduced that allow specific proteins to be tracked by EM via genetic fusion, in a similar manner to how green fluorescent protein (GFP) is used to track proteins by light microscopy (LM). Tagged proteins are revealed by tag-mediated conversion of 3-3’-diaminobenzidine (DAB) into a localized osmiophilic polymer that is readily distinguished under the electron microscope. Available EM tags precipitate DAB either enzymatically through peroxidase activity or via photo-generated singlet-oxygen. While the peroxidase tag “APEX” and photo-oxidizing protein “mini-Singlet Oxygen Generator” (“mini-SOG”) are powerful tools for “painting” individual proteins, analogous tools for marking biochemical processes, or nonproteinaceous molecular species by EM, are lacking. As complements to the existing EM-tags, we describe two new methods: 1) a novel split-protein complementation system based on miniSOG for visualizing protein-protein interactions and kinase activity, and 2) “Click-EM,” a new method for imaging nucleic acids, lipids, and glycans via bio-orthogonal ligation of photo-sensitizing dyes to functionalized metabolic analogs. Using these methods we have visualized protein-protein interactions in the nucleus at nanometer resolution as well as DNA replication and transcription within full context of cellular ultrastructure. POST 03-277 Evaluating The Role Of Peroxidatic Reducing Substrates In An Unusual Catalase Activity Of Catalase-Peroxidases

Olive J. Njuma1, Elizabeth Ndontsa2, Douglas Goodwin1 1 Chemistry and Biochemistry, Auburn University, AL, Auburn, Alabama, US, 2Chemistry and Biochemistry, Scripps Research Institute, La Jolla, California, US Catalase-peroxidase (KatG) is a bifunctional enzyme that decomposes H2O2 in bacteria and lower eukaryotes. By structure, KatG is highly similar to peroxidases like cytochrome c peroxidase. However, KatG exhibits a catalase activity that rivals that of canonical catalases, an ability no other member of its superfamily possesses. We observed the stimulation of catalase activity by peroxidatic electron donors (PxEDs). These results were unexpected because the prevailing paradigm for this enzyme from its discovery is that the two activities of KatG are mutually antagonistic. The synergestic effect of the PxEDs not only points toward a mechanism for much more efficient detoxification of H2O2, but also substantially broadens the conditions under which such a response can be produced. To evaluate the mechanism by which PxEDs increase KatG catalase activity, we produced variants of the enzyme 189

POSTER ABSTRACTS targeting three novel features of the KatG structure. First, the M255I, Y229F, and W107F variants prevent formation of the KatG–unique MYW covalent adduct and eliminate the enzyme’s catalase activity. We observed that PxEDs were unable to restore any catalase activity to these variants, indicating that PxEDs do not act as electron transfer surrogates in place of the MYW adduct during turnover. Second, R418A targets an arginine residue whose conformation is pH dependent, pointing toward the MYW adduct at pH 7.0 but away from it at pH 5.0. R418A showed greatly diminished catalase activity, but this activity could be stimulated by PxEDs to an extent similar to wild-type. The wild-type, and R418A KatGs showed the same propensity to accumulate inactive intermediates in the absence of an appropriate PxED, but the prevention of inactivation required a much more extensive participation of the PxED for R418A than for wild-type. These data suggested that PxEDs prevented accumulation of inactive intermediates produced as a result of off-pathway intramolecular electron transfer. The W321F variant was produced to block a likely route for off-pathway transfer. This variant showed similar catalase activity to the wild-type enzyme and a similar stimulatory effect of PxEDs, indicating that W321 is not part of a conduit for off-pathway electron transfer events. The implications of our results for understanding the virulence and antibiotic resistance of KatG-bearing bacteria (e.g., Mycobacterium tuberculosis) will be discussed. POST 03-278 Pyrrolysine-Inspired Protein Cyclization

Marianne M. Lee1, Tomasz Fekner2, Jia Lu1, Michael K. Chan1 1 The Chinese University of Hong Kong, Shatin, Hong Kong, 2The Ohio State University, Columbus, Ohio, US

The pyrrolysine technology has been used extensively to produce recombinant proteins containing a variety of "site-specific" modifiable unnatural amino acids for both in vitro and in vivo biochemical studies. In this study, we demonstrated its application to the production of branched cyclic protein with a tadpolelike topology. As a proof of concept, we chose the well-studied RGD tripeptide as our subject of interest and fused it to the C-terminus of the reporter protein, mCherry. We evaluated the cellular uptake efficiency of the cyclized mCherry-RGD, the linear mCherry-RGD, and the wild-type mCherry without the RGD appendage using flow cytometry, and showed that the cyclization of RGD did enhance cellular uptake of its cargo protein. Notably, our pyrrolsyine-inspired approach for cyclized protein production circumvents some of the common limitations encountered by current approaches, such as, the confinement of the functionalities to the two ends of proteins, thus producing only head-to-tail cyclized proteins, the linearization of disulfide-based cyclic proteins in the reducing environment of the cytosol, thereby limiting its targets to those of extracellular. Our proposed approach produces cyclic unit that is formed via an isopeptide bond, which is stable inside the reducing cytosol. Significantly, since the pyrrolsyine analog can be incorporated at virtually any position within the protein sequence, this allows for flexible adjustment of the size of the macrocycle and the length of the sidearms. Given the important role of cyclic peptide in therapeutic development, our approach would have great implication in the preparation of cyclic peptide-containing therapeutics. POST 03-279 Functional Sectors Involved In Thermal Stability And Activity In Beta-Glucosidases

Fabio K. Tamaki2, Larissa C. Textor1, Igor Polikarpov1, Sandro R. Marana2 1 Departamento de Física e Informática, Instituto de Física, São Carlos, São Paulo, Brazil, 2Department of Biochemistry, Institute of Chemistry, São Paulo, São Paulo, Brazil

Functional protein sectors were firstly described in serine proteinases through the identification of physically connected positions that are statistically coupled. Each sector contained single positions


POSTER ABSTRACTS involved in enzyme specificity, catalysis or thermal stability. Here it is shown that functional sectors are also present in beta-glucosidases. Indeed, eighteen statistically coupled positions in beta-glucosidase were changed by alanine and allowed us to identify 2 functional sectors, one involved in thermal stability (as demonstrated by Tm changes) and a second one in catalysis and in specificity modulation (determined by kcat/Km changes associated to structural analysis). Differently of that observed for serine proteinases, the sector involved in thermal stability does not contain physically connected positions, but is concentrated in loops and preferentially occupied by prolines. On the other hand, the sector involved in enzyme activity forms a layer surrounding the active site of the beta-glucosidases, so these statistically coupled positions may modulates activity via direct contacts to the active site residues. Supported by FAPESP and CNPq. POST 03-280 A Chemoproteomic Platform To Quantitatively Map Targets Of Lipid-Derived Electrophiles

Chu Wang1, 2, Benjamin F. Cravatt2 1 Chemical Biology, Peking University, Beijing, China, 2Chemical Physiology, The Scripps Research Institute, La Jolla, California, US

Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods. Here, we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify a select set of proteins that constitute “hot spots” for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxynonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by oxidative stress. POST 03-281 Protein Stabilization And Prevention Of Protein Aggregation By Fungal Sucrase Of Termitomyces Clypeatus And Application In Biotechnology And Biomedical Research

Suman Khowala, Sudeshna Chowdhury, Sanjeeta Tamang, Sangita Majumdar, Rajib Majumder Drug Development Diagnostics & Biotechnology, CSIR-Indian Insitute of Chemical Biology, Kolkata, West Bengal, India

The aggregation and stabilization of proteins is of critical importance in a wide variety of industrial and biomedical situations respectively and can be exploited for efficient performances and treatments of abnormal diseases, such as Alzheimer's and Parkinson's diseases. The sucrase of the filamentous fungus Termitomyces clypeatus was purified and characterized as smallest enzyme in the category known so far. In the fungus Termitomyces clypeatus sucrase was co-aggregated with cellobiase, another glycosidase, in extra- and intra-cellular fractions and affected the catalytic efficiency, stability and conformation of the later. Cellobiase activity decreased after disaggregation from the sucrase in intra and extra-cellular preparations and was regained partially on in vitro addition of purified sucrase when assayed at optimum pH and temperature. Sucrase is a well known industrial enzyme, converting sucrose to glucose and fructose, but this observation is not known for any other sucrase. Our work focuses on the altered organization and aggregation of purified sucrase due to changes in coaggregation other enzyme cellobiase in the fungus for better stability and activity and on in vitro prevention of aggregation of insulin and carbonic anhydrase up to more than 83% by the sucrase. Prevention of protein aggregation


POSTER ABSTRACTS by sucrase was stoichiometric and more pronounced after thermal denaturation of the enzymic activity. In presence of sucrase large soluble aggregates of whey protein constituents were reduced in size. The enzyme also interacted with already-aggregated insulin preparation for resolubilisation, as assayed spectrophotometrically. This is the first evidence of an extracellular fungal sucrase from an edible fungus with such low monomeric size and preventing protein aggregation. The study will be useful to explore new uses of the sucrase with applications in biotechnology and biomedical applications. POST 03-282 Using Multiwavelength Collisional Quenching to Investigate the Effects of Arginine and Inhibitors on inducible Nitric Oxide Synthase

Rachel Jones, Ellis Bell University of Richmond, Richmond, Virginia, US

Nitric Oxide Synthase (NOS) plays a critical role in a variety of signaling pathways and responses to cellular challenge. Although the enzyme is well studied, the role of conformational flexibility in inducible Nitric Oxide Synthase (iNOS) activity has received little attention. To understand the role of conformational flexibility; experiments using multi-wavelength collisional quenching with excitation at 280nm (Tyrosine and Tryptophan) and 295nm (Tryptophan only) were performed with differently charged quenchers (iodide, acrylamide, and cesium) were conducted in the presence and absence of the substrate Arginine asc well as a series of know inhibitors of the enzyme, Aminoguanidine hydrochloride, 1400W dihydrochloride and BYK 191023 dihydrochloride, to determine their effects on tyrosine/tryptophan exposure. The collisional quenching data also suggests that Arginine increases the exposure of tyrosine/tryptophans(non-polar regions) of the protein, consistent with Arginine increasing the flexibility of the protein. A comparison of the effects of a number of inhibitors of iNOS on the multi-wavelength collisional quenching suggest different structural basis for their inhibitory effects. Analysis of the amino acid sequence of iNOS using a variety of bioinformatics approaches suggests that these changes in flexibility could be related to the regulation of disordered regions of the protein. POST 03-283 Enzyme Active Sites May Extend Further Than We Thought

Lisa Ngu, Penny J. Beuning, Mary Jo Ondrechen Chemistry & ChemBiol, Northeastern University, Boston, Massachusetts, US

Understanding how nature designs enzymes to catalyze reactions under mild conditions is an important and intriguing problem. To date the catalytic mechanisms of hundreds of enzymes have been investigated. However it is not always obvious, even with a crystal structure with a bound substrate mimic, which residues contribute significantly to catalysis. We have developed computational methods Partial Order Optimum Likelihood (POOL), a machine learning methodology, and Theoretical Microscopic Anomalous Titration Curve Shapes (THEMATICS) to predict the residues that participate in catalysis. These methods require only the 3D structure of the query protein as input and are based on computed electrostatic and chemical properties. Dynamic conformational changes during catalysis, in addition to electrostatic interactions, allow for coupling between remote residues and the canonical active site residues of an enzyme. This suggests that at least some enzyme active sites are spatially extended, with participation by remote residues in catalysis. Guided by computational predictions and using site-directed mutagenesis and kinetics experiments, we have shown that distal residues play significant roles in the catalytic activity of Ps. putida nitrile hydratase, human phosphoglucose isomerase, and the E. coli Y family DNA polymerase DinB. Here we present new computational predictions and kinetics data for E. coli ornithine transcarbamylase (OTC). OTC is reported to undergo induced-fit conformational changes upon binding carbamoyl phosphate, followed by binding of ornithine. POOL predicts OTC to have an


POSTER ABSTRACTS extended triple-layer active site. Kinetics assays of OTC variants Glu299Gln and Arg57Ala show these POOL-predicted remote residues, located in the second and third layer respectively, are important for catalysis. The use of POOL in the prediction of spatially extended active sites is presented. POST 03-284 Nucleotide Pyrophosphatase/Phosphonuclease Possesses The Zeatin Cis-Trans Isomerase Activity In Vitro

Tomáš Hluska1, Michaela Baková1, René Lenobel2, Marek Šebela2, Petr Galuszka1 1 Department of Molecular Biology, Centre of the Region Haná, Olomouc, Czech Republic, 2Department of Protein Biochemistry and Proteomics, Centre of the Region Haná, Olomouc, Czech Republic

Metabolism of isoprenoid cytokinins has been extensively studied in the past years. However, biosynthesis of cis-zeatin remains unknown. Besides isopentenylation of adenine in tRNA there is another option - cistrans-isomeration. In 1993, a zeatincis-trans isomerase was described, purified to near homogeneity and partly characterised. However, the protein nor gene sequences were not identified yet. 20 years later, we have purified the enzyme using several chromatographic columns and gel chromatofocusing. The protein was identified as putative nucleotide pyrophosphatase/phosphonuclease. After heterologous expression we have confirmed FAD hydrolase (nucleotide pyrophosphatase) and zeatin cis-trans isomerase activities of the enzyme in vitro. Nevertheless its contribution to cytokinin metabolism in planta remains to be elucidated. POST 03-285 Exploring the Trigger for Cooperativity at the Subunit Interface of Malate Dehydrogenase

Jacqunae Mays, Ellis Bell University of Richmond, Richmond, Virginia, US

Malate Dehydrogenase is a homodimeric enzyme which shows negative cooperativity in terms of binding its allosteric regulator Citrate, and is presumed to operate via reciprocating subunit mechanism, dependent upon subunit interactions. To explore the triggers of these subunit interactions we have constructed a number of both subunit interface, and active site mutations and are exploring ligand induced conformational changes using a variety of probes of conformation and conformational flexibility including ANS binding and fluorescence, limited proteolysis, and using positional tryptophan mutants (the native protein lacks tryptophan), multiwavelength collisional quenching techniques with excitation at either 280nm (excitation of tyrosine and tryptophan and a sensitive probe of tyrosine to tryptophan resonance energy transfer) or 295nm (excitation of tryptophan only). Correlation of the data on ligand induced conformational changes with previous kinetic data for various mutants suggests that key residues at the interface relay information from one active site, triggered by interaction of carboxylic acid containing ligands with one of the active site arginine residues, to the adjacent active site and are responsible for the subunit interactions involved in both catalytic activity and regulation of this enzyme. This work was supported by NSF Grant MCB 0448905 to EB POST 03-286 Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity

Yalemi Morales, Damon Nitzel, Owen Price, Shanying Gui, Joan Hevel Utah State University, Logan, Utah, US

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with increased markers of reactive oxygen species in subjects with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs), and is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Reports have shown that DDAH


POSTER ABSTRACTS activity is down regulated and PRMT1 expression is upregulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis and decreased degradation. Here we report that recombinant PRMT1, the major PRMT isoform in humans, is susceptible to oxidation. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues. We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity which is readily reversibly under physiological H2O2 concentrations. We also show that increased PRMT1 activity after reduction correlates with a shift towards a smaller oligomeric state and that one or more cysteine residues are responsible for the effects of oxidation on PRMT1. Our results challenge the unilateral view that increased PRMT1 expression results in increased ADMA synthesis, but rather demonstrate that enzymatic activity can be regulated in a redox-sensitive manner. This also raises the possibility that PRMT1 could be a key player in the cellular oxidative stress response. POST 03-287 Structural and Biochemical Characterization of thielavia Terestriscutinase (Ttc)

Abhijit N. Shirke1, 2, Danielle A. Basore3, 2, Evan Baugh4, An Su1, 2, Glen Butterfoss5, George I. Makhatadze3, 1, 2 , Christopher Bystroff3, 6, 2, Richard A. Gross1, 2 1 Chemistry & Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, US, 2Center for Biotechnology & Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 3Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 4Biology, New York University, New York, New York, US, 5Center for Genomics and Systems Biology, New York University, Abu Dhabi, Abu Dhabi, United Arab Emirates,6Computer Science, Rensselaer Polytechnic Institute, New York, New York, US

Cutinase, an esterase found in phytopathogenic organisms, shows potential as an industrial catalyst owing to its ability to hydrolyze esters in rigid chains such as poly(ethyleneterephthalate) and hindered esters in small molecules. Cutinase produced by the thermophilic fungi Thielavia terestris (TtC) displays a distinct characteristic of being stable and active in acidic conditions as compared to other cutinases from Fusarium Solani (FsC), Aspergillus oryzae (AoC) and Humicola insolens (HiC) which are stable and active in alkaline conditions. In order to rationalize TtC's distinct behavior, a homology model was developed and a thorough structural and biochemical characterization was performed based on computational and experimental analyses. The difference stability at acidic pH was rationalized based on surface charge calculations. With respect to enzyme activity, the optimum pH was found to be acidic and basic for polycaprolactone hydrolysis and cellulose acetate deacetylation, respectively. This difference in pH optima is believed to be due to differences in substrate binding. Also, the presence of glycosylation sites and experimental detection of protein glycosylation provides an opportunity to explore the extent that glycosylation influences the stability and activity of TtC. POST 03-288 Investigating Molecular Determinants that Modulate the Preference of IkBs for Specific NFkBs

James D. Marion, Elizabeth A. Komives Biochemistry, UCSD, San Diego, California, US

Cellular responses to internal and external stimuli are the result of cell signaling pathways that mediate events critical for cell survival. Essential to these cascades are transcription factors, such as NFkB, that regulate cellular activity and gene expression. In mammals, the NFkB family consists of different homoand heterodimers of RelA (p65), p50, p52, c-Rel and RelB, which interact with different inhibitors of NFkBs (called IkBs) that render them transcriptionally inactive. Phosphorylation-induced ubiquitination and subsequent degradation of IkBs liberates NFkB dimers that then translocate to the nucleus, bind DNA and participate in transcriptional activation of numerous target genes. While a great deal of research has


POSTER ABSTRACTS focused on the NFkB family, the way in which cells determine which distinct NFkB dimers are present in the cytoplasm, and are then activated to differentially induce specific target genes, has yet to be elucidated. Collaborative research efforts amongst the Komives, Ghosh and Hoffmann labs have recently collected data suggesting that specific IkB proteins bind preferentially to certain NFkB homo- and heterodimers. These preliminary results led to our development of the “chaperone hypothesis” which proposes that IkBs serve as chaperones, maintaining certain NFkB dimer populations in the cytoplasm. My initial investigations using in vitro affinity purification assays combined with quantitative SDS-PAGE gel analysis have revealed that subunit exchange, between NFkB homo- and heterodimers, occurs readily when an IkB protein is not bound. While this suggests that the IkB proteins are critical for determining the latent pool of NFkB isoforms available for activation, further investigations using NFkB constructs we have developed for fluorescence and fluorescence anisotropy measurements, will quantify the dissociation rates and rates of exchange between specific NFkB dimers with and without IkB proteins present. With these data, combined with future in vivo studies, we hope to provide a greater understanding of the underlying molecular mechanisms that modulate NFkB signaling. POST 03-289 The Examination Of The MALDI-TOF-MS Analysis Of The Proteins And The Peptides By Use Of The Sinapic Acid Derivatives As The New Matrix.

Narumi Hirosawa1, Takeshi Sakamoto2, Yasushi Uemura3, Yasushi Sakamoto1 1 Biomedical Research Center, Saitama Medical University, Saitama, Japan, 2Faculty of Pharmaceutical Scieneces, Josai University, sakado, Japan, 3Exploratory Oncology Research & Clinical Trial Center, Kashiwa, Chiba, Japan

To examine the increase in efficiency of the ionization of the proteins and peptides in matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis, we synthesized 15 kinds of sinapic acid derivatives as the candidacy of the matrix and analyzed about the ability as the new MALDI-TOF-MS analytical grade matrix of these derivatives. Some in these derivatives had unique characteristics respectively comparing with general matrices a -Cyano-4-hydroxycinnamic acid (CHCA), 2,5-Dihydroxybenzoic acid (DHBA) and Sinapic acid(SA). A synthesized derivative (A) 2-Cyano-3-(4hydroxy-3,5-dimethylphenyl)prop-2-enoic acid was possible for ionizing of proteins. Especially, it seemed to be effective by the ionization of large proteins but the ionization of peptide was supposed to be weaker than SA. On the other hand, we found that (B)(2E)-3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2enoicacid, (C)(2E)-3-(2-bromo-4-hydoroxy-5-methoxyphenyl)prop-2-enoic acid and (D)(2E)-3-(2-iodo-4hydoroxy-5-methoxyphenyl)prop-2-enoic showed the sensitivity which is stronger than CHCA on proline containing peptide. However, the ability to ionize proteins was weak. Additionally, we performed Quadrupole ion trap MS-MS analysis of the peptides of which the derivative of these (B), (C) and (D) was the matrix. And, these derivatives showed stronger immonium ion intensity than DHBA. Particularly the iodination derivative (D) had the strongest ability about the occurrence of immonium ion. It seems to be extremely useful to suppose the amino-acid sequence of peptides. POST 03-290 Sequential Phosphorylation of SIKE by TBK1

Hyejin Park, SoHo Kim, Jessica Bell, Ellis Bell University of Richmond, Richmond, Virginia, US

Our recent work (Marion et al. J. Biol. Chem. (2013) 288:18612) has demonstrated that SIKE can be multiply phosphorylated by the protein kinase, TBK1, and that phosphomimetic mutants representing differently phosphorylated SIKE forms inhibit TBK1 utilization of its other substrate, IRF3, with significantly different inhibition constants. To establish whether there is a sequence to the phosphorylation of SIKE by


POSTER ABSTRACTS TBK1, we are investigating the time dependence of site-specific SIKE phosphorylation with WT SIKE and several phosphoknockout SIKE mutants. In time-dependent phosphorylation experiments, SIKE with different levels of total phosphorylation was isolated by ion exchange chromatography and unique phosphorylation sites of the separated isoforms identified by fragmentation and MALDI-TOF or tandem mass spectrometry as appropriate. To determine if SIKE phosphorylation proceeds in a site specific manner, six single site phosphoknockout mutants have been constructed and tested for their ability to act as substrates of TBK1. These studies will establish whether certain phosphorylation sites play a dominant role in subsequent multiple phosphorylation. This work is supported by VCU CCTR Endowment Funds, R21AI107447 POST 03-291 Effects of Phosphorylation on the Structure & Stability of SIKE

Clara Kerckhove, Jessica Bell, Ellis Bell University of Richmond, Richmond, Virginia, US

Our recent studies (Marion et al. J. Biol. Chem. (2013) 288:18612) have indicated that SIKE is a substrate for the protein kinase, TBK1, and can be phosphorylated at multiple sites. No 3-dimensional structures of SIKE or related proteins are available. To investigate the impact of phosphorylation on the structure and stability of SIKE we have used a combination of circular dichroism spectroscopy and multiwavelength collisional quenching to investigate the conformation and flexibility of SIKE and the effects of phosphorylation. Three different length constructs of SIKE (SIKE72-207, SIKE1-112, SIKE 113-207) and several phosphomimetic or phosphoknockout (S6E, S6A, S4A, S2A, S185A) mutants have been constructed and used under native conditions to study the impact of phosphorylation. Effects on stability on native and mutant forms has been investigated by thermal melts monitored at an ellipticity of 222nm, and by guanidine or urea unfolding, followed by fluorescence. This work is supported by VCU CCTR Endowment Funds, R21AI107447 POST 03-292 Multiwavelength Collisional Quenching to Study Ligand Protein Interactions in Glutamate Dehydrogenase

Chun Li, Ellis Bell University of Richmond, Richmond, Virginia, US

Collisional quenching approaches using neutral or charged quencher molecules have long been used to indicate conformational changes in proteins Here we introduce a novel approach to collisional quenching using multiwavelength analysis of spectral data collected at two different excitation wavelengths with a variety of small molecule and protein test fluorophores. The results show that not only can detailed information about the local environment of tryptophans in proteins be obtained (even proteins with multiple tryptophans), but information is derived resulting from tyrosine-tryptophan resonance energy transfer in proteins. Using Acrylamide, Iodide and Cesium collisional quenchers with both tyrosine and tryptophan derivatives and with model proteins with one or more tryptophans we demonstrate the utility of the approach for defining the local environment and changes induced by ligand binding, Furthermore, during the course of these studies we revealed that pi-cation effects disrupt resonance energy transfer between tryptophan (acting as either donor or acceptor) and other moieties in the protein. With Glutamate Dehydrogenase we have used this technique to investigate substrate, cofactor and regulatory ligand interactions and found that ligands which support or promote activity lead to increased flexibility of the protein while ligands that inhibit the reaction have the opposite effect. This work was supported by NSF Grant MCB 0448905 to EB


POSTER ABSTRACTS POST 03-293 Mapping The Binding Sites Of Class D beta-Lactamases For Inhibitor Design And Discovery

Joshua M. Mitchell, Rachel Powers Grand Valley State University, Allendale, Michigan, US

Beta-lactams are the most widely prescribed class of antibiotics in clinical use today, but chronic use and misuse has led to the development of widespread resistance to these chemotherapies. Beta-lactam resistance is conferred through the production of beta-lactamase enzymes, which hydrolyze the lactam ring of these drugs, rendering them inactive. To overcome this resistance, beta-lactamase inhibitors have been developed, but these inhibitors also contain a beta-lactam ring. Due to the chemical similarity that exists between b-lactam antibiotics and b-lactamase inhibitors, the discovery and design of novel, non-blactam inhibitors offers a sensible approach to overcome this resistance. Of special concern are the class D beta-lactamases, which are not inhibited by classic beta-lactam-based inhibitors. To address this issue, a structure-based consensus overlay approach was employed to identify and characterize the binding sites of OXA-1 and OXA-24/40. These maps were constructed based on the X-ray crystal structures of twentyone complexes of these enzymes with different ligands. Several binding site hot spots were identified and used to guide the program DOCK to screen the fragment subset of the ZINC database. Twenty-one compounds were selected and tested from the docking hit list. Four of these show inhibition of the OXA enzymes in the low millimolar range. With further improvement and development, we are poised to develop these compounds into the first clinical inhibitors of class D β-lactamases. POST 03-294 Determining the Effects of MitoNEET on Cellular Dehydrogenase Activity

Sarah Banister1, Matthew Woodruff1, Paige Birge1, Michael Menze2, Mary Konkle1 1 Chemistry, Eastern Illinois University, Charleston, Illinois, US, 2Biology, Eastern Illinois University, Charleston, Illinois, US

MitoNEET is a recently discovered mitochondrial [2Fe-2S] protein that is a binding partner of the antidiabetic drug pioglitazonme. MitoNEET contains a unique three cysteines and one histidine ligation of the metal cluster. However, the cellular function of mitoNEET is currently unknown. Several functions have been proposed including a role in cellular respiration, as an iron-sulfur cluster transfer protein, and as an electron-transport protein. Since each of these functions requires a binding partner, pull-down assays using histidine-tagged mitoNEET and cellular lysates from either mouse liver and HepG2 cells were done to capture and identify protein binding partners of mitoNEET. Proteomic analysis identified the cellular dehydrogenase enzymes of malate dehydrogenase 1 (MDH1) and glutamate dehydrogenase 1 (GDH1) as putative binding partners of mitoNEET. Kinetic analysis of GDH1 and MDH1 in the presence and absence of mitoNEET and pioglitazone was accomplished using spectrophotometric methods. POST 03-295 In Silico And Kinetic Studies To Verify The Potency Of α-Glucosidase Inhibitors Isolated From Morus Alba L.

Shakeel Ahmad1, Akash Chaudhary1, Shadab Ahmad2, Mohd. Tashfeen Ashraf1 1 School of Biotechnology, Gautam Buddha University, Greater Noida, India, 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India

Alpha-glucosidase is a carbohydrate-hydrolase that acts upon α-1,4-glycosidic bonds. It breaks down starch and disaccharides to glucose by hydrolyzing the terminal non-reducing 1-4 linked alpha- glucose residues. Our study aims to verify, throughIn silico and kinetic studies, the efficacy of inhibition of αglucosidase by certain phenolic compounds isolated from the polyphenol enhanced extract of mulberry


POSTER ABSTRACTS fruit (Morus alba L.). Our In silico studies confirmed that out of 25 compounds isolated from mulberry fruit, as reported by Yihai Wang et. al, three of them showed significant enzyme binding, and thereby found to be promising for kinetic studies in order to develop effective inhibitors of α* glucosidase. Correspondence: [email protected] POST 03-296 Molecular Dynamic Studies of the Reductase Domain of Polyketide Synthase from the Myxobacterium Stigmatella Aurantiaca

Andrew J. Schaub1, 5, Jesus Barajas2, Ray Luo2, 3, 4, Shiou-Chaun (Sheryl) Tsai2, 5, 6 1 Biological Sciences (Gateway program in Mathematical, Computational and Systems Biology), University of California, Irvine, Irvine, California, US, 2Molecular Biology & Biochemistry, University of California, Irvine, Irvine, California, US, 3Biomedical Engineering, University of California, Irvine, Irvine, California, US, 4Computer Science, University of California, Irvine, Irvine, California, US, 5Chemistry, University of California, Irvine, Irvine, California, US, 6Pharmaceutical Sciences, University of California, Irvine, Irvine, California, US

Polyketides are a large and diverse family of secondary metabolites, and an important source of natural products that include antibiotics, chemotherapeutics, immunosuppressants and toxins. Polyketides are synthesized from various starter units by polyketide synthases (PKSs). Type I modular PKSs are large megasynthase complexes that follow biochemical instructions, which include polyketide start unit selection, chain length control, cyclizations and postprocessing. Type I modular PKSs contain several catalytic domains, each with its own defined function capable of being reused in multiple catalytic cycles. Myxalamids are a class of potent electron transport chain inhibitors, which are produced by myxobacteria (slime bacteria) using PKSs. Myxalamid S is synthesized by a type I modular polyketide synthase and non-ribosomal peptide synthase (PKS-NRPS) system in S. aurantiaca. Molecular dynamic (MD) studies were performed on the reductase (RE) domain of the PKS. The RE domain is responsible for cleaving a thioester bond, and releasing the polyketide product utilizing the cofactor NADPH as a reducing agent. The RE domain of PKS in S. aurantiaca is hypothesized to perform [2 + 2]e reductions. MD was used to model the reductions, as well as conformational changes in the binding pocket of TE leading to the release of the final polyketide product Myxalamid S. POST 03-297 Mechanisms Regulating Ribosome Biogenesis by AKT and c-MYC

Simone Woods, Colin House, Gretchen Poortinga, Ross Hannan, Katherine Hannan Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia Ribosome biogenesis is an energy consuming process essential for cell growth and proliferation, thereby requiring tight regulation. Recently it has become evident that deregulated ribosome biogenesis can underlie cancer susceptibility, and be specifically targeted in cancer cells [1]. The transcription factor cMYC is a master regulator of ribosome biogenesis. We have reported that c-MYC can selectively transcribe a RNA Polymerase I (Pol I) specific regulon [2]. We recently demonstrated that the kinase AKT also mediates Pol I-driven rDNA transcription to a similar extent as c-MYC [3]. Importantly, maximal activation of rDNA transcription and thus cell growth was achieved through the cooperative activities of both c-MYC and AKT [3]. The mechanism by which AKT alone and in cooperation with c-MYC regulates Pol I transcription remains unknown. We hypothesise that AKT may phosphorylate and subsequently activate c-MYC-regulated Pol I regulon components. We have identified nine out of sixty-six of these components and associated factors as putative AKT substrates based on a high stringency bioinformatics analysis using Motif scan. To test this hypothesis, we immunopreciptated the Pol I complex and associated proteins and used high sensitivity LC-MS/MS to characterise the members of the complex 198

POSTER ABSTRACTS and to identify phosphorylated peptides. We find that endogenous nucleolar protein treacle is present in the Pol I complex and phosphorylated at one of the putative AKT phosphorylation sites identified by Motif Scan. Furthermore, immunoprecipitated treacle can be phosphorylated by purified AKT in vitro demonstrating that treacle is a direct AKT substrate. Consistent with our hypothesis, the treacle gene (TCOF1) is also a c-MYC target [2]. We are currently further validating treacle as a target of c-MYC and AKT and assessing the contribution of increased treacle expression and AKTdependent phosphorylation to the cooperation of AKT and c-MYC in driving Pol I transcription and ribosome biogenesis. POST 03-298 Highly Selective Peptide Substrates For The Assay Of Cysteine Peptidases From The C1 Family

Elena N. Elpidina1, Irina Y. Filippova2, Tatiana A. Semashko1, Elena A. Vorotnikova1, Valeriya F. Sharikova2, Konstantin S. Vinokurov3, Lyndsey Fallis4, Brenda Oppert4 1 Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russian Federation, 2Chemical Department, Moscow State University, Moscow, Russian Federation, 3Entomological Institute, Biology Centre AV ĈR, , Ĉeské Budĕjovice, South Bohemian Region, Czech Republic, 4USDA Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, Kansas, US

Cysteine peptidases are important in many biological processes. In this study, we describe the design, synthesis and use of selective peptide substrates for cysteine peptidases of the C1 papain family. The structure of the proposed substrates can be expressed by the general formula Glp-Xaa-Ala-A, where Glp – pyroglutamyl, A = pNA (p-nitroanilide), AMC (4-amino-7-methylcoumaride) or AFC (4-amino-7trifluoromethyl-coumaride); Xaa = Phe, Val. The synthesis of substrates was enzymatic to guarantee the selectivity of the reaction and provided optical purity of the target compounds, thus simplifying the scheme of synthesis and isolation of products. Hydrolysis of substrates was studied by representative cysteine peptidases from the C1 family: plant enzymes papain, bromelain, ficin, and bovine and human lysosomal cathepsins B and L, respectively. The synthesized substrates were selective for cysteine peptidases of C1 family and were not hydrolyzed by peptidases of other classes: serine peptidases trypsin, α-chymotrypsin, subtilisin Carlsberg, aspartic peptidase pepsin and metallopeptidase thermolysin. The substrates were successfully used to monitor cysteine peptidases activity during the chromatographic separation of a multi-component set of digestive peptidases of a beetle, Tenebrio molitor. The role of the proposed selective substrates for the enzyme characterization in multicomponent mixtures were demonstrated also by inhibitory studies of cysteine peptidases in the midgut extracts from T. molitor larvae, as well as larvae of insect pests of the genus Tribolium. The results of the analysis show that the proposed substrates are superior to other commercially available cysteine peptidase substrates in the evaluation of biologically-active enzymes in complex systems. This work was supported by ISTC grant 3455 and RFBR grants 12-04-01562-а, 12-03-01057-a.


POSTER ABSTRACTS POST 03-299 Collagen Determination and Its Application for the Authentication of Tortoise Shell

Linqiu Li1, Hon-Yeung Cheung2 1 Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China, 2Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China

Tortoise shell has been using as medicine for a thousand of years in China for its benefit of nourishing the yin and suppressing the sthenic yang. Modern research reveals that it enhances kidney functions, invigorates the bone and regulates menstruation. Nowadays, it is commonly utilized as an ingredient in various kinds of functional and tonic food, e.g. tortoise jelly (Gui-ling Gao) and Gui Lu Erxian Jiao. However, some fake tonic food is made from gelatination-food or food gums instead of tortoise shell. Even though this tonic food is made from tortoise shell, the proper species is controversial. Therefore, it is necessary to develop a method for authentication of tortoise shell. Since high content of hydroxyproline (hypro) is commonly found in tortoise shell, we speculate that tortoise shell is in abundant in collagen. Therefore, we intend to differentiate this Chinese medicine by determining and charactering the collagen. In this work, a method for extraction and characterization of collagen in tortoise shell was developed. After a series of treatment (removal of lipid, calcium, and non-collagen), the collagen in tortoise shell was extracted by pepsin (in 0.5 M acetic acid, 1000U/mg) and was purified by 4% NaCl (w/v). Subsequently, the pure collagen was determined and characterized by SDS-PAGE gel and capillary electrophoresis (CE). As a comparison, two collagen-containing materials, chicken skin and chicken tendon, were studied at the same time. It was found that the band pattern of SDS-PAGE gel of Cuora trifasciata (CT), Chinemys reevesii (CR), Curoa flavomarginata (CF), and Trachemys scripta (TS) were almost the same. While one more band appeared between 25 - 37 kD in Cuora Aurocapitata (CA) and Hardella Thurjii (HT), which were not found in other species. Nevertheless, only a few bands (around 100 kD) were seen in chicken skin and chicken tendon. The result of CE manifested that the content of collagen varied from 116.986 in CT to 161.333 mg/g in CF, with a range of extraction efficiency of 63.031% ~ 73.330%. By comparing the ratios of hypro/gly, hypro/pro, hypro/hylys and gly/pro, it was concluded that the ratios of these amino acids in collagen extracted from chicken skin and chicken tendon were distinctly different from that of tortoise shell, which suggested that the established method could be applied for the authentication of tortoise shell. * Content of amino acid was determined by capillary electrophoresis; 1 Standard deviation (SD) was calculated by analyzing at least six of each material with triple replicates (Only adults were studied); 2 Hypro in extracted collagen from tortoise shell; 3 Hypro in tortoise shell raw material; extraction efficiency= (yield of extracted collagen/total collagen) ×100%. c(collagen)=c(Hypro)/13% POST 03-300 The B-Domain-Half-Deleted Recombinant Coagulation Factor VIII (FVIII) Shows Much Higher Coagulation Activity Compared With B-Domain-Deleted Recombinant Ones

Zhang Jun, Zhu Chong-yang, Wen Quan, Lin Ming-ming Department of cell biology & genetics, Chongqing Medical University, Chongqing, China

Human coagulation factor VIII (FVIII) consisting of 2332 amino acid residues is a cofactor for activation of FX in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3-C1-C2. It is previously demonstrated that most of B domain can be deleted and the resulting recombinant protein has essentially normal survival in circulation and corrects the bleeding tendency in hemophilia A patients. Replacement therapy using Bdomain-deleted FVIII is the leading scheme in the management of hemophilia A. However approximately 15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein during long-term treatment. Therefore it is a continuous pursuit of lower immunogenicity, higher efficacy and


POSTER ABSTRACTS longer activity of B-domain-deleted recombinant FVIII in recent decades. But so far much importance is attached to the chemical modification (including with polyethylene glycol (PEG), disulfide bond and excipient) and shorter recombinant FVIII. In this investigation, a longer recombinant FVIII with B-domainhalf-deleted (residues 979 through 1458, designated BDHD-FVIII) was constructed, which was in turn transfected and expressed in Chinese hamster ovary cells and purified according to the standard protocol. The activity of BDHD-FVIII was compared with B-domain-deleted FVIII (residues 751 through 1642, designated BDD-FVIII) and two commercial FVIII products (C-FVIII). The activity of FVIII (FVIII: C) was determined using one-stage clotting assay. Although it was inferred that B-domain didn’t play a crucial role in FVIII functions, the experimental results clearly indicated that HBDD-FVIII showed much higher coagulation activity than BDD-VIII and C-FVIII. Addition of purified von Willebrand factor (vWF) did not significantly alter the coagulation activity for all the tested samples. Highly glycosylation in specific regions of B-domain probably involved in the activation processing and stability of FVIII might be responsible for the results. The investigation implies that moderate length of recombinant FVIII with higher activity could reduce the antibody reactions by minimizing the treatment dosage. From a fresh perspective, the primary evidences promise further search for the optimal regions and lengths in Bdomain to be deleted. Key word: Coagulation factor VIII; Recombinant FVIII; activity of FVIII; vWF POST 03-301 Novel Heterotetrameric Enzymes in Cholesterol Metabolism from Mycobacterium tuberculosis

Meng Yang1, Kip Guja2, Miguel Garcia-Diaz2, Suzanne Thomas1, Nicole Sampson1 1 Chemistry, Stony Brook University, East Setauket, New York, US, 2Pharmacological Sciences , Stony Brook University , Stony Brook, New York, US The genome of Mycobacterium tuberculosis (Mtb) contains a disproportionately large number of lipid

metabolizing genes. The ability of this pathogen to metabolize complex lipids like cholesterol, and the roles that this steroid plays in the virulence and pathogenesis of this pathogen are increasingly evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent acad (fadE) genes that are regulated by cholesterol. We demonstrate that FadE26-FadE27 catalyzes the dehydrogenation of an analog of the 5-carbon side chain cholesterol degradation intermediate. Sequence alignment with ACAD homologs and the crystal structure of FadE26-FadE27 show only two FAD cofactors per tetramer and a single active site per dimer, as previously predicted. This is the first structural report of a heterotetrameric acyl-CoA dehydrogenase. The Mtb igr operon plays an essential role during the latent stage of Mtb infection. Infection of macrophages or mice with an igr deletion mutant (Δigr) results in attenuated growth early in infection. In vitro, the igr operon is required for growth on cholesterol, but not fatty acids, as sole carbon source. Here we report the first structures of a heterotetrameric MaoC-like enoyl-CoA hydratase, ChsH1-ChsH2, which is encoded by two adjacent genes from the igr operon. We demonstrate that ChsH1-ChsH2 catalyzes the hydration of a steroid enoyl-CoA, a 3-carbon side chain cholesterol degradation intermediate, in the modified β-oxidation pathway for cholesterol side chain N degradation. The ligand-bound and apoenzyme structures of ChsH1-ChsH2 reveal an unusual, modified hot-dog fold with a severely truncated central α-helix that creates an expanded binding site to accommodate the bulkier steroid ring system. The structures show quaternary structure shifts that accommodate the four rings of the steroid substrate and offer an explanation for why the unusual heterotetrameric assembly is utilized for hydration of this steroid. These unique a2b2 heterotetrameric architectures, absent in humans, provide an opportunity to develop new anti-mycobacterial drugs that target an important steroid metabolic pathway highly specific to Mtb.


POSTER ABSTRACTS POST 03-302 Energetic Contribution of n→π* Interactions to PII Conformations of Model Peptides

Liu He, Zhengshuang Shi School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, Hubei, China

Recent lines of evidence indicate that the backbone conformation of unfolded peptides is predisposed to the polyproline II (PII) conformation, a structure with backbone dihedral angles (φ,ψ) centered at (-75°, [1] +145°) . What interactions are responsible for the predominance of PII in unfolded peptides remains a poorly answered question. Recent lines of evidence from statistical surveys and theoretical calculations * suggest that the n-π interaction, together with other noncovalent interactions, might help to define and [2,3] modulate protein structure . In this interaction, the lone pair on the oxygen of a peptide carbonyl group * overlaps with the antibonding orbital (π ) of the carbonyl group in the subsequent peptide bond. The n* [3-5] π interaction was suggested to play roles in several fundamental structural elements in proteins . In * [6] particular, n-π interactions were suggested to stabilize PII conformation . We sought to examine the * effects of n→π interactions on PⅡconformation in short model peptides: Ac-aa-NMe2, Ac-aa-OMe (aa=Ala, Val, Phe, Leu, Ile, Pro). We reason that substitution on the donor or acceptor group would shift * * the electronic delocalization, therefore the strength of an n→π interaction. If n→π interactions indeed stabilize PⅡ conformation, population of PⅡ will shift upon changing the strength of the interaction; observation of no effect on PⅡ population with changing strength of the interaction would reveal that * * n→π interactions have neglectable effects on PⅡ conformation. Our data show that the n→π interactions can stabilize PⅡ conformation. Reference: [1] Shi, Z. S.; Chen, K.; Liu, Z. G.; Kallenbach, N. R. Chem. Rev. 2006, 106, 1877−1897. [2] Jia-Cherng Horng; Raines, R. T. Protein Sci. 2006, 15, 74-83. [3] Hinderaker, M. P.; Raines, R. T. Protein science. 2003, 12, 1188-1194. [4] Choudhary, A.; Gandla, D.; Krow, G. R.; Raines, R. T. J. Am. Chem. Soc. 2009, 131, 7244–7246. [5] Bartlett, G. J.; Choudhary, A.; Raines, R. T.; Woolfson, D. N. Nature chemical biology, 2010, 6, 615-620. [6] Hodges, J. A.; Raines, R. T. Org. Lett. 2006, 8, 4695–4697

POST 03-303 The Secretion And Expression Of A Series Of B Domain Truncated Recombinant Coagulation Factor VIII In Hepatocyte

Zhang Jun, Zhu Chong-yang, Zhang Qian-ying, Lin Ming-ming, Wen Quan, Li Xin Department of cell biology & genetics, Chongqing Medical University, Chongqing, China

The B domain of the coagulation factor (FVIII) is believed to be important for processing, intracellular transport and secretion of FVIII protein, but the specific regions for these functions still remains mysterious so far. The investigation aims to preliminarily determine these regions by comparing the secretion and expression of five B domain truncated variants with full-length wild type FVIII (WT FVIII). Firstly, six recombinant vectors were contrusted and transiently expressed in hepatocyte (HL-7702 cell line) that don’t express endogenous FVIII. Media and cell lysates were collected after 48 hours following tranfection. The secretion and expression of the five B domain truncated variants were analyzed in comparison to wild type FVIII construct. All six recombinant FVIII constructs were highly expressed in hepatocyte without significant differences at mRNA level. But the levels of FVIII antigens (FVIII: Ag) in media exhibited markedly differences for the four recombinant constructs with truncated regions of 840950aa, 950-1200aa, 1200-1400aa and 1400-1642aa respectively. Only one variant 751-840aa truncated recombinant FVIII demonstrate equivalent relative antigen level, which suggested that the region of 740840aa in B domain seems not to be involved with the FVIII protein transport and extracellular secretion process. All the remaining regions of B domain could, more or less, be associated with the posttranslational transport or secretion and the inferred number of glycosylation sites in each truncated


POSTER ABSTRACTS region show no obvious correlation with the abilities of FVIII protein transport and secretion. Key words: Coagulation factor FVIII; expression; secretion; B domain truncated constructs

POST 03-304 Crystal Structure Studies Of Dipeptidyl Aminopeptidase BII From Pseudoxanthomonas Mexicana WO24.

Yasumitsu Sakamoto1, Yoshiyuki Suzuki2, Ippei Iizuka1, Chika Tateoka1, Saori Roppongi1, Mayu Fujimoto1, Koji Inaka3, Hiroaki Tanaka4, Mika Masaki5, Kazunori Ohta5, HIrofumi Okada2, Takamasa Nonaka1, Yasushi Morikawa2, Kazuo T. Nakamura6, Wataru Ogasawara2, Nobutada Tanaka6 1 Department of Structural Biology, Iwate Medical University, Yahaba, Iwate, Japan, 2Department of BioEngineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan, 3Maruwa Foods Inc., Yamatokoriyama, Nara, Japan, 4Confocal Science Inc., Chiyoda-ku, Tokyo, Japan, 5Japan Aerospace Exploration Agency, Tsukuba, Ibaraki, Japan, 6School of Pharmacy, Showa University, Shinagawa-ku, Tokyo, Japan

Dipeptidyl aminopeptidase cleaves a penultimate positioned peptide bond from the NH2 terminus of peptides. Some asaccharolytic pathogens are known to utilize peptides or protein as a carbon/energy source. Therefore, these microorganisms secrete a variety of peptidases that involved in peptide metabolism. Especially, Porphyromonas gingivalis is known to utilize dipeptides, instead of free amino acids. Thus, dipeptidyl aminopeptidases are very important from the point of view of dipeptides production. The peptidase family S46 that contains dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24 is the only exopeptidase family in clan PA peptidases. DAP BII exhibits a broad specificity for hydrophobic and basic amino acids on P1 position of substrate peptide. Other enzymes that belong to this family are DPP7 and DPP11 from Porphyromonas gingivalis, DPP11 from Porphyromonas endodontalis (periodontal pathogen) and DPP11 from Shewanella putrefaciens (multidrug resistance associated opportunistic pathogen). Moreover, as family S46 peptidases are not found in mammals, we expect our study will be useful for the discovery of specific inhibitors to family S46 peptidases from these pathogens. Our present phylogenetic and experimental studies indicated that the catalytic triad of DAP BII is composed of His 86, Asp 224 and Ser 657 and implied that unknown large helical domains involved in exopeptidase activity[1]. However, threedimensional structure of family S46 peptidases has not yet been reported. Thus, the crystal structure of DAP BII is essential not only to understand the catalytic mechanism of family S46 peptidases but also to clarify the structural origin of the exo-type peptidase activities of these enzymes. Recently, we successfully crystallized DAP BII and collected X-ray diffraction data to 2.3Å resolution from the crystal. This crystal belongs to space group P212121, with unit-cell parameters a = 76.55, b = 130.86, c = 170.87Å . Crystal structure analysis by the multi-wavelength anomalous dispersion method is in progress [2]. Here, we report the first crystallization and structural analysis of DAP BII from P. mexicana WO24 as family S46 peptidases. [1] Suzuki et al., Scientific Reports, 4 4292 (2014) [2] Sakamoto et al., Scientific Reports, 4 4977 (2014) POST 03-305 Protein Recognition by Multivalent Fluorescent Molecular Sensors

David Margulies Organic Chemistry, Weizmann Institute, Rehovot, Israel

Fluorescent molecular sensors have become valuable tools in the analytical biosciences owing to their high sensitivity and their ability to track proteins in their native environment. A major limitation in using


POSTER ABSTRACTS these probes, however, is the lack of a general and easily applicable method for providing them with high selectivity and high signal-to-noise ratios. In addition, many of these sensors are designed according to the "lock and key" paradigm; therefore, they cannot be used to analyze biomolecule combinations. In our 1group, we aim to address these problems by developing novel classes of fluorescent molecular sensors. 5 I will present our recent development of fluorescent switches that light up in the presence of specific enzyme biomarkers, as well as probes that can shed light on analyte combinations in biofluids. In addition, the design of a sensory system that utilizes both specific and non-specific interactions for distinguishing between protein isoforms will be discussed.References (1 Rout, B.; Unger, L.; Armony, G.; Iron, M. A.; Margulies, D. Angew. Chem. Int. Ed. 2012, 21, 12477. (2) Rout, B.; Milko, P.; Iron, M. A.; Motiei, L.; Margulies, D. J. Am. Chem. Soc 2013, 135, 15330. (3) Rout, B.; Motiei, L.; Margulies, D. Synlett, 2014, In press, DOI 10.1055/s-0033-1340639. (4) Motiei, L.; Pode, Z.; Koganitsky, A.; Margulies, D. Angew. Chem. Int. Ed. 2014, In press, DOI: 10.1002/anie.201402501R201402501 (5) Unger-Angel, L.; Rout, B.; Motiei, L.; Margulies, D. 2014, Submitted. POST 03-306 Assessing Student Understanding of Foundational Concepts of Protein Structure and Function

Ellis Bell University of Richmond, Richmond, Virginia, US

In recent years there have been a number of reports and publications emphasizing the importance of student understanding of foundational concepts and core competencies in the context of both disciplinary and interdisciplinary content and skills. For biochemistry and molecular biology the content knowledge, skills and allied fields necessary for the discipline have been discussed in a series of recent papers. Protein Structure and Function was identified as one of the five essential content areas that students must understand. In the current work, principles of scientific teaching and backward design have been used to align six critical components of understanding protein structure function relationships with focused assessment of student understanding and potential teaching strategies. Of the six, two align with key features of understanding structure (Bonding & Dynamics), two with understanding biological function (Catalysis & Regulation) while two (Interactions & Evolution) clearly bridge the other concepts. Integrated with these core concepts of protein structure and function are the essential interdisciplinary concepts of Modularity, Energy, Change over time, Stochasticity and the Use of Mathematical models. Using a modified Bloom’s taxonomy Potential assessments aligned to each of the six foundational concepts of protein structure and function are discussed. In addition to assessing student understanding of the foundational concepts of protein structure and function the goal of such assessments is to incorporate key aspects of both the allied fields and the skills necessary for student success in biochemistry and molecular biology. Finally. potential student centered teaching strategies for introductory, gateway or capstone courses are suggested. This work was funded by NSF RCN-UBE Grant 0957205 - Ellis Bell, Principal Investigator POST 03-307 Dietary Condensed Tannins And Their Effect On Microbial Protein And Theoretical Methane Yield In Ruminal Fermentation On Cattle In The Northern Mexico

Ericka Fabiola Luisillo-Quiñones2, Gerardo A. Pámanes-Carrasco1, 2, Christian A. Hernández-Vázquez2, Zaira J. Romo-Astorga2, Esperanza Herrera3 1 UPIDET, Technological Institute of Durango, Durango, Mexico, 2Ingeniería en Tecnologías Ambientales, Universidad Politécnica de Durango, Durango, Durango, Mexico, 3FMVZ - UJED, Durango, Durango, Mexico


POSTER ABSTRACTS The purpose of this research was to evaluate the effect of dietary condensed tannins (CT) on microbial protein, volatile fatty acids (VFA) and theoretical methane yield in rumen. Four rumen fistulated steers were used in a duplicated 4x4 Latin square design in 21-d adaptation periods. Measured dietary CT were -1 62.93, 77.11 and 114.92 µg g of DM intake for T1, T2 and T3, respectively. Steers were fed twice daily and ruminal samples were obtained before feeding (0h) and every 4h until 12h. Methane proportion (mmol/mol VFA) was calculated from acetate, propionate and butyrate concentrations. Ruminal pH had no significant differences (p 500 residues), multidomain proteins. Luciferase is known to fold co-translationally and require chaperones for refolding but its folding trajectory and the role of chaperones remain unknown. Our results from single-molecule stretching and refolding experiments in the absence and presence of ATP and luciferin ligands, corroborated by computer simulations show that partial unfolding of the C-terminal residues of Luciferase - with the N-terminal domain remaining folded - allow the entire protein to robustly refold. However, complete unfolding causes Luciferase to get trapped in mechanically stable non-native configurations that prevent refolding, and that refolding can be rescued by adding chaperones. These results indicate that the misfolded states only occur when C-terminal and N-terminal unfolded residues interact. This suggests that co-translational folding is effective because it prohibits the N-terminal residues from exploring the configurational space jointly with the C-terminal residues, and its vectorial nature allows the N-terminal domain to fold first. This avoidance mechanism catalyzes then folding of the middle and the C-terminal domain in Luciferase. Chaperones are not needed when only the C-terminal residues are unfolded suggesting that they specifically assist refolding the N-terminal domain to help the folding pathway avoid the kinetic traps.


POSTER ABSTRACTS POST 07-395 Direct Measurement Of The Multimer Stabilization In The Mechanical Unfolding Pathway Of Streptavidin

Zackary N. Scholl, Piotr Marszalek Duke University , Durham, North Carolina, US

Understanding the stability of a protein monomer within a multimer is crucial to the development of biopolymers and the development of protein cages for drug delivery. Here, we use single-molecule force spectroscopy (AFM-SMFS) to measure the unfolding force of a single monomer of Streptavidin and also measure the unfolding force of a monomer within both a tetramer and a dimer. The unfolding force directly relates to the folding stability, and we report two main findings regarding the stability of a monomer of Streptavidin: that is stabilized by 40% upon dimerization, and that it is stabilized an additional 24% upon tetramerization. We also find that biotin increases stability by another 50% and that the protein-ligand complex, rather than being strengthened through intramolecular contacts, is strengthened due to the contacts provided by the biotin-binding loop that crosses the interface between the dimers.

POST 07-396 ATPase domain of DnaK, Escherichia coli Hsp70 Molecular Chaperone, Experiences pH-dependent ATPase Activity Upon Linker Binding Due to Asp194 and Glu171

Rahmi Imamoglu, Umut Gunsel, Bulent Balta, Gizem Dinler-Doganay Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey

Hsp70 is a highly conserved molecular chaperone that play significant role in variety of cellular activities, such as de novo folding of newly synthesized proteins, refolding of misfolded proteins, protein trafficking and translocation to organelles. DnaK,Escherichia coli homolog of Hsp70 molecular chaperone, consists of two domains; an N-terminal ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD), which are connected by highly conserved hydrophobic linker. Conformational changes brought about by substrate binding to SBD causes NBD to adopt a conformation for efficient ATP hydrolysis and in the reverse direction ATP binding allows fast on and off rates for substrate. Previous studies showed that 389 392 allosteric communication between two domains of DnaK is provided by the conserved VLLL sequence on the linker region. In the presence of linker, DnaK(1-392), pH-dependent higher ATPase rates are observed, which mimics the substrate-stimulated activity of full-length protein, whereas in the absence of linker, DnaK(1-388), behaves similar to the substrate-free unstimulated-form of the full-length. However, it has still not been revealed which amino acids are important in the allosteric mechanism underlying the linker binding effects to the ATPase domain. In this study, with molecular dynamic simulations, we found out that the pH-dependent ATPase activity upon linker binding could be related to the well identified 2+ catalytic residues, Glu171, Asp194 and Asp 201, which participate in localization of Mg ion. Further pHdependent ATPase activity measurements revealed that the alkaline arm of the bell-shaped activity is caused by a late deprotonation of Asp194 (almost 5 pH units increased pKa is observed for this Asp than to that of the expected pKa of Asp), upon its susbstitution with Ala, alkaline site of the bell shape was completely lost. In addition, when we mutated Glu171 to alanine we observed that the linker-bound form of the ATPase domain did not reveal any pH-dependence. We think that linker-bound state of the ATPase


POSTER ABSTRACTS domain experiences a pH-dependent ATPase activity due to a proton transfer reaction taking place during catalytic activity and this is only occurring with linker tucking onto the ATPase domain.

Poster Session: Protein Evolution POST 08-397 Structural Evidence for Antigen Receptor Evolution

Romain Rouet1, David Langley1, 2, Daniela Stock2, Daniel Christ1 1 Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia, 2Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia

The adaptive immune system of jawed vertebrates pre-emptively enables specific responses to pathogens and cultivates memory of past encounters. Key components of this system are the B- and T- lymphocytes and their “rearranging” heterodimeric receptors; the B-cell receptor (the soluble form being the canonical “antibody”) and the T-cell receptor. Both are built from the same building block, the immunoglobulin (Ig) domain fold. The phylogenetic pathway for the emergence of these receptors is unclear as they are absent in the genomes of modern jawless vertebrates (hagfish and lampreys), but are present in their jawed cousins, the sharks and rays. Hence, their emergence is believed to have occurred when these chordate lineages diverged, some 500 million years ago. The modern heterodimeric Ig receptors are proposed to have evolved from primordial homodimeric receptors through processes of gene duplication and diversification. We have used laboratory evolution to reconstruct a homodimeric proto-receptor type molecule and investigate how such a symmetric receptor is capable of interacting with protein antigens. Crystal structures of two such Ig receptor-antigen complexes reveal that both of the Ig halves contribute to an extensive interface capable of high affinity interactions with asymmetric targets. Symmetry mismatch is accommodated either by the utilization of different side chain rotamers within otherwise identical complementarity determining regions (CDRs) (conformational plasticity), or by context-dependent differential use of CDRs (selective recruitment). These data provide structural evidence for the evolutionary origins of modern Ig-based antibodies and receptors and support the thesis that these molecules arose from simple Ig-domain precursors. POST 08-398 Computational Prediction And Experimental Characterization Of A “Size Switch Type Repacking” During The Evolution Of Dengue Envelope Protein Domain III (ED3)

Montasir Elahi1, 4, Keiichi Noguchi2, Masafumi Yohda1, Hiroyuki Toh3, Yutaka Kuroda1 1 Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo, Japan, 2Instrumentation Analysis Center, Tokyo University of Agriculture & Technology, Koganei-shi, Tokyo, Japan, 3Computational Biology Research Center, AIST, Tokyo, Japan, 4Dept. of Neurology, Graduate School of medicine, Juntendo University, Tokyo, Japan

Dengue viruses (DEN) are classified into four serotypes (DEN1-DEN4) exhibiting high sequence and structural similarities, and infections by multiple serotypes can lead to the deadly dengue hemorrhagic fever. Here, we aim at characterizing the thermodynamic stability of DEN envelope protein domain III (ED3) during its evolution, and we report a structural analysis of DEN3 and DEN4 ED3 combined with a systematic mutational analysis of residues 310 and 387. Molecular modeling based on our DEN3 and 310 387 DEN4 ED3 structures indicated that the side-chains of residues 310/387, which are Val /Ile and 310 387 Met /Leu in DEN3wt and DEN4wt, respectively, could be structurally compensated, and that a “size


POSTER ABSTRACTS switch type repacking” might have occurred at these sites during the evolution of DEN into its four serotypes. This was experimentally confirmed by a 10 ºC and 5 ºC decrease in the thermal stability of, 310 387 310 387 310 387 respectively, DEN3 ED3 variants with Met /Ile and Val /Leu , whereas the variant with Met /Leu , which contain a double mutation, had the same stability as the wild type DEN3. Namely, the Met310Val mutation should have preceded the Leu387Ile mutation in order to maintain the tight internal packing of ED3 and thus its thermodynamic stability. This view was confirmed by a phylogenetic reconstruction 310 387 indicating that a common DEN ancestor would have Met /Leu , and the intermediate node protein, 310 387 310 387 Val /Leu , which then mutated to the Val /Ile pair found in the present DEN3. The hypothesis was further confirmed by the observation that all of the present DEN viruses exhibit only stabilizing amino acid pairs at the 310/387 sites. POST 08-399 Evolutionary Exploitation of Promiscuous NSAR/OSBS Enzymes

Andrew McMillan, Denis Odokonyero, Mariana Lopez, DaNae Woodard, Ashley Brizendine, Margaret E. Glasner Biochemistry and Biophysics, Texas A&M University, College Station, Texas, US Many enzymes are promiscuous, catalyzing non-biological side reactions in addition to their normal

reactions. New metabolic pathways are thought to evolve by recruiting promiscuous enzyme activities. We are evaluating this hypothesis using one of the few natural pathways that is known to use a promiscuous enzyme. This enzyme’s ancestral activity was o-succinylbenzoate synthesis (OSBS), which is a step in menaquinone synthesis. N-succinylamino acid racemization (NSAR) evolved in one branch of the OSBS family, and it is used in a pathway to convert D-amino acids to L-amino acids in Geobacillus kaustophilus. How does a single active site catalyze two reactions? Promiscuous NSAR/OSBS enzymes differ from nonpromiscuous OSBS enzymes in three ways: orientation of substrate binding, quaternary structure, and identity of catalytically important active site residues. Second, how has the specificity of promiscuous NSAR/OSBS enzymes evolved? Early-branching enzymes whose biological function is OSBS have minimal NSAR activity, while late-branching enzymes whose biological function appears to be NSAR have substantial OSBS activity. This suggests that positive selection increased the efficiency of the NSAR reaction, but there is little disadvantage to retaining the ancestral activity. Third, how were metabolic pathways that use NSAR activity assembled? The operon encoding the known pathway in G. kaustophilus is poorly conserved. Instead, gene fusions and operons of some NSAR/OSBS genes suggest that more than one metabolic pathway evolved to use NSAR activity. NSAR/OSBS genes were acquired by diverse species through horizontal gene transfer, providing a mechanism for introducing NSAR/OSBS genes into a variety of operons and gene fusions. POST 08-400 Active Site Profile-Based Clustering Of Enolase Structures And Sequences

Janelle Leuthaeuser1, Stacy Knutson1, Brian Westwood1, Patricia Babbitt2, Jacquelyn Fetrow1 1 Wake Forest University, Winston Salem, North Carolina, US, 2Pharmaceutical Chemistry, University of California - San Francisco, San Francisco, California, US

The elucidation of protein molecular function lags far behind the rate at which protein sequences are identified; accurate and efficient computational methods that cluster protein sequences in functionally relevant ways are essential. Active site profiling was previously developed to identify and compare details of protein molecular functional sites. The program DASP utilizes a profile-based approach to search sequence databases for proteins containing similar functional sites to the input proteins. The Structure Function Linkage Database (SFLD) contains enzyme superfamilies whose functional subgroups and families have been identified by expert curation. Therefore, validation that DASP can identify discrete


POSTER ABSTRACTS functionally relevant groups corresponding to SFLD-curated groups would provide the foundation for development of accurate and efficient computational methods to functionally cluster the protein sequence universe. To validate the approach, active site profiles were created for each subgroup and family in the well-studied enolase superfamily and used to search the PDB sequences. Results demonstrate high correlation between DASP grouping and SFLD annotation, suggesting that profiling could be used in a process to identify functionally relevant clusters, with no a priori knowledge of those clusters. Such a process, TuLIP, was developed and applied to the enolases, producing 23 groups that correspond well with SFLD subgroups and families. Profiles of these groups were used to search Genbank, and over 15,500 enolase superfamily members were identified and assigned to one of the 23 functionally relevant, discrete clusters. Continued validation and automation of this method could provide a necessary tool to automatically cluster proteins into functionally relevant groups. POST 08-401 Active Site Profile-Based Clustering Of The Peroxiredoxin Superfamily

Angela Harper, Janelle Leuthaeuser, Jacquelyn Fetrow Wake Forest Univeristy, Downingtwn, Pennsylvania, US

Protein function is often predicted based on sequence similarity, leading to misannotations and propagation of false classification of proteins. The Structure Function Linkage Database (SLFD) is considered by many to be the gold standard for protein annotation due to manual curation. However, this process is expensive and time-consuming, so automated processes for annotation and functional clustering are imperative. The Peroxiredoxin (Prx) superfamily contains cysteine-based peroxidases that react with hydrogen peroxide, organic peroxides, and peroxynitrite, and have been extensively studied by experts making this an appropriate baseline. Using the Prx superfamily, parameters for determining discrete and functionally relevant groups of proteins within a superfamily were defined. The Deacon Active Site Profiler (DASP), previously developed by our lab, extracts residues around an active site environment of a protein, creating its unique signature. Using DASP, our lab recently developed a process called TuLIP which functionally clusters all structurally characterized members of a superfamily. The Prx superfamily was split into four distinct TuLIP groups and the signatures from DASP for each group are aligned and GenBank is searched for proteins with similar active site features. This is an iterative process which uses the GenBank searches to gather information about all Prxs. The results of this process were mapped onto a representative network of all members of the Prxs, showing that our method classified a majority of the superfamily. This process created discrete groups in which no protein is assigned to two different groups at significant scores. Additionally, TuLIP groups containing multiple subgroups were split into the appropriate functionally relevant subgroups as defined by the SFLD. This method of functional clustering is both accurate and automatable, which will aid future efforts to create a fully automated process for annotating further superfamilies. POST 08-402 Bioinformatics and Network Analysis of Lipocalin Superfamily

Nardos Sori, Lesley H. Greene Old Dominion University, Norfolk, Virginia, US

Lipocalins are a superfamily of proteins that are found in various organisms with a wide range of functions such as pheromone activity, lipid transport and coloration. Lipocalins share a common three dimensional structure which consists of an antiparallel β-barrel and a C-terminal α-helix. Even though lipocalins are found widely in nature, it was only recently that proteins in Wheat and Arabidopsis were identified as true lipocalins through the elucidation of three structurally conserved regions. The study of these lipocalins is vital as these proteins are believed to help plants tolerate oxidative stress and extreme conditions which


POSTER ABSTRACTS broadens our understanding of plant sustainability in different environments. We focused on Triticum aestivum temperature induced lipocalin (Tatil), found in wheat, an essential crop that is used by the

majority of the world population. We conducted bioinformatics studies and a network analysis of lipocalin superfamily which sheds insight into the structure, function and evolutionary relationships of the Tatil. We made structure-based sequence alignment of lipocalin superfamily which enabled us to calculate conservation of the amino acid positions and elucidate a conserved long range interaction network. We also made a model of the temperature induced lipocalin which gives us an idea of its interaction with the cell membrane. POST 08-403 A New Evolutionary Subclass of HMG-CoA Reductases

Jeffrey Watson, James Palmer Chemistry and Biochemistry, Gonzaga University, Spokane, Washington, US

Isopentenyl diphosphate (IPP) is a key precursor to a diverse range of biomolecules required by all three kingdoms of life. These molecules include prenyl groups for membrane attachment of proteins and small molecules, plant hormones, ether-linked fatty acids in archaeal cell walls and sterols. IPP biosynthesis proceeds via one of two known pathways. Eukaryotes, archaea and some eubacteria utilize the mevalonate pathway, which proceeds through the action of HMG-CoA reductase (HMGR) as the ratelimiting and first committed step. Most eubacteria utilize the DXP pathway, which does not require HMGR at any step. HMG-CoA reductase sequences were shown over a decade ago, prior to the explosion in genome sequencing, to fall into two evolutionary classes. Class I includes eukaryotic and most archaeal HMGRs, while Class II includes eubacterial and some archaeal HMGRs. At the time, with a single exception, all of the eubacteria known to express HMGR also expressed the other enzymes of the mevalonate pathway and therefore functioned biosynthetically. In contrast, HMGR from Pseudomonas mevalonii has been shown to function in an oxidative fashion, allowing the organism to grow on mevalonate as its sole carbon source. We have recently identified a group of bacteria that possess the gene for HMGR, yet only have the genes for DXP pathway enzymes and not the mevalonate pathway enzymes. One HMGR from a member of this group (Burkholderia cenocepacia) has been characterized, and also appears to function oxidatively. Multiple sequence alignments have identified some common characteristics of HMGRs from these organisms that suggest they represent a new evolutionary subclass we refer to as Class II Oxidative, or Class IIox. We are employing phylogenetics, whole-genome comparison and protein evolution tools to explore possible physiological roles for Class IIox HMGRs and how this class of enzymes might have evolved.

POST 08-404 Ebola Protein VP35 Exploits Evolutionary Constraints In Host Defense Kinase PKR to Evade Translational Blockade

Maayan Eitan-Wexler, Raymond Kaempfer Department of Biochemistry and Molecular Biology, Faculty of Medicine, The Hebrew University , Jerusalem, Israel

Antiviral defense depends strongly on activation of the RNA-activated host kinase PKR that inhibits translation by phosphorylating initiation factor eIF2α. To replicate, viruses must counteract PKR. The highly lethal Ebolavirus vigorously inhibits innate immune responses through its VP35 protein. We show that VP35 evades host defense by exploiting evolutionary constraints in PKR. PKR kinase activation requires its homodimerization on the activating RNA. VP35 acts as a decoy of activated PKR that binds


POSTER ABSTRACTS PKR, inhibiting its activation by forming a non-productive complex. VP35 mimicks the kinase activation segment of PKR wherein two highly conserved threonines must undergo phosphorylation to enable PKR activation and eIF2α substrate recognition, both replaced in VP35 by phosphomimetic residues. Mutation of these phosphomimetic residues sufficed to abrogate the PKR-antagonist activity of VP35. By mimicking activated PKR, the kinase-dead VP35 protein presents a unique viral strategy to evade hostmediated translational blockade. We thus demonstrate a novel molecular mechanism of immune evasion and show that evolutionary constraints can render a host target protein powerless vis-à-vis a pathogen. POST 08-405 The Substrate Specificity “Lock;” Evolutionary Epistasis In Apicomplexan Lactate And Malate Dehydrogenases

Brian Beckett1, Michelle Y. Fry2, Douglas L. Theobald1 1 Biochemistry and Biophysics, Brandeis University, Waltham, Massachusetts, US, 2Biophysics, Brandeis University, Waltham, Massachusetts, US

The unusual Apicomplexan lactate dehydrogenase (LDH) evolved from a malate dehydrogenase (MDH) via a six amino acid insertion in the active site loop, which shifts specificity from malate to lactate by over twelve orders of magnitude. When these six amino acids are inserted in the active site loop of the modern MDH, all enzyme activity is lost. However, in ancestrally reconstructed MDH the same insertion creates a highly-active bifunctional enzyme. Evolution since the ancestor has resulted in mutations that “lock in” substrate specificity; residues distal to the active site must have non-trivial epistatic interactions that are changing throughout evolution. To determine the nature of the “lock” in substrate specificity, we generated ancestral intermediates along the Cryptosporidium parvum lineage. We cloned, expressed, and purified the ancestors in E. coli., and characterized their substrate specificities using steady-state Michaelis-Menten kinetics. The number of potential residues responsible for epistasis was narrowed using site-directed mutagenesis. Our data suggest that substrate specificity was only recently “locked” in the C. parvum family tree. The epistatic changes during evolution may be dynamic inter-residue interactions, which can not be observed using x-ray crystallography. Therefore, we generated mutant MDH and LDH amenable to nuclear magnetic resonance (NMR) characterization. Preliminary NMR spectra have been obtained for ancestral MDH. Future NMR studies will inform the dynamics of how epistasis has evolved to lock substrate specificity in Apicomplexa.

POST 08-406 Multi-level Iterative Functional Clustering of Glutathione Transferase Superfamily

Kiran Kumar, Janelle Leuthaeuser, Brian Westwood, Jacquelyn Fetrow Physics, Wake Forest University, Winston-Salem, Virginia, US

High throughput sequencing has resulted in a significant misannotation increase within large protein databases such as GenBank. These incorrect classifications are often due to function annotation at a level beyond what the data currently suggests. Relying on manual curation, however, to categorize the high volume of sequence data is neither cost nor time effective. Therefore, it is imperative to develop automated methods for functionally classifying proteins. To meet this need, our lab is developing parameters for a computational, multi-level, iterative searching procedure to functionally cluster protein sequences. Instead of full sequential or structural analysis, this procedure clusters proteins based on the structural and sequential information within the active site. To test the parameters of this procedure, The Structure Function Linkage Database (SFLD) is used as a gold standard because it contains expertly curated proteins. The Glutathione S-Transferase superfamily, in particular, is being used to explore our ability to match SFLD GST subgroup designations. First, the GST structures are clustered into distinct


POSTER ABSTRACTS groups based on active site similarity. The Deacon Active Site Profiler (DASP), previously developed in our lab, searches GenBank using these groups to generate hits of functionally analogous proteins. Hits can be tracked onto a map of all the GST sequences to visually assess the progression of expanding an initial search, which used only structurally characterized GST members, to the mostly uncategorized sequential universe of the entire superfamily. Results have shown that our methods are successful in matching SFLD annotation in the majority of clusters when searching GenBank. Findings also indicate the potential to extend family level annotation to GST proteins, which have not yet been determined by the SFLD. Overall, results demonstrate that current parameters produce functionally relevant GST clusters, which indicates the feasibility of developing an automated, accurate method to functionally cluster protein superfamilies.

POST 08-407 Structural Analysis of Toxin/immunity Complexes in Contact-Dependent Growth Inhibition Systems

Parker M. Johnson1, Robert P. Morse1, David A. Low2, Christopher S. Hayes2, Celia W. Goulding1 1 Molecular Biology and Biochemistry, UC Irvine, Irvine, California, US, 2Molecular, Cellular, and Developmental Biology, UC Santa Barbara, Santa Barbara, California, US

Bacteria have evolved complex strategies to compete and communicate with one another. A new mechanism of interbacterial competition, termed contact-dependent growth inhibition (CDI) was recently 1,2 discovered . CDI systems are found in a wide variety of gram-negative bacteria, including many 1 important human pathogens such as uropathogenic E. coli strain 536 (UPEC536) and Burkholderia pseudomallei3. CDI allows for competitive advantage in the ability to kill neighboring bacterial cells mediated by contact of the CdiA protein with a target cell, where its C-terminal domain (CdiA-CT) is translocated into the target cytosol and inhibits growth of non-cognate target cells. In order to prevent + susceptibility to their own toxins, CDI bacteria express a cognate immunity protein (CdiI) to specifically 2 bind and inactivate CdiA-CT . Burkholderia pseudomallei environmental isolates E479 and 1026b have 3 been shown to utilize CDI mechanisms to outcompete neighboring bacteria , signifying the pathway’s importance in growth and survival. The toxin and immunity proteins between these two isolates share significant sequence identity, and both toxins exhibit tRNase activity; however the toxins have different tRNA specificities along with cleavage sites, and cognate immunity proteins only bind to cognate toxins. We have solved the structures of these two B. pseudomallei toxin/immunity complexes that shed light on toxin specificity as well as the unique toxin/immunity protein interfaces. The CDI system from UPEC536 is unusual, CdiA-CT exhibits toxic tRNase activity only when activated by binding target cell 4 CysK, a permissive factor involved in cysteine biosynthesis . We have solved the X-ray crystal structure of the toxin/immunity/CysK complex to 2.99 Å. We will discuss the interaction of CysK with toxin and its neutralization by the immunity protein, and the functional implications gleaned from this ternary complex. 1. S.K. Aoki et al., Science 309, 1245 (2005). 2. S.K. Aoki et al., Nature 468, 439 (2010). 3. K. Nikolokakis et al., Mol Microbiol. 84, 516 (2012). 4. E.J. Diner et al., Genes & Dev 26, 515 (2012). POST 08-408 Automated Functional Clustering of the Crotonase Superfamily based on active site motif sequences

Julia Hayden, Janelle Leuthaeuser, Brian Westwood, Jacquelyn Fetrow Wake Forest University , New Cumberland, Pennsylvania, US The increasing ease of protein sequencing has lead to heightened use of computational methods utilizing full protein sequences to rapidly classify the growing number of proteins, leading to rampant misannotation in databases using these computational methods. Manually curated databases, such as 256

POSTER ABSTRACTS the The Structure Function Linkage Database (SFLD) have less annotation errors and can be used as a gold standard. One of the superfamilies in the SFLD, the Crotonase superfamily, contain proteins related by conserved peptidic NH groups that mediate the formation of an oxyanion hole. A process formerly completed, termed TuLIP, iteratively clustered 100 non-redundant structurally represented proteins based on active site from the Crotonase superfamily into 22 distinct protein groups through use of DASP, a program written to compare and score similarity between protein active sites. For all structurally characterized proteins that did not cluster with any groups, a method of profile engineering was utilized to take advantage of structural information to find other sequences with similar active sites. The 22 groups were used in isolated searches of NR Genbank via protein active sites to find other functionally similar proteins. Protein hits resulting from these searches were examined by similarity score and on family level based on the SFLD hierarchy, and plotted on a representative network of proteins in the Crotonase superfamily to examine coverage. This search process showed high correlation with the data presented in the SFLD with protein groups highly discreet at a DASP score cut-off of 1E-12 as well as high coverage of the Crotonase superfamily representative network. The results of this process indicate a step forward in creation of an accurate computational method to separate proteins into functionally related groupings. POST 08-409 Domain Exchange Between Membrane And Soluble Proteins Contributes To Expand Intercellular Communication Network

Hyun Jun Nam1, Inhae Kim2, James U. Bowie3, Sanguk Kim1, 2 1 School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea, Republic of, 2Department of Life Sciences, POSTECH, Pohang, Korea, Republic of, 3Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, US

The transition to multicellularity from protozoa marks one of the most pivotal and poorly understood events in life’s history. We hypothesize that membrane proteins must be key players in the development of multicellularity because they are well positioned to form the cell-cell contacts and to provide the intercellular communication required for the creation of complex organisms. We found that a major mechanism for the necessary increase in membrane protein complexity during the transition to multicellularity was the new incorporation of domains from soluble proteins. Moreover, the membrane proteins that have incorporated soluble domains in metazoans are enriched in the functions unique to multicellular organisms such as cell-cell adhesion, immune defense and developmental processes. They also show enhanced protein-protein interaction (PPI) network complexity and centrality, suggesting an important role in the cellular diversification found in complex organisms. Our finding provides empirical evidence for metazoan evolution process that highlights the importance of domain recombination between membrane and soluble proteins to expand intercellular communication network. POST 08-410 Utilization of an Iterative Clustering Method to group Radical SAMs in Functionally-Relevant Ways

Gabrielle B. Shea, Janelle B. Leuthauser, Brian Westwood, Jacquelyn S. Fetrow Wake Forest University, Remsenburg, New York, US Over the past decade, the use of computational techniques has greatly increased the amount of data gathered on protein sequence, structure, and function; however, this data is often poorly analyzed, resulting in a greater prevalence of functional misannotations. These misannotations make it difficult to identify relationships between proteins, and understanding these relationships is highly useful when attempting to identify new candidates for drug therapies. Although manually-curated databases like the Structure-Function Linkage Database (SFLD) are nearly free of misannotations, hand curation is neither 257

POSTER ABSTRACTS cost- nor time-effective. Our lab is developing a Function Annotation Protocol (FAP) that utilizes computational tools to accurately and efficiently classify proteins based on active site information. This iterative pipeline uses structural information to cluster the proteins in an SFLD-defined superfamily into smaller, functionally-relevant groups. This project focuses on the Radical SAMs, a superfamily of proteins for which there are many more known subgroups and families than there are structures. The goal of the project is to use the active site environment to compare proteins and to search databases in order to find and annotate all of the sequences in this superfamily. We have already used the Deacon Active Site Profiler (DASP), a computational tool developed by our lab, to cluster Radical SAM protein structures into groups that can be deemed functionally relevant based on active site similarity. For those structural representatives that didn’t fit in any group, we engineered a group based on proteins with high sequence similarities. However, DASP has not yet hit all of the proteins that the SFLD claims are members of the Radical SAM superfamily because most of the members do not have known structures, and DASP relies on structural inputs. Thus, this project aims to subcluster in the initial results of GenBank searches in order to create more specific groups of proteins that can therefore be used by DASP to pick up the Radical SAM protein sequences that are more distantly related to the original groups of protein structures. POST 08-411 Directed Evolution of Duplicated Qbeta RNA replicases in liposome

Keisuke Uno1, Takeshi Sunami1, 3, Yasuaki Kazuta3, Norikazu Ichihashi1, 3, Tetsuya Yomo1, 3, 2 1 Graduate School of Information Science and Technology, Osaka University, Osaka, Japan, 2Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan, 3YOMO Dynamical Micro-scale Reaction Environment, ERATO, JST, Suita, Osaka, Japan

Gene duplications play an important role in the evolution of novel gene functions. Many research of gene duplications are based on genomic sequence analysis and population modeling. There is a few research of experimental evolution and analysis of its evolutionary process. We have tried experimental directed evolution of duplicated genes in vitro, and analyzed the functional changes of the two genes. “Translation-coupled RNA replication (TcRR) system” (H. Kita, 2008), in which the genetic information (RNA) is replicated by self-encoded replicase, was used as a model reaction for the directed evolution. In this system, the catalytic subunit of Qbeta replicase is synthesized from the template RNA that encodes the protein, and the replicase replicates the template RNA. To detect the RNA replication, we used a reporter RNA (GUS(-) RNA) which encodes an antisense sequence of beta-glucuronidase (GUS) gene. After replicase synthesis in TcRR system, the complementary strand of the reporter RNA (GUS(+) RNA) is synthesized by the replicase, and fluorogenic substrate is hydrolyzed by GUS translated from the GUS(+) RNA . “Liposome-based IVC” (T. Nishikawa, 2012) was used as a method for the directed evolution. Briefly, a DNA library of duplicated and mutated beta-subunit of replicase was compartmentalized into liposomes. After 3 hours reaction, liposomes exhibiting the strongest fluorescence signals were sorted by a fluorescence-activated cell sorter. The genes obtained were amplified in a test tube, and transferred to the second round of screening. The DNA library after 5th selection round (G1R5) showed about 3 times higher reaction activity than original DNA (WILD). For next round, G1R5 was mutated by error-prone PCR to G2R1. G2R9 showed about 6 times higher reaction activity than WILD after 9th selection round. We have showed that the duplicated genes can evolve using liposomes as microreactors.


POSTER ABSTRACTS POST 08-412 Comparative Analysis of Post-translational Modification Hot Spots in Phylogenetically Distant Heterotrimeric G proteins

Shilpa Choudhury, Henry Dewhurst, Matthew P. Torres Biology, Georgia Institute of Technology, Atlanta, Georgia, US G protein signaling (GPS) systems composed of G protein coupled receptor (GPCR) and heterotrimeric G proteins (Gαβγ) are accountable for transducing a myriad of extracellular stimuli including light, neurotransmitters and hormones. Emerging evidence suggests that heterotrimeric G proteins are regulated by a variety of post-translational modifications (PTMs). Recent bioinformatics analyses of mammalian G protein PTMs using an in-house toolkit (Structural Projection of PTMs (SPoP)) shows that Gα, Gβ and Gγ each exhibit PTM “hot spots” within their respective structures. These hot spots are most frequently encountered at GPS protein interfaces or in catalytic regions, suggesting that they play an important role in regulating signaling. Here, we tested the hypothesis that similar hot spots would be modified in the phylogentically distant Saccharomyces cerevisiae, which harbors a single canonical GPS system sensitive to mating pheromones. To test the hypothesis, we identified PTMs on each of the yeast G proteins using affinity purification mass spectrometry (AP-MS). Since yeast G proteins are highly modified under conditions of GPCR activation or nutrient stress, we assayed each G protein under both conditions independently. AP-MS resulted in identification of all modification sites currently known as well as additional sites of modification. Phosphorylation is the predominant PTM found on yeast G proteins, with the Gα subunit (Gpa1) exhibiting the greatest number of identified sites. While Gpa1 was highly phosphorylated at multiple sites under nutrient stress conditions (low glucose), multi-site phosphorylation of Gβ (Ste4) was stimulated upon GPCR activation with mating pheromone. To estimate conservation of structural PTM hotspots between yeast and mammals, we performed predictive structure threading of yeast G proteins, and compiled the AP-MS results into a structural topology. We then surveyed the degree of overlap between yeast and mammalian G protein topologies using a range of criteria including conservation of modified amino acid residue, structure, and protein interface residence. We find that residues involved in guanine nucleotide binding and in interactions between G protein subunits are structurally conserved PTM hotspots. These data suggest the existence of a conserved mechanism of regulation for G protein signal transduction by post-translational modification of heterotrimeric G proteins. POST 08-413 The Evolution Of Caffeine Synthases In Theobroma Cacao

Craig D. Thulin Chemistry, Utah Valley University, Orem, Utah, US

How novel protein-coding genes evolve is a question of great interest. The accepted paradigm is that of gene duplication followed by divergent mutations that lead to the sequence of a new protein with new function. An example of this mechanism of protein evolution was sought among the caffeine synthases. Several caffeine synthases have been characterized from Coffea (coffee) and Camellia (tea) plants. These appear to be the results of gene duplication events because all of the caffeine synthases are closely related within (but not between) each of these two somewhat distantly related genera. One gene encoding caffeine synthase has been characterized from the cocoa plant Theobroma cacao, which does not closely cluster with either Coffea or Camellia caffeine synthases. The genome of T cacao has been sequenced, and a number of closely related putative caffeine synthase genes are found in the genomic area on chromosome ten where the known caffeine synthase gene is located. We find that elsewhere in the T cacao genome there are four other genes encoding more distantly related N-methyltransferase


POSTER ABSTRACTS genes. Comparison of non-coding regions of these genes with the genes on chromosome ten enables us to deduce a probable evolutionary history of the caffeine synthase genes in T cacao, which do appear to be the result of gene duplication followed by divergence. POST 08-415 Quantitative Imaging with Amersham™ Imager 600

Erik Bjerneld, Erika Svensson, Johan Johansson, Maria Winkvist, Hakan Roos GE, San Francisco, California, US Electrophoretic based protein- and DNA separation are well established techniques that are widely used in Life Sciences. However, the development of highly sensitive detection reagents together with advanced imaging techniques has shifted the obtained results from qualitative towards quantitative. To obtain quantitative results, the whole workflow has to be considered. Here we have focused on the importance of the CCD imager system, suitable applications and important considerations when working quantitatively. The Amersham Imager 600 QC or RGB, which is autocalibrated for accurate and reliable measurements of optical density, generates linear quantitative measurements of proteins stained with colorimetric stains such as Coomassie or silver. Amersham Imager 600 QC is a dedicated configuration for densitometry applications in a QC environment. Quantitative Western blotting requires a signal response that is proportional to the amount of protein. A broad dynamic range with linear response allows you to simultaneously quantitate both high and low levels of proteins with a signal response that is proportional to the amount of protein. Furthermore, the minimal crosstalk of Amersham Imager 600, and the spectrally resolved dyes Cy™2, Cy3, and Cy5, makes it a suitable system for a wide range of multiplexing applications, such as the detection of several proteins at the same time or different proteins of similar size. POST 08-416 Solution NMR Structure of the SLED Domain from Scml2 (Sex Comb on Midleg-like 2) - a New Domain on Epigenetic Landscape.

Irina Bezsonova University of Connecticut Health Center, Farmington, Connecticut, US

Scml2 is a member of the Polycomb group of proteins involved in epigenetic gene silencing. Human Scml2 is a part of a multi-subunit protein complex, PRC1, which is responsible for maintenance of gene repression, prevention of chromatin remodeling, preservation of the “stemness” of the cell and cell differentiation. While the majority of PRC1 subunits have been recently characterized, the structure of Scml2 and its role in PRC1-mediated gene silencing remain unknown. We have identified a conserved protein domain within human Scml2 and determined its structure by solution NMR spectroscopy. We named this module Scm-Like Embedded Domain, or SLED. Evolutionarily, the SLED domain emerges in the first multicellular organisms, consistent with the role of Scml2 in cell differentiation. Furthermore, it is exclusively found within the Scm-like family of proteins, often accompanied by MBT and SAM domains. The domain adopts a novel alpha/beta fold with no structural analogues found in the Protein Data Bank. We also examined the ability of the SLED domain to bind double-stranded DNA, and show that the isolated domain interacts with DNA in a sequence-specific manner. Since PRC1 complexes localize to the promoters of a specific subset of developmental genes in vivo, the SLED domain of Scml2 may provide an important link connecting the PRC1 complexes to their target genes


POSTER ABSTRACTS POST 08-417 Monitoring the Effect Of Extensive Genetic Drift on the Evolution Of Novel Enzyme Function

Linda Jäger, Peter Kast, Donald Hilvert ETH Zürich, Zürich, Switzerland Enzymes evolve through neutral genetic drift creating a panoply of different proteins with varying stabilities and promiscuous activities. It is conceivable that today’s spectrum of highly active catalysts arose from the natural selection of such promiscuous precursor proteins. Here we chose to study EcCM, the chorismate mutase (CM) domain of the Escherichia coli P-protein, responsible for the pericyclic rearrangement of chorismate into prephenate, a branchpoint reaction in the biological synthesis of phenylalanine and tyrosine. Interestingly, EcCM strongly resembles PchB, an enzyme from Pseudomonas aeruginosa, in its structure, active site organization, and transition state analog binding. PchB is an isochorismate pyruvate lyase (IPL), catalyzing the concerted conversion of isochorismate to salicylate and prephenate. While PchB has some residual CM activity in addition to its main function, EcCM shows no promiscuous IPL activity. These observations jointly suggest that both enzymes stem from a common ancestor. Nevertheless, decades of research employing state-of-the art protein engineering strategies have failed in creating IPL activity in EcCM. Therefore, we chose to recapitulate in the lab by a neutral drift experiment what presumably has occurred in nature before. We drifted EcCM and PchB over 20 generations of error-prone PCR mutagenesis followed by low-stringency selection for CM activity to maintain the overall structural fold thereby increasing the likelihood of finding novel catalysts. The 107-108 variants generated this way each contained 1-25 nucleotide mutations (up to 10% of the full length gene) thus representing a significantly genetically diversified population considering the low spontaneous mutation rates acting on contemporary genomes (ranging in the order of 10-9 substitutions per base per bacterial replication cycle). Initial data suggest that the genetic drift of either enzyme predominately decreases the native catalytic efficiency while possibly promoting increased substrate promiscuity. Deep sequencing experiments are currently underway and are expected to provide further clues as to which residues to target for further biochemical characterization of their prospective CM or IPL activity. POST 08-418 Synuclein and the Coelacanth

James M. Gruschus NHLBI, NIH, Bethesda, Maryland, US

The alpha-synuclein member of the synuclein family appears to have arisen in an early Sarcopterygian, the common ancestor of land vertebrates and the coelacanth, a lobe-finned fish. Compared to ray-finned fish, the sequences of alpha- and beta-synuclein show significantly more conservation among land vertebrates and coelacanths, suggesting they have evolved more important biological roles involving additional functions and/or interaction partners. Two potential partners of alpha-synuclein are synaptobrevin-2 (VAMP-2), part of the SNARE complex involved in presynaptic vesicle release, and glucocerebrosidase (GCase), a lysosomal enzyme in which mutations have been linked to Parkinson disease. If these proteins are truly biological partners of alpha-synuclein, then they must have co-evolved, that is, their interaction must be subject to evolutionary selection. To test the co-evolution hypothesis, correlated mutation analyses of alpha-synuclein with VAMP-2 and GCase have been performed using up to 57 species, including mammals, birds, reptiles, amphibians and coelacanth. While the number of species is too few to generate robust correlations via the analyses alone, when combined with experimental results a number of intriguing correlated mutations emerge. For instance, the highest correlations with VAMP-2 occur in its first 28 residues, truncation of which is known to result in loss of


POSTER ABSTRACTS interaction with alpha-synuclein. For GCase, the correlation between H374 and alpha-synuclein E132 could explain why alpha-synuclein only interacts with GCase at the acidic pH found in lysosomes. This study highlights the benefits of combining bioinformatics with experiment, a partnership whose importance will continue to grow as more and more genomes are sequenced.

Poster Session: Proteins in Altered States POST 09-419 Storage of Environmental Information into Biological Structures: Temperature-dependent Fibrillar Polymorphism of α-Synuclein

Ghibom Bhak, Seung Ryeoul Paik Seoul National University , Seoul, Korea, Republic of

Molecular-level storage of environmental information into tangible structures and their subsequent inheritance to next generations have been studied with a biological phenomenon of amyloidogenesis. Amyloidogenesis defines a biochemical condition producing highly ordered amyloid fibrils from soluble proteins via molecular disorder-to-order transition. α-Synuclein is an amyloidogenic protein responsible for the Lewy body formation in Parkinson’s disease (PD). Based on the accelerated amyloidogeneses of αsynuclein in its oligomeric state with shear force and hexane, we have proposed a double-concerted fibrillation mechanism where two successive concerted assemblies of monomers and oligomers have led to the amyloid fibril formation. By employing the α-synuclein oligomers as an environment sensing agent, an intangible information of temperature has been stored into the tangible supra-structures of amyloid fibrils in various morphologies through the oligomeric unit-assembly process. The stored information, then, has been perpetuated by self-propagating the diversified fibrillar structures with distinctive molecular characteristics to next generations through the nucleation-dependent fibrillation process. This oligomer-mediated molecular inscription process of environmental information, therefore, may not only represent influx of information into biological system but also be applied in the areas of nanobiotechnology to process various external signals.

POST 09-420 Reversible Polymeric Fibers made from Low-Complexity Sequences Function as a Foundation of RNA Metabolism

Masato Kato, Steven McKnight Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, US Low-complexity (LC) sequences have been believed to perform important biological roles by unknown mechanisms. We have discovered that purified liquid samples of the LC domains of RNA-binding proteins, such as FUS, hnRNPA2 and TDP-43, made the transition into a gel-like phase in a concentration-dependent manner1,2. The hydrogels are composed of morphologically uniform polymers endowed with an underlying cross-β structure. Unlike pathogenic amyloid fibers, our polymeric fibers are reversible. Hydrogel has capability to capture LC domains of RNA granule proteins via copolymerization of the LC domain forming the hydrogel and the test LC domain into a single polymeric fiber. The C-terminal domain (CTD) of RNA polymerase II3 and SR domains of splicing factors also bind to the hydrogel, yet these interactions are reversible by phosphorylation of CTD and SR domains by their 262

POSTER ABSTRACTS respective kinases. The outcome of these studies has led to a conceptual framework hypothesizing the structural underpinnings of how dynamic puncta, including RNA granules, transcription factories and nuclear speckles, form in eukaryotic cells. Many mutations in these LC domains found in ALS and FTLD patients have been reported to facilitate fibril aggregate formation of these proteins and thereby to possibly exert toxicity on neuronal cells. We hypothesize, as mechanisms for the aggregate formation, that disease-causing mutations in LC domains tip the equilibrium of reversibility of polymeric fiber formation such that the fibers become unacceptably stable, resulting in accumulation of irreversible polymers. References 1. Kato, M., Han, T. W., Xie, S., Shi, K., Du, X., Wu, L., Mirzaei, H., Goldsmith, E., Longgood, J., Pei, J., Grishin, N., Frantz, D., Schneider, J., Chen, S., Li, L., Sawaya, M., Eisenberg, D., Tycko, R. and McKnight, S. L. Cell-Free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers Within Hydrogels, Cell 149,753-767 (2012) 2. Han, T. W., Kato, M., Xie, S., Wu, L., Mirzaei, H., Pei, J., Chen, M., Xie, Y., Allen, J., Xiao, G. and McKnight, S. L. Cell-Free Formation of RNA Granules: Bound RNAs Identify Features and Components of Cellular Assemblies, Cell 149, 768-779 (2012) 3. Kwon, I., Kato, M., Xiang, S., Wu, L., Theodoropoulos, P., Mirzaei, H., Han, T., Corden, J. L. and McKnight. S. L. Phosphorylation-regulated Binding of RNA Polymerase II to Fibrous Polymers of LowComplexity Domains, Cell 155, 1049 (2013) POST 09-421 Flavone Derivatives As Inhibitors Of Insulin Amyloid-Like Fibril Formation

Ricardas Malisauskas, Akvile Botyriute, Vytautas Smirnovas Vilnius University Institute of Biotechnology, Vilnius, Lithuania

A number of diseases are associated with formation of amyloid fibrils. Both, mature fibrils and oligomers or protofibrils, which can exist on pathway of fibril formation, may be responsible for the pathogenesis. Thus any molecules, able to change kinetics or alter the pathway of protein aggregation, can potentially interfere with diseases. A number of compounds, including several natural and synthetic flavone derivatives were reported as inhibitors of amyloid fibril formation. Moreover, it was reported that the inhibition potential depends on the number and the position of hydroxyl groups across the flavone backbone. In most of studies the main value used to rate the inhibition potential of flavones is fluorescence intensity of Thioflavin T (ThT) binding assay. We studied impact of 260 commercially available flavone derivatives on insulin fibril formation. In addition to ThT fluorescence intensity we also compared halftime of aggregation (t50). Almost one third of studied flavone derivatives changed final ThT fluorescence at least two fold, however most of these derivatives had much smaller impact on t50, with no clear correlation between these two values. POST 09-422 Phosphorylation Releases Constraints to Domain Motion in ERK2

Yao Xiao1, Thomas Lee1, 2, Michael P. Latham1, 4, Lisa R. Warner1, 5, Akiko Tanimoto1, 6, Arthur Pardi1, Natalie G. Ahn1, 2, 3 1 Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, US, 2HHMI, University of Colorado, Boulder, Colorado, US, 3BioFrontiers Institute, University of Colorado, Boulder, Colorado, US, 4Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, 5Institute of Structural Biology, Neuherberg, Germany, 6Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US Protein dynamics has been shown to be important for regulating protein function, such as allostery and enzyme catalysis. It remains a challenging question what is the direct role of protein dynamics in enzyme catalysis. Here, we present NMR data on millisecond ILV methyl dynamics of MAP kinase ERK2 in its inactive and active forms, using 13C relaxation dispersion experiments. ERK2 is activated by dual263

POSTER ABSTRACTS phosphorylation. Inactive, unphosphorylated ERK2 shows only local ILV methyl dynamics. Upon activation, the dispersion data demonstrate that ERK2 undergoes global conformational dynamics. The observed dynamics in active ERK2 is consistent with inter-conversion between two major conformations that affect residues throughout the kinase core, with kex ≈ 300 s-1 (kAB≈240 s-1/kBA≈60 s-1) and pA/pB ≈ 20%/80%. A mutant of ERK2 was designed to increase backbone flexibility at the hinge region, the pivot point connecting N- and C-terminals. The methyl dynamics data of this mutant of inactive ERK2, also indicate inter-conversion between conformations, with kex ≈ 500 s-1 (kAB≈15 s-1/kBA≈485 s-1) and pA/pB ≈ 97%/3%. Thus, activation of ERK2 by phosphorylation leads to a dramatic shift in global conformational exchange, which is likely through release of constraints at the hinge. POST 09-423 Using Mass Spectrometry To Define How The Small HSP Chaperones Protect Substrates From Aggregation

Keith Ballard2, Heather O'Neill1, Wenzhou Li1, Vicki Wysocki3, Elizabeth Vierling2 1 Department of Chemistry and Biochemistry, University of Arizona, Tuscon, Arizona, US, 2Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Amherst, Massachusetts, US, 3Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US

Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to diseases of protein misfolding. They comprise a conserved core αcrystallin domain (ACD) flanked by an evolutionarily variable N-terminal arm (NTA) and semi-conserved Cterminal extension. sHSPs are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that make substrate available to the ATP-dependent chaperones for refolding. Understanding sHSP-substrate interactions may provide insight into developing potential therapeutic strategies for treating protein-folding diseases such as Alzheimer’s and Parkinson’s disease. To derive common features of sHSP-substrate recognition, we compared the chaperone activity and specific sites of interaction with substrate for three different sHSPs: an unusual dimeric sHSP from Arabidopsis, At18.5, and more typical oligomeric sHSPs from pea (Ps18.1) and wheat (Ta16.9), for which the atomic structure is available. We used the homobifunctional amine-reactive, 7 Å crosslinker 2 bis(sulfosuccinimidyl)glutarate (BS G) to test for interaction sites between each of the three sHSPs and the model substrate malate dehydrogenase (MDH), identifying crosslinking by mass spectrometry using an LTQ-Orbitrap. MDH exhibits major rearrangement upon heat-denaturation and association with sHSPs as evidenced by detection of new MDH-MDH crosslinks incompatible with the MDH native structure. The NTA of all three sHSPs was a major site of crosslinking to substrate, and was found linked to multiple Lys residues on MDH, consistent with the heterogeneity of the sHSP-MDH complexes. Not all sHSP Lys residues were involved in crosslinking, although they reacted with the crosslinker, demonstrating preferred sites of interaction. In total, the data support an important role for the NTA in substrate recognition and indicate that an early unfolding intermediate of the substrate is recognized. POST 09-424

Mechanically resistant conformations in amyloid β and α-synuclein Sigurdur Æ. Jònsson2, Simon Mitternacht1, Anders Irbäck2 1 Science Library, University of Bergen, Bergen, Norway, 2Astronomy and Theoretical Physics, Lund University, Lund, Sweden Unstructured proteins such as the amyloid β-peptide (Aβ) and α-synuclein (αS) display remarkable mechanical resistance in a significant fraction of unfolding traces in single-molecule pulling experiments. The rupture forces are comparable to those of mechanically stable folded proteins. The nature of these force-resistant states, and their relation to amyloid aggregation is unknown. We have performed all264

POSTER ABSTRACTS atom pulling simulations on broad ensembles of Aβ and αS conformations to identify the mechanically resistant states. The simulated unfolding trajectories have rupture events similar to experiments both in molecular extension and distribution of rupture forces. We find that the most resistant states share a common architecture, which in both cases can be related to that of amyloid fibrils. We also find that the disease-linked arctic mutation of Aβ increases the occurrence of highly force-resistant structures. We propose that similar structures cause the high experimental rupture forces and that they could play an important role in the amyloid aggregation pathway. POST 09-425 Structural Determinants of Amyloid Fibril Formation in Triosephosphate Isomerase.

Edson N. Carcamo-Noriega, Gloria Saab-Rincón IBT-UNAM, cuernavaca, Mexico

The amyloid fibril is one of the most biologically important protein structures due to its implication in numerous degenerative diseases. Although all proteins have the potential to form these aggregates, because their formation relies on main chain interactions, not all of them can form fibrils under physiological conditions. The structural determinants that promote or inhibit the formation of amyloid fibril remain unknown. This study aims to find these determinants in the human enzyme triosephosphate isomerase, whose ability to form fibrils according to recent reports may be associated with Alzheimer's disease. Aggregation kinetics under destabilizing conditions followed by thioflavin T show that triosephosphate isomerase form cross-beta structures reaching saturation within 72 hrs of incubation. The presence of a cross-beta core in non-fibrillar morphology was also confirmed with the antibody WO1 in dot-blot assays and fluorescence microscopy. The cross-beta region was found by a prediction analysis using several predictors. The three regions with the highest score were synthesized and tested under physiological conditions. The region 183-213 was able to form fibrils confirmed with thioflavin T binding, WO1 dot-blot assay and transmission electron microscopy. Despite the presence of a cross-beta region within the structure of triosephosphate isomerase the enzyme formed non-fibrillar aggregates, this may be due to protection by stable modules βα adjacent to the region cross-beta that could work like steric hindrance, preventing the elongation of the fibril and stopping the aggregation in a protofibrillar state. POST 09-426 Developing Quantitative NMR Methods For Predicting Residue Specific Helicity Of MetO-λ Unfolded State

Kan Li, Roy Hughes, Terrence G. Oas Biochemistry, Duke University, Durham, North Carolina, US Numerous studies have provided evidences for residual compactness in protein unfolded state. Studies of unfolded state conformational ensemble will greatly contribute to understanding the starting point of protein folding reaction. NMR techniques are powerful in detecting residue specific information. Nascent helicity, local hydrophobic collapse and long range interactions have been observed in the unfolded state of several proteins by NMR techniques. We use λ repressor N-terminal domain, a helical protein, to study mainly the residual helicity of the unfolded state ensemble along with other possible structural preferences. A variant of the λ repressor N-terminal domain has been constructed by oxidizing protein core methionine residues along with other mutations (MetO-λ), allowing it to populate unfolded state ensemble under nondenaturing conditions. NMR backbone dynamics study of MetO-λ unfolded state shows residual helicity and possible hydrophobic collapse. In order to develop methods to quantitatively determine residue specific helicity of MetO-λ unfolded state, we expressed residue 830 of λ repressor protein as an ioslated peptide (LRH1x), which corresponds to extended first helix of the protein. The first helix is important in domain folding and intrinsically helical in isolation. Analysis of CD 265

POSTER ABSTRACTS spectra and backbone chemical shift (CS) indices of LRH1x at neutral pH both show evidences of significant helicity. However, to predict residue specific helicity, we developed a complementary method in addition to CS indices method due to its intrinsic limitation on the quality of prediction. The protection factors (PFs) of backbone amide hydrogens contain averaged information of residue specific helicity. By comparing PFs from measured amide proton exchange rates and theoretically calculation based of helix-coil transition theory, we are aiming to use quantitative residue specific observation to optimize the helix-coil transition theory to better prediction and understanding of residue specific helicity. We implemented this method for LRH1x and was able to observe discrepancies between experimentally and theoretically extracted PFs, which indicates the need to optimize helix-coil transition theory parameters. Combining with other NMR techniques, such as CS indices and NOE, this method will allow us to gain valuable insights on the unfolded state ensemble of MetO-λ and possibly other protein systems. POST 09-427 Towards in vivo NMR: Structural Studies Of Prion Fibrils Assembled In Native Environments at Endogenous Levels

Kendra K. Frederick1, Vladimir K. Michaelis2, Bjorn Corzilius2, Ta-Chung Ong2, Angela C. Jacavone2, Robert G. Griffin2, Susan Lindquist1, 3, 4 1 Whitehead Institute, Cambridge, Massachusetts, US, 2Chemistry & Francis Bitter Magnet Lab, MIT, Cambridge, Massachusetts, US, 3Biology, MIT, Cambridge, Massachusetts, US, 4HHMI, Cambridge, Massachusetts, US

Nearly all biology happens within the boundary of a cell, and intracellular space is highly crowded and contains a large number of macromolecules with the potential to interact. Yet structural investigations of biomolecules are typically confined to simplified in vitro systems. Solid-state nuclear magnetic resonance (NMR) spectroscopy can yield atomic-level information and has no requirement for crystalline samples, but the ability to investigate low-concentration analytes within complex biological mixtures is in practice unattainable due to impedingly long acquisition times. Dynamic nuclear polarization (DNP) is able to dramatically increase the sensitivity of magic angle spinning (MAS) solid-state NMR. Here we apply DNP MAS NMR to examine an isotopically labeled prion protein, Sup35, assembled into its amyloid form in the presence of unlabeled cellular lysates. We find that we can specifically observe the protein at endogenous levels in a complex background in reasonable experimental time, something that would take several decades to do using traditional MAS NMR. Moreover, we find that while assembly of the amyloid form in the presence of cell lysates does not perturb the structure of the amyloid core, it results in large changes in the chemical environment for a portion of the protein that is dynamically disordered in purified systems. This suggests that the flexible regions of amyloid fibrils may interact with cellular components that can facilitate assembly into macromolecular structures and inheritance of this protein based element of inheritance. POST 09-428 Structural and Ligand Binding Properties of Dimeric Horse Myoglobin

Satoshi Nagao1, Hisao Osuka1, Takuya Yamada1, Takeshi Uni1, Yasuhito Shomura2, 3, Kiyohiro Imai4, Yoshiki Higuchi2, 3, Shun Hirota1 1 Nara Institute of Science and Technology, Ikoma, Nara, Japan, 2Department of Life Science, Graduate School of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, Japan, 3RIKEN SPring-8 Center, Sayo-cho, Sayo-gun, Hyogo, Japan,4Department of Frontier Bioscience, Faculty of Bioscience and Applied Chemistry, Hosei University, Koganei, Tokyo, Japan


POSTER ABSTRACTS Myoglobin (Mb) stores dioxygen in muscles, and is a fundamental model protein widely used in molecular design. The presence of dimeric Mb has been known for more than forty years, but its structural and oxygen binding properties remain unknown. From an X-ray crystallographic analysis at 1.05 Å resolution, we found that dimeric metMb exhibits a domain-swapped structure with two extended α-helices. Each new long α-helix is formed by the E and F helices and the EF-loop of the original monomer, and as a result the proximal and distal histidines of the heme originate from different protomers. The heme orientation in the dimer was in the normal mode as in the monomer, but regulated faster from the reverse to normal orientation. The dimer possessed the oxygen binding property, although it exhibited a slightly higher oxygen binding affinity (∼1.4 fold) compared to the monomer and showed no cooperativity for oxygen binding. The oxygen binding rate constant (kon) of the dimer ((14.0 ± 0.7) × 106 M−1 s−1) was similar to that of the monomer, whereas the oxygen dissociation rate constant (koff) of the dimer (8 ± 1 s−1) was smaller than that of the monomer (12 ± 1 s−1). We attribute the similar kon values to their active site structures being similar, whereas the faster regulation of the heme orientation and the smaller koff in the dimer are presumably due to the slight change in the active site structure and/or more rigid structure compared to the monomer. These results show that domain swapping may be a new tool for protein engineering. Reference Nagao, S., Osuka, H., Yamada, T., Uni, T., Shomura, Y., Imai, K., Higuchi, Y., and Hirota, S., Dalton Trans. 41, 11378−11385 (2012) POST 09-429 Study of Interactions of amyloidogenic Regions of Streptococcus mutans adhesin P1 by Nuclear Magnetic Resonance

Wenxing Tang, L. Jeannine Brady, Joanna R. Long Unviersity of Florida, Gainesville, Florida, US Streptococcus mutans is an established etiologic agent of human dental caries, the most common infectious disease in the world. S. mutans is particularly effective at colonizing hard tissues of the human

oral cavity. Its adherence is mediated by sucrose-dependent and independent mechanisms. In the absence of sucrose, the extracellular cell wall-associated adhesin P1, also known as Antigen I/II or PAc, facilitates attachment to the acquired pellicle on teeth. This multifunctional adhesin interacts with the high molecular weight glycoprotein scavenger receptor gp340, as well as with other oral bacteria and host cell matrix proteins. Our previous work showed that P1 self-aggregates to form amyloid fibrils and demonstrates common biophysical properties ascribed to amyloids including uptake of Thioflavin T and Congo Red (CR) as well as CR-induced birefringence. Amyloid formation is evident within S. mutans biofilm cultures and known inhibitors of amyloid fibrillization inhibit biofilm formation. P1 has an unusual structure in which the protein folds back on itself such that an alanine-rich alpha helix intertwines with a polyproline type II helix to form an extended helical stalk. Beta sheet-rich globular structures corresponding to the adhesive domains lie on either end of the stalk. Segments containing these globular regions (A3VP1 and C123) are associated with uptake of amyloidophilic dyes during self-aggregation, and can also interact with one another. Since P1 was originally identified as a dual antigen comprising antigen II (C123), and the remainder of the molecule (antigen I), it is likely that interactions between protein fragments are biologically relevant. The detailed process of amyloid formation is not fully understood. Here we describe our preliminary studies using high resolution Nuclear Magnetic Resonance to evaluate the previously crystallized C123 fragment. Shown in Figure 1 is the Transverse Relaxation-Optimized 15 SpectroscopY (TROSY) spectrum of uniformly N labeled C123 in Tris buffer carried out at 25°C with 64 scans and 256 t1 points. By assigning the corresponding residues to these resonance peaks, we will establish the foundational basis by which to identify key residues and assess conformational changes associated with intermolecular interactions of C123 with itself during amyloid aggregation, and with A3VP1. Similar experiments are in process for A3VP1, for which a crystal structure is also available.



POST 09-430 Identification and Characterization of Functional Amyloids in Streptococcus mutans

Richard N. Besingi, L. Jeannine Brady Oral Biology, University of Florida, Gainesville, Florida, US

Functional amyloid formation is increasingly recognized as a mechanism utilized by microorganisms (viruses, bacterial, fungi) to facilitate establishment and persistence within their respective environmental niches. Recently it has been shown that secreted proteins produced by the cariogenic bacterium S. mutans are capable of amyloid fibrillization. These include but are not limited to adhesin P1 (Ag I/II). Amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of S. mutans. Known inhibitors of amyloid fibril formation can inhibit biofilm formation by amyloidogenic microbes such as S. mutans suggesting a potential target for therapeutic intervention. Although S. mutans lacking P1 demonstrates residual amyloid forming properties and forms biofilms, a mutant lacking the transpeptidase enzyme sortase, which covalently links a number of proteins to the peptidoglycan cell wall, is defective in biofilm formation as well as cell-associated amyloid-like properties. The objective of this study is to identify additional potential amyloid forming proteins of S. mutans and to determine the biophysical conditions under which these proteins form amyloids, as well as determine their individual or combined roles in biofilm formation. To identify amyloid forming proteins in S. mutans, a P1-deficient mutant strain was grown to stationary-phase in defined minimal media and secreted proteins were concentrated from spent culture supernatants, followed by fractionation by ion exchange chromatography. Partially purified protein fractions were tested for binding of the amyloidophilic dyes Congo Red (CR) and Thioflavin T, and for characteristic birefringent properties following staining with CR and visualization under a crossed set of polarizing filters. Proteins from fractions that tested positive using these assays were separated by SDS PAGE, and identified by LC/MS. Proteins identified from chromatography fractions with amyloid-like properties included WapA, GbpA, GbpB, SMU_2147c and SMU_63c. Genes encoding these proteins were cloned and recombinant proteins expressed and purified for confirmation and characterization of individual amyloidogenic properties in vitro. In several instances, amyloid formation was significantly increased under acidic conditions. This finding is consistent with the acidogenic and aciduric nature of S. mutans. (Supported by NIH R01DE021789). POST 09-431 Achieving Selectivity In The Hippo Pathway: An Investigation Of The Inter-Domain Communication In TEAD Transcription Factors

Priyanka Rauniyar, Sudha Veeraraghavan Pharmaceutical Sciences, Univ Maryland School Pharmacy, Baltimore, Maryland, US

TEAD is the downstream transcriptional regulator of the Hippo pathway, which plays an important role in normal organ development, growth, cell proliferation, and apoptosis. Protein co-factors of TEAD include YAP and TAZ in mammals; defect in TEAD-YAP/TAZ interactions are linked to uncontrolled cell proliferation, resulting in tumors and cancers. Our group established the first three-dimensional structure of the DNA-binding TEA domain, which consists of three alpha helices. The N- terminal domain (TEA) is


POSTER ABSTRACTS followed by a 'Linker' domain and then the C-terminal protein-protein interaction domain (CTD). It is not yet understood how the binding of a protein co-factor at the CTD might influence DNA-binding by the TEA domain. Here, as part of my undergraduate research internship, I over-expressed and purified the TEA and CTD and tested for interactions between the domains. Our preliminary results indicate that interaction between the isolated domains is weak or undetectable suggesting either a role for the linker domain or for protein cofactors in determining specificity of TEAD proteins. Further biophysical and biochemical studies on these domains and on full-length TEAD and the DNA complexes are underway. POST 09-432 Structural Projection of PTMs (SPoP): A Toolkit For Providing Structural And Functional Context For Sequence-Specific Protein Features

Henry Dewhurst, Matthew P. Torres Biology, Georgia Institute of Technology, Atlanta, Georgia, US

While structure is accepted as the key determinant of protein function, the majority of site-specific feature data (e.g., post-translational modifications (PTMs), mutations) is strictly sequence-linked and lacks structural context. Here we describe a novel toolkit, Structural Projection of PTMs (SPoP), an application that bridges this gap by examining protein features in three-dimensional context. SPoP provides a platform for quantitative and visual analysis of protein features. SPoP is written in Perl, Java, and PyMol script and is modular in design to allow for rapid integration of additional feature data (e.g. interface residence and surface accessibility). The toolkit is capable of projecting any feature-set onto a known or modeled protein structure. The native operating mode employs a composite MySQL database of PTMs from 12 sources – including user-defined novel PTM sites. Multiple feature-sets may be layered to generate composite projections. SPoP utilizes either sequential or structural homology for projection of feature frequencies onto three-dimensional models within PyMol. In addition to visualizations, SPoP’s analytical core generates raw data on homologous feature detection and conservation of feature sites. Layering of feature maps allows SPoP to detect overlapping feature-rich regions and identify potential regulatory sites. POST 09-433 Investigating the Molecular Basis of Curli Amyloid Inhibition by Protein and Chemical Chaperones

Neha Jain, Margery L. Evans, Matthew R. Chapman Molecular Cellular and Developmental Biology, University of MIchigan, Ann Arbor, Michigan, US

Proteins can readily adopt stable, ordered aggregates called amyloids. Amyloid formation can be cytotoxic and is the hallmark of several neurodegenerative diseases. Recently, a new class of amyloids has been described called ‘functional amyloids’ that are not cytotoxic and instead capitalize on the unique properties of amyloids to fulfil cellular functions. Functional amyloids are ubiquitous in nature. Curli are bacterially-produced functional amyloids that are important structural components of bacterial biofilms. CsgA is the major curli subunit and the assembly of CsgA into extracellular amyloid fibers is a highly orchestrated process. Therefore, the curli system provides a unique platform to understand the process of amyloid formation and how it might be inhibited. Here, we present the modulation of curli amyloid assembly by two classes of molecules: protein and chemical chaperones. We have discovered that the Escherichia coli periplasmic protein CsgC is an efficient and selective inhibitor of CsgA amyloid formation. CsgC inhibits CsgA amyloid formation at substoichiometric molar ratios by preventing early stages of oligomerization in vitro. CsgC also inhibits aggregation of curli amyloid forming proteins from closely related bacterial species. Furthermore, deletion of CsgC in vivo results in the accumulation of toxic intracellular CsgA amyloid aggregates. We have also identified and characterized 2-pyridone peptidomimetic compounds that modulate CsgA amyloid assembly in vivo and in vitro. When added in


POSTER ABSTRACTS combination with another protein chaperone, CsgE, the 2-pyridone compounds act synergistically to inhibit CsgA amyloid assembly. The 2-pyridone compounds therefore inhibit amyloid assembly through different mechanisms than protein chaperones. Combined studies of amyloid inhibition by protein and 2pyridone compounds will continue to provide insights into the development of highly specific and efficient anti-amyloid therapeutics. POST 09-434 The Effects Of Mutations On The Aggregation Propensity Of Human Prion-Like Domains

Eric D. Ross, Kacy Paul, Sean Cascarina Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, US

Numerous human proteins contain prion-like domains (PrLDs), defined as domains with similar amino acid composition to the yeast prion domains. In the past few years, mutations in a number of these PrLDcontaining proteins have been linked to various degenerative diseases. Evidence is emerging that these PrLDs may play an important role in normal cellular physiology. For example, many of the diseaseassociated PrLDs are found in RNA binding proteins, and result in formation of large cytoplasmic inclusions. However, formation of naturally-occurring RNA-protein granules is also mediated in part by PrLDs, suggesting that disease may result from perturbations in normal RNA dynamics. We are using two proteins, hnRNPA1 and hnRNPA2, as a model system to explore the relationship between PrLD sequence, aggregation, and pathogenesis. Point mutations in the PrLDs of hnRNPA1 and A2 cause either IBMPFD (inclusion body myopathy associated with Paget’s disease of the bone and fronto-temporal dementia) or ALS, and result in formation of large cytoplasmic inclusions. Expression of these proteins causes mutation-dependent muscle degeneration in Drosophila. In yeast, the mutant prion-like domains are able to support prion activity, while the wild-type prion-like domains are not. To explore the relationship between amino acid sequence and aggregation propensity, we have tested approximately 80 hnRNPA1 and A2 mutants in yeast. For the vast majority, the effects of the mutations are accurately predicted by PAPA (Prion Aggregation Prediction Algorithm), a prediction algorithm designed to predict yeast prion propensity, suggesting that aggregation propensity can be manipulated in a rational manner. These mutants will provide a powerful model system to explore the relationship between aggregation propensity and pathogenesis. POST 09-435 Competition Or Forced Collaboration? An Unusual Pattern Of Self-Propagating Polymorphism Of Insulin Amyloid Fibrils Upon Seeding With Mixed Templates.

Wojciech Dzwolak1, 2, Weronika Surmacz-Chwedoruk1, 3 1 Institute of High Pressure Physics, Polish Academy of Sciences, Warsaw, Poland, 2Department of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland, 3Institute of Biotechnology and Antibiotics, Warsaw, Poland B31


Cross-seeding of fibrils of bovine insulin ([BI]) and Lys -Arg human insulin analog ([KR]) induces selfpropagating amyloid variants with infrared features inherited from mother seeds. Here we report an unexpected outcome of seeding experiments with mixed [BI] and [KR] amyloid templates. When native BI (or KR) insulin is seeded with mixed equal portions of preformed [BI] and [KR] templates, phenotype of the resulting daughter fibrils is the same as in the case of purely homologous seeding – i.e. in the absence of foreign seeds. The selection bias toward homologous daughter amyloid was found to be exceptionally strong: more than 200-fold excess of heterologous seed was required to imprint its structural phenotype upon mixed seeding. In the presence of both templates, the fibrillation-promoting and structureimprinting properties of heterologous seeds become uncoupled – heterologous templates still accelerate fibrillation but have no say in determining phenotype of daughter amyloid (Figure 1). Hence the bias


POSTER ABSTRACTS toward copying the homologous phenotype cannot be explained by kinetic factors only. A hypothetical explanation is put forward in which: [i] docking of soluble insulin with amyloid tips proceeds through several steps and [ii] is under thermodynamic control; [iii] free energy of spontaneously formed fibrils is below energy levels of amyloid variants induced by cross-seeding. POST 09-436 Nanoscale Organization of Protein Molecules within Amyloids and Prions

Samrat Mukhopadhyay Department of Biological Sciences and Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Mohali, Punjab, India

Amyloids are ordered protein aggregates that are implicated in a variety of debilitating human disorders such as Alzheimer's, Parkinson's and prion diseases. The transition from a normal functional protein to an altered (misfolded) form involves a profound conformational change that triggers the aberrant protein assembly resulting in a wide variety of nanostructures including amyloid oligomers, pores and fibrils. My laboratory utilizes a diverse array of methodologies to unravel the key molecular events that are crucial in amyloid formation from a number of proteins. Using Raman spectroscopy in combination with atomic force microscopy (AFM), we have been able to delineate the key structural transitions during amyloid formation [1]. The AFM images revealed a progressive morphological transition from spherical oligomers to nanoscopic annular pores (Fig. 1a), whereas, the Raman data indicated the protein structural changes during amyloid assembly and pore formation. Recently, we have used the combination of AFM and Raman to monitor the structural transition of human prion protein into protease-resistant amyloid oligomers that assemble into ordered fibrils (Fig. 1b) [2]. Additionally, we have adapted a super-resolution nanophotonic technology that allows us to optically image individual amyloids at the nanoscopic spatial resolution [3-5]. Due to the optical diffraction-limit, conventional optical microscopy does not allow us to monitor the nanoscale organization at a high spatial resolution. Therefore, we have utilized near-field scanning fluorescence microscopy to optically map the amyloid fibrils far beyond the diffraction-limit. Interrogation of individual fibrils by simultaneously monitoring both nanoscale topography (Fig. 1c) and fluorescence brightness (Fig. 1d) revealed heterogeneous packing of the cross-β architecture within amyloids. Our results provide structural underpinnings of diverse amyloid polymorphs that underlie the strain phenomenon in prion and amyloid biology. [1] M. Bhattacharya, N. Jain, P. Dogra, S. Samai & S. Mukhopadhyay (2013) J. Phys. Chem. Lett. 4 ,480. [2] V. Dalal & S. Mukhopadhyay (unpublished). [3] V. Dalal, M. Bhattacharya, D. Narang, P.K. Sharma & S. Mukhopadhyay (2012) J. Phys. Chem. Lett. 3, 1783. [4] M.Bhattacharya & S. Mukhopadhyay (2014) Nanophotonics, 3, 51. [5] V. Dalal, S. Arya & S. Mukhopadhyay (2013) in Bionanoimaging: Protein Misfolding & Aggregation (Elsevier; Eds. Y. Lyubchenko & V. Uversky). POST 09-437 A Temperature Sensitive Parkinsonian Mutation in DJ-1 Enhances Protein Dynamics in a Metaldependent Fashion

Nicole M. Milkovic1, Steven Halouska2, Jonathan Catazaro2, Sara Basiaga3, Robert Powers2, Mark A. Wilson1 1 Biochemistry and the Redox Biology Center, University of Nebraska-Lincoln, Lincoln, Nebraska, US, 2Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska, US, 3Chemistry Research and Instrumentation Facility, University of Nebraska-Lincoln, Lincoln, Nebraska, US Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the midbrain. DJ-1 is a small (20 kDa) conserved protein whose absence or inactivation causes rare forms of inherited parkinsonism. The native function of DJ-1 is cytoprotective, particularly against oxidative stress and mitochondrial damage. The M26I mutation causes loss of DJ-1 stability and 271

POSTER ABSTRACTS diminished protein levels in the cell. The crystal structure of M26I DJ-1 is similar to that of wild-type (WT) DJ-1, showing only minor perturbations near the site of mutation. Thus, it is unclear why this missense mutation is pathogenic. We have investigated the solution dynamics of WT and M26I DJ-1 using multidimensional NMR spectroscopy. We find that fast timescale dynamics are largely unchanged between WT and M26I DJ-1; however, longer timescale dynamics are enhanced by M26I DJ-1 at physiological temperature (37°C), particularly within the hydrophobic core of the protein. Moreover, M26I DJ-1 shows a marked increase in transition metal-facilitated aggregation in vitro at 37°C, suggesting that the mutation is pathogenic because it results in a thermally-activated metal-dependent change in protein dynamics that reduces DJ-1 stability. We propose that the increased conformational dynamics of M26I DJ-1 lead to protein instability, resulting in a temperature-sensitive and metaldependent loss-of-function phenotype that causes parkinsonism at physiological temperature. POST 09-438 Analytical Tools in Decision Making of Suitability of Monoclonal Antibodies for Immunodiagnostic Assays

Kevin R. Rupprecht, Tracey D. Rae, Svetoslava D. Gregory, Na Yang, Janet M. Bergsma, Ryan M. Bonn, Martin R. Lopez, Panfilo F. Ozaeta, Cheng Zhao, Carol S. Ramsay, Jeffrey R. Fishpaugh Diagnostic Analytical Chemistry R&D, Abbott Laboratories, Waukegan, Illinois, US

Over the past two decades there has been a rapid and significant rise in the number and types of analytical methods available for analysis of proteins. These methods are designed to give particular information about biological molecules such as size, charge characteristics, post transcriptional modifications and other characteristics. This availability of multiple techniques has led to a realization that alteration of bioactive molecules can have a profound effect on the activity of those molecules. This is particularly important when trying to induce cellular systems to produce large amounts of a particular protein, such as antibodies or antigens for use in immunodiagnostic tests. For example, changes in growth conditions have been shown to affect glycosylation of monoclonal antibodies. In some cases these changes to molecular structure may not be easily recognized if one is using methods that only measure a limited number of protein characteristics, or even using only one method to measure a particular characteristic. In the immunodiagnostic industry an analytical group is challenged to analyze large numbers of different antibodies for different antigens in a single year. In this work we detail our experiences with two particular monoclonal antibodies. In both cases initial analyses did not indicate extensive variability within or between lots of the monoclonals. There were however subtle clues in the data that something was amiss with each of these antibodies. By using multiple methods, measuring multiple characteristics a detailed understanding of the monoclonal structure was obtained for each. In one case it was discovered that an endogenous light chain was being produced and incorporated into the monoclonal and in the other case a variable glycosylation of a light chain CDR was found. Both of these types of changes impact potency and ability of the monoclonal to recognize their respective antigenic targets. The decisions made that affected recognition of these modifications are discussed. POST 09-439 Spectroscopic Investigation of the Structural Perturbations of Tau Microtubule Binding Domains at the Golgi Membrane as Illustrated by Membrane Mimics

Lauren E. Sparks, Larry R. Masterson Chemistry, Hamline University, St. Paul, Minnesota, US

The demand for understanding certain types of dementia are underscored by limitations for mechanistic details of the development of Alzheimer’s disease (AD), the only disease in the top ten leading causes of death that has seen a rise in fatality over the past decade. Hallmarks of AD include plaque formation


POSTER ABSTRACTS created by the amyloid beta protein, and neurofibrillary tangles (NFTs) of the protein, tau. The conserved microtubule binding domains (MBDs) of tau nucleate the formation of NFTs and have been the subject of a number of investigations. In vivo studies have shown that NFTs are found in high concentration at the Golgi membrane (GM) but do not associate with the plasma membrane (PM), suggesting that the GM may catalyze fibril formation. In order to understand how these regions in tau contribute to the formation of aggregates, we used peptide fragments corresponding to each of the four conserved repeat segments in the MBD to investigate their potential interactions with lipid membrane mimics of the GM. To illustrate that the peptides were sensitive to membrane type, we studied their structural perturbations in the presence of negatively charged (a mixture of DMPC and DMPG) and uncharged (DMPC only) small unilamellar vesicles (SUVs) via CD spectroscopy. Introduction of DMPG lipids enhanced the β-sheet composition of the peptides, a conformational change that is required for NFT formation. Since the GM surface is more rich in negative charge and potential hydrogen bonding groups than the PM, the structural changes with PG lipids sheds light on a possible mechanism for protein misfolding events that lead to NFTs. Further details will be presented on the interaction of the peptides with vesicles based on lipids that constitute the PM and GM, as well as the influence that these membranes have on the kinetics of aggregation. These results will help contribute a better understanding for the events that drive tau aggregation.



Abe, Ryota POST 10-113 Abedini, Andisheh POST 10-59 Abrol, Ravinder POST 06-367 Adams, Stephen R. POST 03-276 Adams, Paul D. POST 11-140 Adams, Joseph A. POST 03-308 Aebersold, Ruedi POST 06-349 Affandi, Trisiani POST 11-124 Agyekum, Boadi POST 02-229 Ahmad, Shakeel POST 03-295, POST 12-199 Ahmad, Shadab POST 03-295, POST 12-199 Ahn, Sae Ryun POST 06-337 Ahn, Kwang H. POST 06-367 Ahn, Natalie G. POST 09-422 Ahn, Natalie POST 03-261 Ahuja, Lalima G. POST 10-67 Allison, Timothy M. POST 06-359 Allison, Brittany POST 12-195 Alon, Assaf POST 12-208 Altshuler, Rachel POST 12-183 Anada, Masahiro POST 06-357 Andersson, Ken POST 12-171 André, Ingemar POST 12-197 Anorma, Chelsea POST 10-116 Arase, Hisashi POST 06-357 Archbold, Julia K. POST 03-233 Arias, Cesar A. POST 10-96 Arreola-Barroso, Rodrigo A. POST 12-178 Arroyo Verástegui, Rossana POST 07-384 Ashraf, Mohd. Tashfeen POST 03295, POST 12-199 Atienza, Carmen POST 12-189 Ausar, Salvador F. POST 10-95 Auton, Matthew POST 10-103 Axe, Jennifer M. POST 03-268 Ayna, Adnan POST 03-243


Babbitt, Patricia POST 05-328, POST 08-400 Baek, Kwang-Hyun POST 04-315 Bah, Alaji POST 12-151 Baker, David POST 03-240 Baker, Tania POST 04-318 Baker, Tania A. POST 04-319 Baker, Dr. David POST 12-214 Baková, Michaela POST 03-284 Balasubramaniam, Deepa POST 10-85 Balch, William POST 10-120

Baldwin, Andrew POST 11-132 Ballard, Keith POST 09-423 Balta, Bulent POST 07-396 Baltz, Morgan R. POST 12-211 Bandak, Diana POST 10-116 Bandi, Swati POST 07-390 Banerjee, Shounak POST 12-183, POST 12-218 Banister, Sarah POST 03-294 Bao, Ju POST 10-72 Barajas, Jesus POST 03-296 Bardwell, James C. POST 11-126 Barkdoll-Weil, Forest POST 10-90 Barkho, Sulyman POST 03-308 Barragan-Galvez, Juan C. POST 06-336 Bartle, Emily POST 10-89, POST 10-93 Basiaga, Sara POST 09-437 Basore, Danielle A. POST 12-180 Basore, Danielle A. POST 03-287 Batjargal, Solongo POST 03-239 Baugh, Evan POST 03-287 Baxa, Michael C. POST 07-389 Baytshtok, Vladimir POST 04-319 Beatty, Kimberly POST 03-272 Beauregard, Marc POST 03-252, POST 03-254, POST 03-257, POST 03-259 Beckett, Brian POST 08-405 Beck-Sickinger, Annette G. POST 06-333, POST 10-106 Beeby, Morgan SYMP 05-26 Beelen, Steven POST 07-371 Bell, Anthony POST 12-184 Bell, Ellis POST 03-282, POST 03285, POST 03-290, POST 03-291, POST 03-292, POST 03-306, POST 03-311, POST 04-322, POST 04323, POST 10-89, POST 10-90, POST 10-93 Bell, Jessica POST 03-290, POST 03-291 Bell, Jessica K. POST 02-228 Benesch, Justin POST 11-132 Benítez, Claudia POST 10-98 Benitez Cardoza, Claudia Guadalupe POST 03-274 Benítez Cardoza, Claudia G. POST 07-384 Benner, W. Henry POST 06-370 Bennett, Eric POST 05-327 Berger, Edward A. POST 12-161 Berger, Edward POST 06-343 Berger, Edward A. POST 10-91 Berghuis, Albert POST 03-250

Bergsma, Janet M. POST 09-438 Berkamp, Sabrina POST 06-360, POST 06-362 Bernal, Ana POST 11-134 Bernal-Perez, Lina POST 12-209 Berning, Karsten POST 11-128 Besingi, Richard N. POST 09-430 Beuning, Penny J. POST 03-283 Beuron, Fabienne POST 04-321 Bezsonova, Irina POST 08-416 Bhak, Ghibom POST 09-419 Bi, Jing POST 12-159 Bieschke, Jan POST 10-97 Birge, Paige POST 03-294 Biswas, Himadri POST 10-105 Biverstål, Henrik POST 10-86 Bjerneld, Erik POST 08-415 Bjorkman, Pamela SYMP 06-46 Blackledge, Martin POST 10-83 Blackwell, Tim POST 12-222 Blanchette, Craig D. POST 06-368 Blank, Volker POST 12-147, POST 12-148 Blankenship, Robert E. POST 06345 Blois, Tracy POST 06-369 Boassa, Daniela POST 03-276 Bobik, Thomas A. POST 02-225 Bobik, Thomas A. POST 03-310 Boehr, David D. POST 03-248 Boehr, David D. POST 03-268 Bogyo, Matthew SYMP 11-20 Bolduc, Jill M. POST 03-240 Bolon, Dan SYMP 05-25 Bonn, Ryan M. POST 09-438 Bonsor, Daniel A. POST 10-58 Borchers, Christoph H. POST 11126 Borgo, Ben POST 05-329 Botyriute, Akvile POST 09-421 Bourguet, Feliza A. POST 06-368 Bouyain, Samuel E. POST 06-340 Bouyain, Samuel POST 02-229 Bowie, James U. POST 08-409 Bowie, James U. POST 02-230 Bowie, James POST 06-369 Bowie, James U. POST 06-363 Boyd, Courtney POST 03-247 Brady, Jeannine POST 07-387 Brady, L. Jeannine POST 09-429, POST 09-430 Brajcich, Michelle POST 03-247 Brar, Gloria SYMP 03-01 Brautigam, Chad POST 06-332 Brautigam, Chad A. POST 03-234 Brieba, Luis G. POST 10-98


AUTHOR/SPEAKER INDEX Brieba-Castro, Luis POST 06-336 Brieba de Castro, Luis G. POST 07384 Britton, Robert POST 07-381 Brizendine, Ashley POST 08-399 Broersen, Kerensa POST 10-78 Brooks, Teresa M. POST 10-53 Brooks, Cory L. POST 10-53 Brooks, Cory POST 10-54 Brooks, Shayla A. POST 11-142 Brown, Breann POST 04-318 Brown, Zuben POST 12-185, POST 12-216 Brown, Kyle L. POST 12-222 Bucher, Denis POST 11-129 Budhathoki, Pradeep POST 12-207 Buenavista, Maria Teresa POST 10-104 Buhrman, Jason S. POST 10-114 Bundoc, Virgilio POST 10-91, POST 12-161 Burban, David J. POST 07-392 Bushweller, John POST 10-99 Bustos, Cheene POST 03-310 Butterfoss, Glen POST 03-287, POST 12-180 Bystroff, Christopher POST 03-287, POST 12-174, POST 12-180, POST 12-183, POST 12-218


Caaveiro, Jose POST 07-386, POST 10-87 Caaveiro, Jose M. POST 06-358 Caaveiro, Jose M. POST 10-113 Cabaleiro-Lago, Celia POST 10-68 Caballero, Diego POST 07-393 Cade, Nicholas POST 02-227 Cao, Ping POST 10-59 Capovilla, Alexio POST 06-338 Capraro, Dominique POST 07-392 Carballo-Amador, Manuel A. POST 12-146 Carcamo-Noriega, Edson N. POST 09-425 Carraway, Kermit POST 10-57 Carrillo-Ibarra, Normande POST 03274 Carroll, Candace POST 10-72 Carver, John POST 11-132 Cascarina, Sean POST 09-434 Cascio, Duilio POST 02-230, POST 03-310, POST 11-132 Cash, Jennifer POST 10-55 Catazaro, Jonathan POST 09-437

Cavicchioli, Ricardo POST 11-123 Cerutti, Nichole M. POST 06-338 Cesnekova, Jana POST 04-320 Chacon, Kelly M. POST 06-335 Chadwick, Alexandra POST 03-241 Chakravarty, Suvobrata POST 02226, POST 11-131 Chan, Yan M. POST 03-248 Chan, Michael K. POST 03-278 Chand, Subhash POST 03-267 Chandrashekar, Reena POST 11140 Chánez, Maria E. POST 07-391 Chang, Dennis POST 10-57 Chang, Roger POST 05-331 Chang, Yonggang POST 11-133 Chang, Yu-Chu POST 06-363 Chao, Frank A. POST 08-414 Chapman, Matthew R. POST 09433 Chapman, Matthew SYMP 04-05 Chatani, Eri POST 07-385, POST 10-80 Chattopadhyaya, Rajagopal POST 10-105 Chaudhary, Nidhee POST 03-267 Chaudhary, Akash POST 03-295, POST 12-199 Chen, Yiliang POST 03-241 Chen, Yao POST 10-69 Chen, Xi POST 12-219 Chen, Liqing POST 06-366 Cheung, Hon-Yeung POST 03-299 Chiang, Chu-Harn POST 07-388 Chica, Roberto A. POST 12-173 Chmielewski, Jean POST 12-144 Chmielewski , Jean POST 10-50 Choi, Angela POST 12-183 Chong-yang, Zhu POST 03-300, POST 03-303 Choudhury, Shilpa POST 06-351, POST 08-412 Chowdhury, Chiranjit POST 02-225 Chowdhury, Sudeshna POST 03281 Christ, Daniel POST 08-397 Chuang, Kuo-Hsiang POST 12-193 Chun, Sunny POST 02-225 Chung, Hokyung K. POST 03-242 Church, George POST 05-331 Churchfield, Lewis POST 08-414 Churchman, L. Stirling SYMP 03-02 Clancy, Shonda POST 10-117 Clarke, Jane POST 12-169, SYMP 01-23 Clasman, Jozlyn R. POST 03-263

Colelli, Kathryn POST 03-231 Coleman, Matthew A. POST 10-57 Coleman, Matthew A. POST 06-368 Coleman, Matt POST 06-370 Collier, Miranda POST 11-132 Collyer, Charles A. POST 10-77 Colon, Selene POST 12-222 Corbo, Claudia POST 06-355 Correa-Basurto, Jose POST 03-274 Cortajarena, Aitziber L. POST 12189 Corzilius, Bjorn POST 09-427 Couleaud, Pierre POST 12-189 Cox, Dezerea POST 11-132 Craig, Connor P. POST 03-253 Cravatt, Benjamin F. POST 03-280 Cravatt, Benjamin SYMP 07-13 Crawford, Terry POST 04-316 Crone, Donna E. POST 12-183, POST 12-218 Crosby, Christine EW II-48 Crowhurst, Karin A. POST 11-142 Crowley, Paula POST 07-387 Crump, Matthew . POST 12-150 Cui, Li POST 03-254 Cukalevski, Risto POST 10-65 Culp, Megan POST 10-112 Curmi, Paul M. POST 11-123 Curnow, Paul POST 06-344, POST 06-346, POST 06-348, POST 12166


da Fonseca, Paula POST 04-321, SYMP 11-19 Daman, Andrew POST 12-191 Das, Bibhuti POST 06-360, POST 06-362 Dattelbaum, Jonathan POST 12167 Davey, James A. POST 12-173 Davis, Ellen POST 10-55 Davis, Joseph H. POST 07-381 Davlieva, Milya POST 10-96 Day , Richard POST 12-200 De Angelis, Anna POST 06-360, POST 06-362 Deb, Arpan POST 06-366 Deerinck, Thomas J. POST 03-276 Deka, Swapnav POST 06-332 Deka, Ranjit K. POST 03-234 DeLisa, Matthew P. POST 12-153 DeLisa, Matthew P. POST 12-211 Dembinski, Holly E. POST 11-137 Demir, Ozlem POST 10-101


AUTHOR/SPEAKER INDEX Dennis, Edward A. POST 11-129 Derewenda, Zygmunt POST 12-188 Deroo, Stéphanie POST 06-349 Deshayes, Kurt POST 04-316 De Sutter, Delphine POST 05-326 Dewhurst, Henry POST 06-351, POST 08-412, POST 09-432 Dey, Moul POST 11-131 Dey, Barna POST 10-91 Dickson, Alan J. POST 12-146 Di Lello, Paola POST 04-316 DiMaio, Daniel POST 06-335 Dinler-Doganay, Gizem POST 07396 Dobson, Renwick C. POST 03-236 Dodard-Friedman, Isadore POST 12-148 Dolfe, Lisa POST 10-86 Donald, Bruce R. POST 11-130 Dordick, Jonathan S. POST 12-183, POST 12-218 Doucet, Nicolas POST 11-139 Dowhan, William SYMP 12-38 Doyle, Shannon POST 11-135 Drobnick, Joy POST 04-316 Drummond, Jake POST 04-316 Duda, David POST 10-69 Duellberg, Christian POST 02-227 Duhr, Stefan POST 10-92 Dvir, Hay POST 10-81 Dzwolak, Wojciech POST 09-435


Ebert, Maximilian POST 03-250 Ecroyd, Heath POST 11-132 Eda, Kazuo POST 07-385 Efremov, Rouslan POST 06-349 Eisenberg, David POST 11-132, POST 12-188 Eisenmesser, Elan Z. POST 10-71 Eitan-Wexler, Maayan POST 08404 Elahi, Montasir POST 08-398 Eller, Chelcie H. POST 10-110 Ellisman, Mark H. POST 03-276 Elpidina, Elena N. POST 03-298 Elpidina, Elena POST 03-251 Engler , David A. POST 06-355 Engler , Anthony C. POST 06-355 Ernst, James POST 04-316 Escalante, Carlos R. POST 10-115 Essel, Francisca POST 02-226, POST 11-131 Euler, Christian K. POST 12-173

Evangelopoulos , Michael POST 06-355 Evans, Margery L. POST 09-433 Expression group, Structural Biology POST 04-316 Eyckerman, Sven POST 05-326


Fairlie, David P. POST 03-233 Fallis, Lyndsey POST 03-298 Fan, Jin-Yuan POST 02-229 Faraggi, Eshel POST 07-383 Farmer, Rohit POST 12-150 Farrell, Eric POST 11-138 Fekner, Tomasz POST 03-278 Fetrow, Jacquelyn POST 05-328, POST 08-400, POST 08-401, POST 08-408 Fetrow, Jacquelyn POST 08-406 Fetrow, Jacquelyn S. POST 08-410 Filippova, Irina Y. POST 03-298 Filippova, Irina POST 03-251 Fischer, Nicholas O. POST 06-368 Fishpaugh, Jeffrey R. POST 09-438 Fleetwood, Filippa POST 12-171 Fleishman, Sarel J. POST 12-208 Fleishman, Sarel POST 12-170 Floyd, Sally POST 05-330 Ford, Nicole POST 10-92 Forman-Kay, Julie POST 12-151, SYMP 04-08 Forouhar, Farhad POST 03-240 Forsaith, Marc POST 06-348 Frank, Matthias POST 06-370 Fraser, Keith POST 12-183 Frederick, Kendra K. POST 09-427 Freed, Karl F. POST 07-389 Friedler, Assaf POST 10-58 Frohm, Birgitta POST 10-65 Fromme, Petra POST 06-366 Fry, Michelle Y. POST 08-405 Fuchs, Stephen POST 02-224 Fuglebakk, Edvin POST 03-256 Fujii, Satoshi POST 12-210 Fujii, Yuki POST 12-205 Fujimoto, Mayu POST 03-304 Fukakusa, Shunsuke POST 10-107 Fukamizo, Tamo POST 03-244, POST 03-245 Furuie, Yoshito POST 07-385 Furukawa, Atsushi POST 06-357 Fusa, Shuhei POST 07-380


Galuszka, Petr POST 03-284

Gao, Jinlong POST 10-77 Garcia, Jorge D. POST 03-269 Garcia, Ponciano POST 10-98 Garcia, Chris SYMP 12-37 Garcia-Diaz, Miguel POST 03-301 Gardner, Nathan POST 07-372 Garreta, Luis POST 12-183 Gassaway, Brandon POST 12-164 Gemeinhart, Richard A. POST 10114 Gene, Robert POST 10-54 Geng, Xi POST 12-175 Gevaert, Kris POST 05-326 Ghanem, Mustafa POST 10-91 Ghribi, Manel POST 03-252 Gilbert, Erin POST 12-183 Gilmore, Petra E. POST 06-354 Gintner, Lucas POST 12-194 Glasner, Margaret E. POST 08-399 Gleaton, Jeremy POST 12-144 Glukhova, Alisa POST 10-52 Goddard III, William A. POST 06367 Goldman, Adrian POST 10-118 Goldschmidt, Luki POST 12-188 Gonzalez, Teresa POST 12-189 Goodwin, Douglas POST 03-277 Goptar, Irina POST 03-251 Goto, Natalie K. POST 12-213 Goulding, Celia W. POST 08-407 Govinda Remesh, Soumya POST 10-115 Greene, Lesley H. POST 08-402 Gregory, Svetoslava D. POST 09438 Griffin, Michael D. POST 03-236 Griffin, Robert G. POST 09-427 Gross, Richard POST 12-180 Gross, Richard A. POST 03-287 Grove, Tijana POST 12-175 Grove, Tijana Z. POST 12-177 Gruschus, James M. POST 08-418 Gruszka, Dominika T. POST 12-169 Gui, Shanying POST 03-286 Guja, Kip POST 03-301 Gunsel, Umut POST 07-396 Gurrola Acosta, MIsrain E. POST 07-391 Guzman, Carol POST 10-90


Hackel, Benjamin POST 12-143 Hackel, Benjamin J. POST 12-182 Haddadian, Esmael POST 07-389 Hadi, Masood Z. POST 06-368


AUTHOR/SPEAKER INDEX Hahn, Klaus SYMP 10-36 Hailey, Kendra POST 10-100 Haimovich, Adrian POST 12-164 Haines, Anthony S. POST 12-150 Hajduczki, Agnes POST 12-161 Halili, Maria A. POST 03-233 Halouska, Steven POST 09-437 Hamakubo, Takao POST 07-386 Hams, Nicole POST 06-365 Handel, Tracy POST 06-353 Handley, Lindsey D. POST 10-119 Hannan, Ross POST 03-297 Hannan, Katherine POST 03-297 Harada, Erisa POST 11-127 Harper, Angela POST 08-401 Harry, David POST 04-322, POST 04-323 Hashimoto, Shunichi POST 06-357 Haugner, John C. POST 08-414 Hau-Riege, Stefan POST 06-370 Havranek, Jim POST 05-329 Hayden, Julia POST 08-408 Hayes, Christopher S. POST 08407 Hayre, N. Robert POST 12-219 He, Wei POST 06-368, POST 10-57 He, Liu POST 03-302 Heald, Rebecca SYMP 08-17 Hébert-Ouellet, Yannick POST 03259 Hecht, Michael SYMP 09-29 Heddle, Jonathan G. POST 12-185 Heddle, Jonathan G. POST 12-216 Hefzi, Hooman POST 05-331 Heim, Erin N. POST 06-335 Heim, Kyle P. POST 07-387 Heine, Holger POST 10-79 Heirbaut, Michelle POST 07-371 Henderson, Paul POST 10-57 Henderson, Rory POST 11-141 Henry, Nicolas POST 06-349 Henry, Ralph POST 11-141 Hermansson, Erik POST 10-86 Hernández-Vázquez, Christian A. POST 03-307 Herrera, Esperanza POST 03-307 Hess, Sara K. POST 06-350 Hevel, Joan POST 03-286 Heyes, Colin D. POST 11-140 Heyes, Colin POST 11-141 Higuchi, Yoshiki POST 09-428 Hilvert, Donald POST 08-417 Hinkovska-Galcheva, Vania POST 10-52 Hinsen, Konrad POST 03-256 Hirosawa, Narumi POST 03-289

Hirota, Shun POST 09-428 Hluska, Tomáš POST 03-284 Hoang, Quyen POST 10-87 Hoang-Phou, Steven POST 10-57 Hochberg, Georg POST 11-132 Hockla, Alexandra POST 12-217 Holden, Jeffrey K. POST 10-70 Holden, Lauren POST 06-353 Holec, Patrick POST 12-143 Holme, Rebecca L. POST 03-241 Holmes, David POST 02-227 Holton, Thomas POST 12-188 Honda, Shinya POST 12-160 Honda, Takeshi POST 10-107 Hongo, Kunihiro POST 07-380, POST 07-382 Hoppe, Adam POST 11-131 Hori, Hiroshi POST 07-385 Horiuchi, Masataka POST 10-108 Horiuchi, Rumi POST 10-108 Horng, Jia-Cherng POST 07-388 Horowitz, Scott POST 11-126 Hoskins, Joel POST 11-135 Houk, Kendall N. POST 02-225 House, Colin POST 03-297 Houstek, Josef POST 04-320 Hsu, Chih-An POST 03-237 Hu, Jiancheng POST 10-67 Huang, Yao-ming POST 12-174, POST 12-183 Huang, Yao-Ming POST 12-218 Huang, Tai-huang POST 04-324 Huber, Thaddaus R. POST 12-221 Hubin, Ellen POST 10-78 Hudson, Billy POST 12-222 Hughes, Roy POST 09-426 Huh, Ian POST 10-54 Hui, Elliot E. POST 03-273 Hunter, Mark POST 06-370 Huo, Yunwen POST 03-242 Hussain, Rohanah POST 10-104 Hyde, Alaina POST 10-93


Ichihashi, Norikazu POST 08-411, POST 12-162 Ieong, Pek POST 10-101 Iida, Tetsuya POST 10-107 Iizuka, Ippei POST 03-304 Ikeda, Masashi POST 07-380 Imai, Kiyohiro POST 09-428 Imamoglu, Rahmi POST 07-396 Inaba, Kenji POST 03-233 Inaka, Koji POST 03-304 Iosub-Amir, Anat POST 10-58

Irbäck, Anders POST 09-424 Isaacs, Farren POST 12-164 Ishizuka-Katsura, Yoshiko POST 06-356 Issaian, Aaron V. POST 11-124 Iwamoto, Aikichi POST 06-341 Iwasaki, Kenji POST 12-185, POST 12-216 J Jacavone, Angela C. POST 09-427 Jackman, Brianna M. POST 03-263 Jackson, Sophie E. POST 07-375 Jackson, Paul J. POST 06-368 Jacobs, Conor POST 03-242 Jacobsen, Michael T. POST 12-156 Jäger, Linda POST 08-417 Jaikaran, Anna S. POST 12-151 Jain, Nikhil POST 07-381 Jain, Nitin POST 11-134 Jain, Neha POST 09-433 Jamin, Marc POST 10-83 Jarvik, Nick POST 12-212 Jefferson, Robert POST 06-369 Jeng, Wen-Yih POST 03-237 Jen-Jacobson, Linda POST 03-270 Jennings, Patricia A. POST 03-308, POST 10-100, POST 10-121 Jennings, Patricia POST 07-392 Jensen, Malene R. POST 10-83 Jez, Joseph M. POST 03-260 Jiang, Zhiping POST 10-75 Jo, Hyunil POST 12-202 Joachimiak, Andrzej POST 10-117 Johansson, Jan POST 10-86 Johansson, Johan POST 08-415 Johnson, Parker M. POST 08-407 Johnson, Lucas POST 12-194 Johnson, Troy POST 10-96 Johnson, Colin P. POST 06-365 Johnson, William POST 06-366 Jomaa, Admad POST 07-381 Jones, Krysten A. POST 03-273 Jones, Victoria POST 12-183 Jones, Rachel POST 03-282 Jones, Bryan E. POST 10-100 Jònsson, Sigurdur Æ. POST 09-424 Jorda, Julien POST 03-310 Jugnarain, Vinesh POST 06-338 Jun, Zhang POST 03-258, POST 03-300, POST 03-303, POST 10-94 June, Cynthia M. POST 03-263



Kadumoori, Rajashekar V. POST 12-201 Kaempfer, Raymond POST 08-404 Kailasan, Shweta POST 07-387 Kakita, Kosuke POST 06-357 Kalastavadi, Tejas POST 04-318 Kamali-Moghaddam, Masood POST 10-109 Kamer, Kimberli J. POST 03-232 Kamiya, Narutoshi POST 12-163 Kang, Soosung POST 10-70 Kannan, Meenakshi B. POST 12147 Kantserova, Nadezda P. POST 1061, POST 10-64 Kao, Tang-Chun POST 07-388 Karamitros, Christos S. POST 1066 Karonicolas, John POST 12-183 Karsai, Arpad POST 12-219 Kast, Peter POST 08-417 Kategaya, Lorna POST 04-316 Kato, Masato POST 09-420 Kaur, Nameet POST 03-267 Kawahara, Kazuki POST 10-107 Kawata, Yasushi POST 07-380, POST 07-382 Kay, Michael S. POST 12-156 Kay, Brian K. POST 06-368 Kazuta, Yasuaki POST 08-411, POST 12-162, POST 12-210 Kehr, Andrew D. POST 03-270 Keillor, Jeffrey W. POST 12-213 Kelly, Robert J. POST 10-52 Kendall, Debra E. POST 06-367 Kendrick, Agnieszka A. POST 1071 Kerckhove, Clara POST 03-291 Kerstetter, Nicole E. POST 03-268 Kesavardhana, Sannula POST 12165 Kessans, Sarah A. POST 03-236 Khare, Sagar D. POST 03-240 Khatri, Vinay POST 03-259 Khersonsky, Olga POST 12-170 Khowala, Suman POST 03-281 Kim, So-Ra POST 04-315 Kim, Jin-Ok POST 04-315 Kim, Hyun-Sook POST 10-60 Kim, Seungkyung POST 03-255 Kim, Inhae POST 08-409 Kim, Sanguk POST 08-409 Kim, SoHo POST 03-290 Kimoto, Hisashi POST 03-244

Kines, Kelsey POST 04-322, POST 04-323 Kingdom, Grace POST 03-311 Kleanthous, Colin SYMP 02-40 Klein, Mark A. POST 12-215 Klinger, Neil V. POST 03-266 Kloczkowski, Andrzej POST 07-383 Kluber, Alexander J. POST 12-219 Knight, Michael POST 06-344, POST 06-346, POST 12-166 Knight, Mary J. POST 02-230 Knowles, Tuomas POST 10-65 Knox, Curtis POST 12-196 Knutson, Stacy POST 05-328, POST 08-400 Kobayashi, Yuji POST 10-107 Kodama, Tatsuhiko POST 07-386 Koide, Akiko POST 12-176 Koide, Shohei POST 12-176 Köllisch, Gabriele POST 10-79 Kolodziejczyk, Robert POST 10-118 Komives, Elizabeth POST 10-85, POST 11-137 Komives, Elizabeth A. POST 03288 Komives, Elizabeth A. POST 10119 Konkle, Mary POST 03-262, POST 03-294 Konrad, Manfred W. POST 10-66 Konuma, Tsuyoshi POST 11-127 Kornev, Alexandr P. POST 10-67 Kosel, David POST 06-333 Kovacich, Chelsea POST 12-196 Krammer, Eva-Maria POST 06-349 Kratzke, Marian POST 12-215 Krendel, Mira POST 12-159 Krishnan, Krish POST 11-136 Krishnan, Yamuna SYMP 07-12 Krumm, Stefanie A. POST 03-242 Kruziki, Max POST 12-143 Krylov, Vyacheslav V. POST 10-64 Kudo, Shota POST 07-386 Kumar, Anil POST 12-151 Kumar, Aditya A. POST 12-165 Kumar, Kiran POST 08-406 Kumar, Suresh POST 11-141 Kumaran, Jyothi POST 10-54 Kumeta, Msahiro POST 07-377 Kung, Chia-Hsiuan C. POST 04-324 Kuntimaddi, Aravinda POST 10-99 Kuroda, Yutaka POST 08-398 Kuroki, Kimiko POST 06-357 Kurpiewski, Michael R. POST 03270 Kusaoke, Hideo POST 03-244

Kwok, Michael POST 04-316 Kwong, Peter SYMP 06-44 Arroyo, Rossana POST 10-98 Cox, Daniel L. POST 12-219 DeGrado, William F. POST 12-202 Geli Fernandez-Penaflor, Maria Isabel SYMP 10-32 Ladbury, John POST 10-96


Laffoon, Megan POST 03-262 Laganowsky, Arthur POST 11-132 Laganowsky, Art POST 06-359 Lai, Yen-Ting POST 12-155 Lam, Stephanie S. POST 03-232 Lam, Kit S. POST 06-368 Lamberson, Colleen POST 12-183 Lamoureux, Guillaume POST 03250 Landau, Mark SYMP 07-10 Langley, David POST 08-397 Latham, Michael P. POST 09-422 Laurence, Ted A. POST 06-368 Lawrence, Kenneth F. POST 02228 Lazic, Ana EW II-48, POST 10-92 Lee, Cheng-Chung POST 03-237 Lee, Thomas POST 09-422 Lee, Soon Goo POST 03-260 Lee, Ho-Jin POST 10-72 Lee, Heewon POST 10-74 Lee, Jennifer C. POST 10-75 Lee, Jennifer C. POST 06-350 Lee, Marianne M. POST 03-278 Leettola, Catherine N. POST 02230 Lee†, Kyunglim POST 10-74 Leimer, Tobias POST 06-333 Leket-Mor, Tsafrir POST 06-366 Lenobel, René POST 03-284 Leonard, David A. POST 03-263, POST 03-264 Leonard, Paul POST 10-96 Lermyte, Frederik POST 07-371 Leung, Isabel POST 12-212 Leuthaeuser, Janelle POST 05-328, POST 08-400, POST 08-401, POST 08-406 Leuthaeuser, Janelle POST 08-408 Leuthauser, Janelle B. POST 08410 Lev-Ram, Varda POST 03-276 Lew, Scott POST 03-240 Lewis, Nathan E. POST 05-331


AUTHOR/SPEAKER INDEX Leyrat, Cédric POST 10-83 Li, Linqiu POST 03-299 Li, Wenzhou POST 09-423 Li, Nan POST 10-77 Li, Kan POST 09-426 Li, David POST 03-273 Li, Chun POST 03-292 Li, Xiao-dan POST 06-370 Liao, Xiaoli POST 12-179 Liddle, Jen POST 03-261 Lim, Nicole C. POST 07-375 Lin, Michael Z. POST 03-242 Lin, Tao POST 02-226, POST 11131 Lin, Pen-Jen POST 04-325 Lin, Eugene POST 06-361 Lin, Qingsong POST 06-364 Lindahl, Fredrik POST 03-233 Lindquist, Susan POST 09-427 Linse, Sara POST 10-65, POST 1068 Lipper, Colin H. POST 10-121 Liu, Wei Z. POST 03-234 Liu, Chang POST 12-149 Liu, Wei-Chun POST 03-237 Liu, Cong POST 11-132 Liu, Li POST 10-91 Liu, Gang-yu POST 12-219 LiWang, Andy POST 11-133 Lobner, Elisabeth POST 12-203 Löf, Liza POST 10-109 Löfblom, John POST 12-171 Loh, Stewart N. POST 12-159 Lokensgard, Melissa E. POST 12181 Lomax, Jo E. POST 10-110 Long, Joanna R. POST 09-429 Lopes, Nicholas POST 11-134 Lopez, Mariana POST 08-399 Lopez, Javier POST 12-189 Lopez, Martin R. POST 09-438 Love, John J. POST 03-313, POST 03-314, POST 12-181 Low, David A. POST 08-407 Low, David SYMP 02-42 Lu, Jia POST 03-278 Luisillo-Quiñones, Ericka Fabiola POST 03-307 Luo, Ray POST 03-296 Lu-yu, Zhang POST 10-94 Ly, Cuong POST 04-316 Lysenko, Liudmila A. POST 10-61, POST 10-64


Mabbutt, Bridget C. POST 12-186 MacInnes, Katherine A. POST 06348 Madde, Pranathi POST 10-103 Maenaka, Katsumi POST 06-357 Maeng, Jeehye POST 10-74 Maestre-Reyna, Manuel POST 03237 Maffitt, Mark POST 05-330 Majumdar, Sangita POST 03-281 Majumder, Erica L. POST 06-345 Majumder, Rajib POST 03-281 Makepeace, Karl A. POST 11-126 Makhatadze, George I. POST 03287 Malay, Ali A. POST 12-185 Malay, Ali D. POST 12-216 Malisauskas, Ricardas POST 09421 Mallela, Krishna POST 07-390 Mallon, Ann-Marie POST 10-104 Mamat, Uwe POST 12-196 Manea, Francesca POST 12-186 Mann, Stephen POST 12-166 Marada, Suresh POST 10-72 Marana, Sandro R. POST 03-279 Marcos, Enrique POST 12-214 Marcotte, Ed SYMP 03-04 Marean-Reardon, Carrie POST 12158 Margulies, David POST 03-305 Marieni, Michelle POST 03-231 Marion, James D. POST 03-288 Marston, Jez L. POST 06-335 Marszalek, Piotr POST 07-394, POST 07-395 Martell, Jeffrey D. POST 03-232 Martin, Jennifer L. POST 03-233 Martin, Esther POST 07-371 Martin, Jeffrey W. POST 11-130 Martin, Rachel W. POST 10-116 Martínez-François, Juan Ramón POST 03-249 Masaki, Mika POST 03-304 Mascola, John R. POST 12-165 Masson, Laura POST 12-167 Masterson, Larry R. POST 03-271, POST 09-439 Masterson, Larry POST 10-112 Masuda, Yuuki POST 10-80 Masuda, Akemi POST 12-192 Matsuda, Zene POST 06-341 Matsuura, Tomoaki POST 12-210 Matsuura, Tadashi POST 07-386 Mattern, Andreas POST 10-106

Maurer, Till POST 04-316 Mays, Jacqunae POST 03-285 McAteer, Kathleen POST 12-158 McCammon, J. Andrew POST 11129 McEvoy, Megan M. POST 11-124 McFarland, Benjamin J. POST 12191 McGuffin, Liam James POST 10104 McKenna, Robert POST 07-387 McKinley, Sean W. POST 02-228 McKnight, Steven POST 09-420 McMillan, Andrew POST 08-399 McNamara, Dan E. POST 03-310 Mead, David POST 05-330, POST 12-196 Meddeb, Fatma POST 03-254 Meddeb-Mouelhi, Fatma POST 03252, POST 03-257, POST 03-259 Mehl, Ryan POST 06-365 Meiler, Jens POST 12-195 Meisl, Georg POST 10-65 Mejias, Sara H. POST 12-189 Mendoza-Cozatl, David G. POST 03-269 Meng, Fanling POST 10-59 Menze, Michael POST 03-262, POST 03-294 Mercedes-Camacho, Ana POST 12177 Metz, Carlos POST 10-93 Meyer, Matthew R. POST 06-347, POST 06-352 Meyer, Matthew R. POST 06-354 Michaelis, Vladimir K. POST 09-427 Middleton, Chris T. POST 10-59 Mikula, Kornelia M. POST 10-118 Milkovic, Nicole M. POST 09-437 Millan-Pacheco, Cesar POST 07374 Miller, Liz SYMP 01-21 Mills, Kenneth POST 03-231 Mills, Jeremy POST 03-240 Minardi, Luke POST 12-194 Ming-ming, Lin POST 03-238, POST 03-258, POST 03-300, POST 03-303 Mir, Stephan POST 10-85 Mirasol Melendez, Elibeth POST 03-274 Mirasol Meléndez, Elibeth POST 07-384 Mirzaee, Nima POST 12-219 Mitchell, Joshua M. POST 03-264 Mitchell, Joshua M. POST 03-293


AUTHOR/SPEAKER INDEX Mitternacht, Simon POST 09-424 Mittler, Ron POST 10-121 Mizobata, Tomohiro POST 07-380, POST 07-382 Mizuta, Toshifumi POST 07-382 Mizutani, Kenji POST 06-356 Moisan, Jessica K. POST 03-257 Molinaro, Roberto POST 06-355 Mongeon, Rebecca POST 03-249 Montelongo, David M. POST 10116 Moody, Peter POST 03-243 Moon-Tasson, Laurie POST 10-103 Mootha, Vamsi K. POST 03-232 Mootz, Henning D. POST 03-246 Morales, Yalemi POST 03-286 Morante, Koldo POST 10-113 Morelli, Aleardo POST 08-414 Moreno-Sánchez, Rafael POST 03269 Morgan, Richard POST 10-91 Morikawa, Yasushi POST 03-304 Moriwaki, Yoshitaka POST 10-113 Mörl, Karin POST 06-333 Morrill, Summer M. POST 02-224 Morris, Edward POST 04-321 Morris, Kyla POST 11-140 Morse, Robert P. POST 08-407 Mosaheb, Mohammad POST 02224 Mou, Yun POST 12-206 Mouchlis, Varnavas POST 11-129 Moulick, Roumita POST 07-378 Movahedin, Reza POST 10-53 Mueller, Karl T. POST 06-339 Mukhopadhyay, Samrat POST 09436 Mulligan, Vikram K. POST 03-240 Mundorff, Emily POST 12-190 Mura, Cameron POST 07-379 Murata, Takeshi POST 06-356 Murray, Jeremy POST 04-316


Nagai, Kazuma POST 12-187 Nagao, Satoshi POST 09-428 Nagatoishi, Satoru POST 07-386 Naik, Mandar T. POST 04-324 Najnin, Tahria POST 11-123 Nakamura, Haruki POST 12-163 Nakamura, Kazuo T. POST 03-304 Nakamura, Shota POST 10-107 Nakane, Shuhei POST 06-341 Naldrett, Michael J. POST 04-317 Nam, Hyun Jun POST 08-409

Nasir, Irem POST 10-68 Naumann, Todd A. POST 04-317 Ndontsa, Elizabeth POST 03-277 Ndubaku, Chudi POST 04-316 Nechushtai, Rachel POST 10-121 Nemova, Nina N. POST 10-61, POST 10-64 Nepal , Manish POST 10-50 Newberry, Robert W. POST 07-373 Ngo, John T. POST 03-276 Ngo, Alice POST 12-219 Ngu, Lisa POST 03-283 Nguyen, Nhung T. POST 11-139 Nguyen, Long POST 12-184 Nicastri, Michael POST 03-231 Nikolaienko, Roman M. POST 06340 Nishida, Takamasa POST 07-385 Nishiyama, Kotaro POST 12-162 Nishiyama, Hiroto POST 12-187 Nitzel, Damon POST 03-286 Njuma, Olive J. POST 03-277 Noel, Joseph P. SYMP 05-27 Noguchi, Keiichi POST 08-398 Nomura, Takao POST 06-357 Nomura, Wataru POST 12-192 Nonaka, Takamasa POST 03-304 Norgard, Michael V. POST 03-234 North, Rachel A. POST 03-236 Numata, Tomoyuki POST 03-245 Nutt, David POST 10-104


O'Hara, Danielle N. POST 03-253 O'Hern, Corey S. POST 07-393 O'Neill, Heather POST 09-423 O'Rourke, Kathleen F. POST 03268 Oas, Terrence G. POST 09-426, POST 11-130 Ochiai, Masanori POST 10-108 Oda, Takashi POST 11-127 Odokonyero, Denis POST 08-399 Ogasawara, Wataru POST 03-304 Ogden, Stacey POST 10-72 Ohkubo, Tadayasu POST 10-107 Ohnuma, Takayuki POST 03-244, POST 03-245 Ohta, Kazunori POST 03-304 Okada, HIrofumi POST 03-304 Oki, Hiroya POST 10-107 Olajuyigbe, Folasade M. POST 1056 Oliveir, Felipe POST 10-109 Ollis, Anne A. POST 12-153

Omolu, Abbie POST 12-200 Ondrechen, Mary Jo POST 03-283 Ong, Ta-Chung POST 09-427 Onuchic, José N. POST 10-121 Oosaka, Fumina POST 06-357 Opella, Stanley J. POST 06-360, POST 06-362 Opella, Stanley POST 06-361 Opgenorth, Paul POST 12-204 Oppert, Brenda POST 03-251, POST 03-298 Ortega, Joaquin POST 07-381 Ortega, Jaime POST 10-98 Ortiz-Navarrete, Vianney POST 06336 Ose, Toyoyuki POST 06-357 Oshiro, Satoshi POST 12-160 Osuka, Hisao POST 09-428 Ozaeta, Panfilo F. POST 09-438 Ozaki, Makoto POST 12-187


Paddock, Mark L. POST 10-121 Padmanarayana, Murugesh POST 06-365 Pai, Emil F. SYMP 10-35 Paik, Seung Ryeoul POST 09-419 Palei, Shubhendu POST 03-246 Palida, Sakina F. POST 03-276 Palmer, James POST 08-403 Palsson, Bernhard POST 05-331 Pámanes-Carrasco, Gerardo A. POST 03-307 Panda, Dulal POST 10-73 Panecka, Joanna POST 07-379 Pardi, Arthur POST 09-422 Paredes, Diana I. POST 12-218 Parish, Carol POST 10-89 Park, Chiwook POST 07-372 Park, Tai Hyun POST 06-337 Park, Jiyong POST 02-225 Park, Hyejin POST 03-290 Park, Sang Ho POST 06-360 Park, Sang Ho POST 06-362 Parker, Rachael POST 12-177 Parnell, Jonathan POST 10-85 Parodi, Alessandro POST 06-355 Pastan, Ira POST 10-51 Pastor, Nina POST 07-374 Pastor, Rich POST 04-316 Patel, Bhavik POST 10-91 Paul, Kacy POST 09-434 Peacock, Riley POST 03-247 Pedchenko, Vadim POST 12-222 Pedrini, Bill POST 06-370


AUTHOR/SPEAKER INDEX Pelletier, Joelle POST 03-250 Pentelute, Bradley POST 12-179 Peralta, Maria D.R. POST 12-219 Pernstich, Christian POST 06-348 Perriman, Adam POST 12-166 Petersson, E. James POST 03-239 Petrotchenko, Evgeniy V. POST 11126 Petti, Lisa M. POST 06-335 Pielak, Gary J. POST 03-312 Pierce, Levi C. POST 03-308 Pita, Liz EW I-47 Pitchai, Ganesha p. POST 10-88 Pitman, Derek J. POST 12-174 Pitman, Derek POST 12-183 Pitman, Derek J. POST 12-188 Plemper, Richard K. POST 03-242 Plesner, Annette POST 10-59 Polikarpov, Igor POST 03-279 Pooe, Ofentse J. POST 10-79 Poortinga, Gretchen POST 03-297 Portnoff, Alyse D. POST 12-211 Potts, Jennifer R. POST 12-169 Poulos, Thomas L. POST 10-70 Powers, Rachel A. POST 03-263, POST 03-265 Powers, Rachel A. POST 03-266 Powers, Robert POST 09-437 Powers, Rachel POST 03-293 Pozzi, Ambra POST 12-222 Prashek, Jennifer POST 03-255 Prescher, Jennifer A. POST 03-273 Presto, Jenny POST 10-86 Prevost, Martine POST 06-349 Price, Neil P. POST 04-317 Price, Owen POST 03-286 Price, Jeffrey POST 02-229 Prieto, Judith H. POST 10-102 Pritchard, Kirkwood A. POST 03241 Prokopenko, Khristina N. POST 1061 Przybyla, David POST 12-144 Pyun, Haejun POST 10-74


Qi, Yang POST 11-130 Qian-ying, Zhang POST 03-238, POST 03-258, POST 03-303 Qin, Lingyun POST 10-69 Qin, Ling POST 06-353 Quan, Shu POST 11-126 Quan, Wen POST 03-300, POST 03-303, POST 10-94


Rabideau, Amy POST 12-179 Radisky, Evette S. POST 12-217 Radoicic, Jasmina POST 06-360, POST 06-362 Rae, Tracey D. POST 09-438 Rahman, Nausheen POST 10-95 Raines, Ronald T. POST 07-373, POST 10-110 Raleigh, Daniel P. POST 10-59 Raman, Bakthisaran POST 10-76 Rämisch, Sebastian POST 12-197 Ramsay, Carol S. POST 09-438 Rana, Rajashree POST 10-72 Ranscht, Barbara POST 06-333 Rao, Gururaj A. POST 06-347 Rao, Ch M. POST 10-76 Rao, Gururaj POST 06-352 Rao, Aragula G. POST 06-354 Rapaport, Hanna POST 12-152 Rape, Michael SYMP 11-18 Rappsilber, Juri SYMP 03-03 Rasche, Madeline E. POST 03-310 Ratcliffe, Sarah POST 06-344, POST 06-346, POST 12-166 Rathore, Ujjwal POST 12-165 Ratnayake, Punsisi Upeka POST 06-342 Rauniyar, Priyanka POST 09-431 Raussens, Vincent POST 06-349, POST 10-78 Ray, Shashikant POST 10-73 Rayahin, Jamie E. POST 10-114 Reading, Eamonn POST 06-359 Reardon, Patrick POST 12-158 Reardon, Patrick N. POST 06-339 Reck-Petersen, Sam SYMP 10-34 Rees, Douglas SYMP 12-39 Regan, Lynne POST 07-393, POST 12-157, POST 12-164 Regan, Lynne J. POST 12-200 Reid, Korey M. POST 11-136 Reingewertz, Tali H. POST 10-58 Reinke, Aaron POST 05-327 Reitter, Julie N. POST 03-231 Rendakov, Nikolay L. POST 10-61 Reuter, Nathalie POST 03-256 Reyes-Lopez, Cesar Augusto Sandino POST 03-274 Rinehart, Jesse POST 12-164 Ripa, Perry POST 12-223 Roberts, Sue A. POST 11-124 Robinson, Carol POST 11-132 Robinson, Carol V. POST 06-359 Rogers, William E. POST 10-62 Rogers, Zachary POST 06-368

Rojo, Arturo POST 10-98 Romo-Astorga, Zaira J. POST 03307 Roos, Hakan POST 08-415 Roppongi, Saori POST 03-304 Roque, Cristopher POST 10-95 Rosario, Rosa POST 10-59 Rosas Trigueros, Jorge L. POST 07-384 Rosenberg, Steven A. POST 10-91 Rosenman, David POST 12-183 Ross, Eric D. POST 09-434 Ross, Gary POST 12-223 Rossjohn, Jamie SYMP 06-45 Rouet, Romain POST 08-397 Rouge, Lionel POST 04-316 Roy, Craig SYMP 02-41 Royappa, Grace POST 10-72 Rubert-Perez, Charles M. POST 12144 Ruigrok, Rob POST 10-83 Rule, Gordon S. POST 03-270 Rupprecht, Kevin R. POST 09-438 Rusek, Marta POST 10-111 Ryu, Youngha POST 12-207, POST 12-209


Šebela, Marek POST 03-284


Saab-Rincón, Gloria POST 09-425, POST 12-178 Saha, Piyali POST 12-165 Sahoo, Daisy POST 03-241 Saibil, Helen SYMP 04-07 Saitoh, Takashi POST 06-357 Sakamoto, Yasumitsu POST 03304 Sakamoto, Jiro POST 06-357 Sakamoto, Takeshi POST 03-289 Sakamoto, Yasushi POST 03-289 Saldana, Matthew POST 10-57 Salvatore, Francesco POST 06-355 Sampson, Nicole POST 03-301 Samyn, Noortje POST 05-326 Sanagavarapu, Kalyani POST 1068 Sandal, Priyanka POST 06-354 Sandros, Marinella POST 10-63 Sangani, Sahil POST 03-270 Sankar, Revathi POST 10-84 Sarroukh, Rabia POST 10-78 Sato, Mamoru POST 11-127 Sauer, Robert T. POST 04-319


AUTHOR/SPEAKER INDEX Sawaya, Michael POST 11-132 Sawyer, Nicholas POST 12-164 Saylor, Benjamin D. POST 03-314 Scaria , Shilpa POST 06-355 Scharadin, Tiffany POST 10-57 Schaub, Andrew J. POST 03-296 Schenkelberg, Christian D. POST 12-174 Scheraga, Harold A. POST 07-376 Schertler, Gebhard POST 06-370 Schiffer, Jamie POST 10-85 Schloss, Ashley POST 12-157 Schloss, Ashley C. POST 12-200 Schmidt, Ann Marie POST 10-59 Scholl, Zackary N. POST 07-394, POST 07-395 Schulman, Brenda POST 10-69 Scott, Caitlin E. POST 06-367 Scott, Brandon POST 11-131 Sebesta, Jacob POST 12-221 Seedorff, Jennifer POST 06-343 Seelig, Burckhard POST 08-414 Seelig, Burckhard SYMP 05-24 Seetharaman, Jayaraman POST 03-240 Segelke, Brent W. POST 06-370 Sellmyer, Mark A. POST 03-273 Semashko, Tatiana A. POST 03298 Sen, Saurabh POST 05-330, POST 12-196 Senapati, Sanjib POST 10-84 Senior, Laura POST 06-344, POST 06-346, POST 06-348, POST 12166 Serpell, Louise C. POST 10-78 Serrano, Crystal POST 12-184 Sezerman, Osman U. POST 12-168 Shah, Shweta POST 06-347, POST 06-352 Sham, Yuk POST 12-215 Shamoo, Yousif POST 10-96 Sharikova, Valeriya F. POST 03298 Sharikova, Valeriya F. POST 03251 Shastry, Shankar POST 11-135 Shaw, Andrey S. POST 10-67 Shayman, James A. POST 10-52 Shea, Gabrielle B. POST 08-410 Shen, Wei POST 11-128 Sheung, Anthony POST 10-95 Shi, Zhengshuang POST 03-302 Shi, Yiwen POST 10-96 Shih, Hsiu-Ming POST 04-324 Shimba, Noriko POST 12-163

Shinya, Shoko POST 03-244 Shirke, Abhijit POST 12-180 Shirke, Abhijit N. POST 03-287 Shirouzu, Mikako POST 06-356 Shlaifer, Irina POST 03-235 Shoichet, Brian SYMP 07-11 Shomura, Yasuhito POST 09-428 Shonhai, Addmore POST 10-79 Shyur, Lie-Fen POST 03-237 Siddiqui, Khawar S. POST 11-123 Sidhu, Sachdev POST 12-212 Siegel, Justin B. POST 12-172 Silverman, Richard B. POST 10-70 Simonyan, Lusine POST 11-142 Singh, Surinder POST 07-390 Singh, Rajiv R.P. POST 12-219 Singh, Jay POST 10-120 Sinha, Kaustubh POST 03-270 Sljoka, Adnan POST 06-334 Smirnova, Yulia POST 03-251 Smirnovas, Vytautas POST 09-421 Smith, Amber M. POST 11-125 Smith, Austin E. POST 03-312 Smith , Janet L. POST 11-125 Snow, Christopher POST 12-194, POST 12-221 Sobott, Frank POST 07-371 Solamo, Felix EW III-49 Song, Fei POST 10-59 Sori, Nardos POST 08-402 Sosnick, Tobin R. POST 07-389 Sot, Begoña POST 12-189 Souvignier, Chad POST 12-196 Sparks, Lauren POST 03-271 Sparks, Lauren E. POST 09-439 Squier, Thomas POST 12-158 Srinivas, Raja R. POST 12-203 Srinivas, Dustin POST 06-366 Ståhl, Stefan POST 12-171 Steinmetz, Eric POST 05-330, POST 12-196 Stengel, Florian POST 06-349 Stephens, Erin A. POST 12-211 Stiburek, Lukas POST 04-320 Stock, Daniela POST 08-397 Stoddard, Barry L. POST 03-240 Strelkov, Sergei POST 07-371 Strmiskova, Miroslava POST 12213 Stroud, James POST 11-132 Strutz, Wyatt POST 10-92 Su, Zhengding POST 10-69 Su, An POST 03-287 Sudou, Yukio POST 07-386 Sugase, Kenji POST 11-127, POST 12-154

Sulkowska, Joanna POST 07-392 Sultan, Abdullah POST 10-76 Sunami, Takeshi POST 08-411, POST 12-210 Sundberg, Eric J. POST 10-58 Suon, Peter POST 03-313 Surmacz-Chwedoruk, Weronika POST 09-435 Surrey, Thomas POST 02-227, SYMP 08-16 Suzuki, Hironori POST 03-236 Suzuki, Yoshiyuki POST 03-304 Suzuki, Kano POST 06-356 Svensson, Erika POST 08-415


Taira, Toki POST 03-245 Takahashi, Satoshi SYMP 01-22 Takeyasu, Kunio POST 07-377 Tallman, Katie POST 03-272 Tamaki, Fabio K. POST 03-279 Tamamura, Hirokazu POST 12-192 Tamang, Sanjeeta POST 03-281 Tanaka, Hiroaki POST 03-304 Tanaka, Nobutada POST 03-304 Tanaka, Koji POST 06-358 Tang, Wenxing POST 09-429 Tang, Mary POST 10-114 Tangirala, Ramakrishna POST 1076 Taniguchi, Tooru POST 10-107 Tanimoto, Akiko POST 09-422 Tantama, Mathew POST 03-249 Tarekegn, Meron POST 04-322, POST 04-323 Tasciotti, Ennio POST 06-355 Tashiro, Shinya POST 10-87 Tata, Angela POST 03-311 Tateoka, Chika POST 03-304 Tavernier, Jan POST 05-326 Taylor, Susan S. POST 10-67 Terada, Takaho POST 06-356 Tesmer, John J. POST 10-52 Tesmer, John J. POST 10-55 Textor, Larissa C. POST 03-279 Tezcan, Akif SYMP 09-30 Theobald, Douglas L. POST 08-405 Theodorakis, Emmanuel A. POST 10-121 Thery, Manuel SYMP 08-14 Thomas, Christopher . POST 12150 Thomas, Suzanne POST 03-301 Thulin, Craig D. POST 08-413 Tidor, Bruce POST 12-203


AUTHOR/SPEAKER INDEX Timmerman, Evy POST 05-326 Timucin, Emel POST 12-168 Ting, Alice Y. POST 03-232 Ting, Alice SYMP 07-09 Tischer, Alexander POST 10-103 Titeca, Kevin POST 05-326 Toh, Hiroyuki POST 08-398 Tolbert, Blanton S. POST 10-82 Tomchick, Diana R. POST 03-234 Tomizaki, Kin-ya POST 12-187 Tompa, Peter SYMP 04-06 Toney, Michael POST 12-219 Tong, Liang POST 03-240 Torres, Matthew P. POST 06-351, POST 08-412, POST 09-432 Totsingan, Filbert POST 12-184 Townsend, Ried POST 06-354 Trans, Denise POST 10-57 Traxlmayr, Michael W. POST 12203 Troemel, Emily POST 05-327 Truscott, Kaye SYMP 08-15 Trylska, Joanna POST 07-379 Tsai, Ching-Ju POST 06-370 Tsai, Shiou-Chaun (Sheryl) POST 03-296 Tseng, Roger POST 11-133 Tsenkova, Roumiana POST 10-80 Tsien, Rodger Y. POST 03-242 Tsien, Roger Y. POST 03-276 Tsubaki, Motonari POST 07-385 Tsuchisaka, Yutaro POST 10-80 Tsui, Vickie POST 04-316 Tsumoto, Kouhei POST 06-358, POST 07-386, POST 10-113, POST 10-87, POST 12-154 Tsuruoka, Takaaki POST 12-187 Tu, Ling-Hsien POST 10-59 Turnbull, Joanne L. POST 03-235


Udgaonkar, Jayant B. POST 07-378 Uemura, Tatsuya POST 07-382 Uemura, Yasushi POST 03-289 Ueno, Takamasa POST 12-154 Ulusu, Nuray N. POST 03-275 Umemoto, Naoyuki POST 03-245 Uni, Takeshi POST 09-428 Uno, Keisuke POST 08-411 Urbach, Adam POST 03-309 Ushakova, Natalia V. POST 10-64 Usui, Kenji POST 12-187


Vadrevu, Ramakrishna POST 12201 Valdes-Garcia, Gilberto POST 07374 Vance, Stephen POST 10-63 Vandebussche, Guy POST 06-349 van Nuland, Nico A. POST 10-78 Vanquickelberghe, Emmy POST 05-326 Varadarajan, Raghavan POST 12165 Varner, Jeffrey D. POST 12-211 Varnes, Philip POST 10-89, POST 10-90 Vazquez Contreras, Edgar POST 07-391 Veeraraghavan, Sudha POST 09431 Veglia, Gianluigi POST 08-414 Venkatesan, Anandakrishnan POST 02-229 Verchere, Bruce POST 10-59 Verhee, Annick POST 05-326 Verschueren, Tim POST 07-371 Vicari, Daniele POST 10-77 Vierling, Elizabeth POST 09-423 Vieux, Ellen POST 04-318 Vinokurov, Konstantin S. POST 03298 Vique, José L. POST 10-98 Voigt, Chris SYMP 09-28 Volker, Mark POST 03-271 Vorotnikova, Elena A. POST 03-298 Voss, John C. POST 06-368 Vu, Nhuan POST 10-113


Walden, Patricia M. POST 03-233 Wang, Peter Yingxiao POST 12-145 Wang, Andrew H.-J. POST 03-237 Wang, Lili POST 06-364, POST 11126 Wang, Hui POST 10-59 Wang, Weiping POST 10-69 Wang, Chu POST 03-280 Wang, Ruiying POST 12-217 Wang, Tzu-Chi POST 03-310 Ward, Todd J. POST 04-317 Warner, Lisa R. POST 09-422 Warwicker, Jim POST 12-146 Watson, Jeffrey POST 03-247, POST 08-403 Weeks, Stephen POST 07-371 Weiner, Zachary POST 11-138

Weininger, Ulrich POST 12-197 Weinstock, Matthew T. POST 12156 Weldon, John E. POST 10-51 Wells, Robert POST 12-176 Wen, Tuan-Nan POST 03-237 Werner, Josephine P. POST 03-265 Wertz, Ingrid POST 04-316 Westwood, Brian POST 05-328, POST 08-400, POST 08-406, POST 08-408, POST 08-410 Whelan, Fiona POST 12-169 Wicklow, Donald T. POST 04-317 Wickner, Sue POST 11-135 Wik, Lotta POST 10-109 Williams, Jennie POST 03-231 Williams, Danielle POST 12-157 Williams, Blake POST 12-220 Williamson, James R. POST 07-381 Williamson, James R. POST 10-122 Wilson, Mark A. POST 09-437 Wilson, Ian SYMP 06-43 Winkvist, Maria POST 08-415 Winn, Peter J. POST 12-150 Wismer, Kevin POST 11-137 Wittrup, Karl D. POST 12-203 Woldring, Daniel R. POST 12-182 Woodard, DaNae POST 08-399 Woodruff, Matthew POST 03-294 Woods, Simone POST 03-297 Woolfson, Dek SYMP 09-31 Woolley, G. Andrew POST 12-151 Wu, Chun-Xiang POST 10-87 Wu, Di POST 10-109 Wu, Ruiying POST 10-117 Wu, Xiuifeng POST 12-198 Wyman, Leslie A. POST 03-266 Wysocki, Vicki POST 09-423


Xia, Yan POST 12-183 Xiao, Yao POST 09-422 Xin, Li POST 03-238, POST 03258, POST 03-303, POST 10-94


Yabukarski, Filip POST 10-83 Yachnin, Brahm POST 03-250 Yamada, Takuya POST 09-428 Yamada, Aoi POST 12-187 Yamato, Ichiro POST 06-356 Yan, Junhong POST 10-109 Yanaka, Saeko POST 12-154 Yang, Jianyi POST 11-126 Yang, Jin POST 03-242


AUTHOR/SPEAKER INDEX Yang, Heehong POST 06-337 Yang, Weitao POST 07-394 Yang, Meng POST 03-301 Yang, Chen POST 05-331 Yang, Xiaoting POST 10-65 Yang, Na POST 09-438 Yao, Xiaolan POST 03-255 Yasuhara, Kazuma POST 12-202 Yeates, Todd O. POST 03-310, POST 12-155 Yeates, Todd O. POST 02-225 Yellen, Gary POST 03-249 Yezdimer, Eric M. POST 03-268 Yohda, Masafumi POST 08-398 Yokoyama, Atsuro POST 10-108 Yokoyama, Misaki POST 07-385 Yokoyama, Shigeyuki POST 06-356 Yomo, Tetsuya POST 08-411, POST 12-162, POST 12-210 Yoshida, Takuya POST 10-107 Yoshimura, Shigehiro POST 07-377 Yousef, Mohammed POST 12-223 Yuan, Xianrui POST 03-268 Yun-hong, Wang POST 03-238, POST 03-258, POST 10-94


Zanni, Martin T. POST 10-59 Zarzhitsky, Shlomo POST 12-152 Zatsepin , Nadia POST 06-370 Zeman, Jiri POST 04-320 Zhang, Yang POST 11-126 Zhang, Sheng POST 12-153 Zhang, Jun POST 03-238 Zhang, Jinghua POST 10-59 Zhang, Huashan POST 10-69 Zhang, Yu POST 10-114 Zhang, Zhiwen J. POST 12-220 Zhang, Hua POST 06-361 Zhang, Shao-Qing POST 12-202 Zhang, Yuanming POST 11-138 Zhao, Ray POST 04-316 Zhao, Chunxia POST 06-353 Zhao, Cheng POST 09-438 Zhao, Mitchell J. POST 06-362 Zheng, Huimei POST 12-159 Zheng, Jie POST 10-72 Zheng, Yi POST 06-353 Zheng, Zhilli POST 10-91 Zhou, Zijian POST 03-312

Zhou, Li POST 10-122 Zhou, Hu POST 06-364 Zianni, Michael POST 10-96 Ziemba, Brian SYMP 10-33 Zobel, Kerry POST 04-316


Abstracts of the 28th Annual Symposium of The Protein Society, July 27-30, 2014, San Diego, CA, USA.

Abstracts of the 28th Annual Symposium of The Protein Society, July 27-30, 2014, San Diego, CA, USA. - PDF Download Free
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