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Cite this: Chem. Commun., 2013, 49, 11403 Received 14th August 2013, Accepted 10th October 2013
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Flagella as a novel alignment medium for the measurement of residual dipolar couplings in proteins† Himanshu Singh,a Manish Shukla,ab Basuthkar J. Raoa and Kandala V. R. Chary*ab
DOI: 10.1039/c3cc46233a www.rsc.org/chemcomm
The two flexible rod-like flagella (B500 nm in diameter and 5–15 lm long) of Chlamydomonas reinhardtii, a unicellular green alga, can weakly align molecules in an external magnetic field, thereby enabling the measurement of various residual dipolar couplings in solution NMR spectroscopy.
Measurement of residual dipolar couplings (RDCs) between different pairs of NMR active nuclei has been an important stride in macromolecular three-dimensional structure determination by NMR.1–4 These couplings can be used to refine or/and validate 3D structures. The knowledge of RDCs has enabled speedier structure calculation, at times even with very few or no nOes.5 They have also been shown to improve the accuracy and increase the quality of three-dimensional (3D) structures dramatically.1,2 Unlike the shortrange restraints such as inter-proton distances and torsion angles derived from nOes and J couplings, respectively, RDCs provide unique data on long-range restraints. In particular, this type of information is essential when the molecule under investigation is a multi-domain protein.6 Besides, these couplings are very useful to describe the macromolecular dynamics, chemical shift anisotropies and even in the measurement of bond lengths.7 RDCs arise from the weak alignment of molecules in an external magnetic field.8,9 The utility of RDCs was first demonstrated by Saupe and Englert in their work on small organic molecules.10 The weak alignment of a biomolecule was first reported by mixing it with large particles that form stable liquid crystals at a concentration less than 5% wt/vol.8 Bax and Tjandra were the first to demonstrate this method on proteins using an admixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dicaproyl-sn-glycero-3-phospho-cholinebicelles (DHPC),11 which orient themselves in an external magnetic field by virtue of intrinsic anisotropy of the magnetic susceptibility.12 Following this, a
Tata Institute of Fundamental Research, Mumbai, India. E-mail: [email protected]; Web: http://www.tifr.res.in/Bchary/; Fax: +91 22 2280-4610; Tel: +91 22 2278-2489 b Tata Institute of Fundamental Research, Center for Interdisciplinary Sciences, Hyderabad, India † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc46233a
This journal is
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The Royal Society of Chemistry 2013
a number of alignment media were proposed. They include bicelles (6 nm 12 nm),13 filamentous phage (6.5 nm 2000 nm),14,15 cellulose crystallites (7 nm (100–300) nm),16 an admixture of polyethylene glycol and hexanol,17 a ternary mixture of hexanol, cetylpyridinium Cl/Br and sodium Cl/Br18,19 and a highly hydrated and anisotropically compressed polyacrylamide gel.20,21 More recently, detergent-compatible collagen (6.5 nm 2000 nm),22–25 DNA nanotubes (7 nm 800 nm)26 and nucleic acid G-tetrads with a diameter of 2.5 nm27 were used successfully for weak alignment. All the above-mentioned alignment media are molecular specific and most of them are suitable to work with water-soluble proteins, although few are compatible with membrane proteins dissolved in detergents.28,29 Besides, as the effective size of the alignment media gets larger, the alignment order increases significantly which in turn leads to extensive line broadening of the NMR signals due to strong 1 H–1H long-range dipolar couplings.22 This problem is effectively overcome by reducing the alignment order. This dictates scaling down the concentration of the alignment media. The caveat here is that most of the media are unstable at lower concentrations. For example, bicelles are mostly unstable when their concentration is less than 2.5% wt/vol. Besides, they are stable only at well-defined ranges of temperature, pH and salt concentration. Another example is polyacrylamide gel, which phase separates when the concentration is less than 3% wt/vol. We demonstrate here for the first time the potential use of the flagella of Chlamydomonas reinhardtii, a unicellular green alga of about 10 mm in diameter that leads the photoautotrophic plant cell life-style30 as well as the heterotrophic animal cell life-style,31 as a novel alignment medium that can weakly align molecules in an external magnetic field. Such an alignment enables the measurement of various RDCs using solution NMR spectroscopy. The two flagella with which the algal cell swims are flexible rodlike structures, B500 nm in diameter and 5–15 mm long. These flagella are cellular membrane protrusions encompassed within a circle of nine doublet microtubules surrounding the two central microtubules, which stabilize them.32 Each of these microtubules is made up of a and b-tubulin monomers.33 These tubulins form different periodic units inside microtubule doublets and cause an uneven surface charge distribution on the outside of the doublets. Chem. Commun., 2013, 49, 11403--11405
11403
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Communication Furthermore, the electric dipole moment of the a and b-tubulin monomers and the dimer (ab) were reported to be |pa| = 552 D, |pb| = 1193 D and |pab| = 1740 D, respectively.34 Thus the size and organization of the flagella lead to a net intrinsic dipole moment to align them in an external field.35 The flagella thus cause alignment of molecules by a combination of both electrostatic and steric effects. We have detached flagella from C. reinhardtii cells by adopting a low-pH sucrose method36,37 and purified it for our study. These cells deflagellate after an acid-induced flagellar abscission, which provides a robust method to obtain the flagella, free of cells. This essentially involved subjecting the concentrated cell suspension at pH 7.5 to a brief shock with a low pH of 4.5 for 45–60 s, during which all the cells in the cell culture undergo deflagellation without losing any cell-viability. The cells were brought back to neutral pH and centrifuged to separate the cell-free flagella. The pelleted deflagellated cells, upon resuspension in the neutral pH medium, exhibit efficient regeneration of their flagella (Fig. 1), thereby rendering the cellular system ready for additional rounds of flagella preparation. This deflagellation procedure can be repeated at least three times with the same culture. In the present study, however, we used one round of deflagellation and needed 4 liters cell culture with inexpensive media components to prepare one NMR sample. A step-wise procedure adapted from Witman’s recent protocol is given in the ESI† (Fig. S1).37 The flagella thus isolated and purified appear intact and mostly rod-like, though flexible (ESI,† Fig. S2). To demonstrate the utility of the flagella as an alignment medium we have recorded 1D 2H spectra at 25 1C, without and with different concentrations of flagella taken in an admixture of 90% H2O and 10% 2H2O with 50 mM sodium acetate buffer (pH 5.0) containing 100 mM NaCl. As expected we observed the quadrupolar splitting of the 2H NMR signal arising from the solvent. The splitting increased with increasing flagellar concentration (Fig. 2), indicating that the flagella indeed induce ordering effect, which could be tuned by adjusting their concentration. In the presence of flagella the solution becomes mildly opaque white (ESI,† Fig. S3). Furthermore, a suspension of 12 mg mL 1 of flagella and 200 mM of uniformly 15N-labeled ubiquitin was prepared by dissolving it in a final concentration of 50 mM sodium acetate buffer (pH 5.0) and 100 mM NaCl to measure RDCs arising from the weak alignment of the protein. It is worth mentioning here that the minimum flagella concentration required is 0.6% w/v, which is considerably less compared to the minimum concentrations required by media reported earlier. For example, the minimum concentrations required by bicelles and polyacrylamide gel are
Fig. 1 Bright field images showing flagellar regeneration at different time points following pH shock.
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Fig. 2 1D 2H spectra recorded without and with different concentrations of flagella taken in an admixture of 90% H2O and 10% 2H2O with 50 mM sodium acetate buffer (pH 5.0) and 100 mM NaCl. These spectra were recorded at 25 1C with four scans and a sweep width of 300 Hz on a Bruker Avance 800 MHz NMR spectrometer.
2.5% w/v and 3.0% w/v, respectively. Panels (A) and (B) in Fig. 3 show illustrative examples of the 2D [15N–1H]-IPAP-HSQC spectra for the u-15N labeled ubiquitin protein sample in the absence and presence of 12 mg mL 1 of flagella, respectively. As illustrated in panels (A) and (B) of Fig. 3, we could measure individual RDCs for almost all the 15N–1H amide pairs, and they ranged from 11.0 to + 11.0 Hz. Panels (C) and (D) show the measured DNH values for ubiquitin and a correlation plot between the measured and calculated DNH values based on its reported structure (pdb id: 1ubq)38 respectively. The calculated alignment tensor (Da) and rhombicity (R) for ubiquitin in the presence of flagella were 7.38 Hz and 0.127, respectively. Euler angles a, b and g, which define the orientation of the alignment frame with respect to the pdb frame of ubiquitin (PDB ID: 1UBQ) were 43.691, 54.391 and 28.571, respectively. These alignment tensors calculated for ubiquitin in the presence of flagella were found to be quite comparable to the alignment tensors (a = 49.801, b = 33.431, g = 39.441, Da = 9.15 Hz and R = 0.17) derived for ubiquitin using the DMPC–DHPC mixture39 as an alignment medium (see ESI,† Table S4). Furthermore, flagella as an alignment medium showed good pH, temperature and detergent tolerance (ESI,† Fig. S5 and S6). It is worth noting here that the mixture of flagella (12.0 mg mL 1) and the protein was quite stable at low pH (4.0) or in a 100 mM dodecylphosphocholine (DPC) solution for more than seven days. No changes were noticed in HSQC recorded during this period. Unaltered 2H splitting or changes in the stability or lack of precipitation over seven days suggests the stability of this system. The highly organized arrangement of nine doublet microtubules surrounding the two central single microtubules, enclosed by membranes, render the flagella, a biological product, as a novel medium for the measurement of RDCs over a wide range of temperature, pH, high ionic strength or in the presence of detergents. No variation was observed in different This journal is
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The Royal Society of Chemistry 2013
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Fig. 3 Selected regions of 2D [15N–1H]-IPAP-HSQC spectra for 200 mM of uniformly 15N labeled ubiquitin in 50 mM sodium acetate buffer (pH 5.0), 100 mM NaCl without (A) and with (B) 12 mg mL 1 flagella isolated from C. reinhardtii. (A) JNH splittings seen for the isotropic sample and (B) (J + D)NH splittings seen in the presence of flagella along the 15N axis are shown with dotted vertical lines. (C) Range of measured DNH values for ubiquitin. (D) A correlation plot between the measured DNH values for ubiquitin in a sample containing 12 mg mL 1 flagella and those calculated based on its reported structure (pdb id: 1ubq). Deviations in the correlation plot correspond to the flexible C-terminal of ubiquitin.
flagellar preparations. Thus, it provides a robust, cost-effective and reliable alternative to align biomolecules in an external magnetic field and measure RDCs, for structure determination, refinement and validation. The facilities provided by the National Facility for High Field NMR, supported by the Department of Science and Technology, New Delhi, Department of Biotechnology, New Delhi, Council of Scientific and Industrial Research, New Delhi, and Tata Institute of Fundamental Research, Mumbai, India, are gratefully acknowledged. KVRC and BJ thank DST for their JC Bose Fellowships of Department of Science and Technology, New Delhi, India. This manuscript is dedicated to Late Professor V. S. R. Rao.
Notes and references ¨nger, P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, 1 A. T. Bru R. W. Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges and N. S. Pannu, Acta Crystallogr., Sect. D, 1998, 54, 905–921. 2 A. Bax, G. Kontaxis and N. Tjandra, in Methods in Enzymology, ed. V. D. Thomas L. James and S. Uli, Academic Press, 2001, vol. 339, pp. 127–174.
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3 C. Tripathy, A. K. Yan, P. Zhou and B. R. Donald, Res. Comput. Mol. Biol., 2013, 271–284. 4 N. Tjandra, in Encyclopedia of Biophysics, ed. G. K. Roberts, Springer Berlin Heidelberg, 2013, pp. 2213–2221. 5 F. Delaglio, G. Kontaxis and A. Bax, J. Am. Chem. Soc., 2000, 122, 2142–2143. 6 G. Bouvignies, P. Bernado and M. Blackledge, J. Magn. Reson., 2005, 173, 328–338. 7 G. M. Clore and A. M. Gronenborn, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 5891–5898. 8 J. R. Tolman, J. M. Flanagan, M. A. Kennedy and J. H. Prestegard, Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 9279–9283. 9 N. Tjandra and A. Bax, Science, 1997, 278, 1111–1114. 10 A. Saupe and G. Englert, Phys. Rev. Lett., 1963, 11, 462–464. 11 A. Bax and N. Tjandra, J. Biomol. NMR, 1997, 10, 289–292. 12 N. Tjandra and A. Bax, J. Magn. Reson., 1997, 124, 512–515. 13 S. Gaemers and A. Bax, J. Am. Chem. Soc., 2001, 123, 12343–12352. 14 M. Zweckstetter and A. Bax, J. Biomol. NMR, 2001, 20, 365–377. 15 M. R. Hansen, L. Mueller and A. Pardi, Nat. Struct. Mol. Biol., 1998, 5, 1065–1074. 16 A. Y. Denisov, E. Kloser, D. G. Gray and A. K. Mittermaier, J. Biomol. NMR, 2010, 47, 195–204. ¨ckert and G. Otting, J. Am. Chem. Soc., 2000, 122, 7793–7797. 17 M. Ru 18 R. S. Prosser, J. Losonczi and I. Shiyanovskaya, J. Am. Chem. Soc., 1998, 120, 11010–11011. 19 L. G. Barrientos, C. Dolan and A. M. Gronenborn, J. Biomol. NMR, 2000, 16, 329–337. ¨ussinger and S. Grzesiek, J. Biomol. NMR, 2002, 24, 20 S. Meier, D. Ha 351–356. 21 T. Cierpicki and J. H. Bushweller, J. Am. Chem. Soc., 2004, 126, 16259–16266. 22 J. Ma, G. I. Goldberg and N. Tjandra, J. Am. Chem. Soc., 2008, 130, 16148–16149. 23 U. Eliav and G. Navon, J. Am. Chem. Soc., 2006, 128, 15956–15957. 24 K. Kobzar, H. Kessler and B. Luy, Angew. Chem., Int. Ed., 2005, 44, 3145–3147. 25 C. Naumann and P. W. Kuchel, J. Phys. Chem. A, 2008, 112, 8659–8664. 26 S. M. Douglas, J. J. Chou and W. M. Shih, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 6644–6648. 27 J. Lorieau, L. Yao and A. Bax, J. Am. Chem. Soc., 2008, 130, 7536–7537. 28 H.-J. Sass, G. Musco, S. Stahl, P. Wingfield and S. Grzesiek, J. Biomol. NMR, 2000, 18, 303–309. 29 Y. Ishii, M. A. Markus and R. Tycko, J. Biomol. NMR, 2001, 21, 141–151. 30 D. Stern, The Chlamydomonas sourcebook: organellar and metabolic processes, Academic Press, 2009. 31 E. H. Harris, The Chlamydomonas sourcebook: introduction to Chlamydomonas and its laboratory use, Academic Press, 2009. 32 J. Lin, T. Heuser, K. Song, X. Fu and D. Nicastro, PLoS One, 2012, 7, e46494. 33 M. D. Weingarten, A. H. Lockwood, S.-Y. Hwo and M. W. Kirschner, Proc. Natl. Acad. Sci. U. S. A., 1975, 72, 1858–1862. 34 A. Mershin, A. A. Kolomenski, H. A. Schuessler and D. V. Nanopoulos, Biosystems, 2004, 77, 73–85. 35 M. Washizu, M. Shikida, S.-I. Aizawa and H. Hotani, IEEE Trans. Ind. Appl., 1992, 28, 1194–1202. 36 G. B. Witman, K. Carlson, J. Berliner and J. L. Rosenbaum, J. Cell Biol., 1972, 54, 507–539. 37 B. Craige, J. M. Brown and G. B. Witman, Curr. Protoc. Cell Biol., 2013, 3.41. 38 S. Vijay-Kumar, C. E. Bugg and W. J. Cook, J. Mol. Biol., 1987, 194, 531–544. ¨schweiler, J. Am. Chem. 39 J.-C. Hus, W. Peti, C. Griesinger and R. Bru Soc., 2003, 125, 5596–5597.
Chem. Commun., 2013, 49, 11403--11405
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Published on 10 October 2013. Downloaded by University of Illinois at Chicago on 25/10/2014 11:02:09.
COMMUNICATION
Cite this: Chem. Commun., 2013, 49, 11403 Received 14th August 2013, Accepted 10th October 2013
View Article Online View Journal | View Issue
Flagella as a novel alignment medium for the measurement of residual dipolar couplings in proteins† Himanshu Singh,a Manish Shukla,ab Basuthkar J. Raoa and Kandala V. R. Chary*ab
DOI: 10.1039/c3cc46233a www.rsc.org/chemcomm
The two flexible rod-like flagella (B500 nm in diameter and 5–15 lm long) of Chlamydomonas reinhardtii, a unicellular green alga, can weakly align molecules in an external magnetic field, thereby enabling the measurement of various residual dipolar couplings in solution NMR spectroscopy.
Measurement of residual dipolar couplings (RDCs) between different pairs of NMR active nuclei has been an important stride in macromolecular three-dimensional structure determination by NMR.1–4 These couplings can be used to refine or/and validate 3D structures. The knowledge of RDCs has enabled speedier structure calculation, at times even with very few or no nOes.5 They have also been shown to improve the accuracy and increase the quality of three-dimensional (3D) structures dramatically.1,2 Unlike the shortrange restraints such as inter-proton distances and torsion angles derived from nOes and J couplings, respectively, RDCs provide unique data on long-range restraints. In particular, this type of information is essential when the molecule under investigation is a multi-domain protein.6 Besides, these couplings are very useful to describe the macromolecular dynamics, chemical shift anisotropies and even in the measurement of bond lengths.7 RDCs arise from the weak alignment of molecules in an external magnetic field.8,9 The utility of RDCs was first demonstrated by Saupe and Englert in their work on small organic molecules.10 The weak alignment of a biomolecule was first reported by mixing it with large particles that form stable liquid crystals at a concentration less than 5% wt/vol.8 Bax and Tjandra were the first to demonstrate this method on proteins using an admixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dicaproyl-sn-glycero-3-phospho-cholinebicelles (DHPC),11 which orient themselves in an external magnetic field by virtue of intrinsic anisotropy of the magnetic susceptibility.12 Following this, a
Tata Institute of Fundamental Research, Mumbai, India. E-mail: [email protected]; Web: http://www.tifr.res.in/Bchary/; Fax: +91 22 2280-4610; Tel: +91 22 2278-2489 b Tata Institute of Fundamental Research, Center for Interdisciplinary Sciences, Hyderabad, India † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc46233a
This journal is
c
The Royal Society of Chemistry 2013
a number of alignment media were proposed. They include bicelles (6 nm 12 nm),13 filamentous phage (6.5 nm 2000 nm),14,15 cellulose crystallites (7 nm (100–300) nm),16 an admixture of polyethylene glycol and hexanol,17 a ternary mixture of hexanol, cetylpyridinium Cl/Br and sodium Cl/Br18,19 and a highly hydrated and anisotropically compressed polyacrylamide gel.20,21 More recently, detergent-compatible collagen (6.5 nm 2000 nm),22–25 DNA nanotubes (7 nm 800 nm)26 and nucleic acid G-tetrads with a diameter of 2.5 nm27 were used successfully for weak alignment. All the above-mentioned alignment media are molecular specific and most of them are suitable to work with water-soluble proteins, although few are compatible with membrane proteins dissolved in detergents.28,29 Besides, as the effective size of the alignment media gets larger, the alignment order increases significantly which in turn leads to extensive line broadening of the NMR signals due to strong 1 H–1H long-range dipolar couplings.22 This problem is effectively overcome by reducing the alignment order. This dictates scaling down the concentration of the alignment media. The caveat here is that most of the media are unstable at lower concentrations. For example, bicelles are mostly unstable when their concentration is less than 2.5% wt/vol. Besides, they are stable only at well-defined ranges of temperature, pH and salt concentration. Another example is polyacrylamide gel, which phase separates when the concentration is less than 3% wt/vol. We demonstrate here for the first time the potential use of the flagella of Chlamydomonas reinhardtii, a unicellular green alga of about 10 mm in diameter that leads the photoautotrophic plant cell life-style30 as well as the heterotrophic animal cell life-style,31 as a novel alignment medium that can weakly align molecules in an external magnetic field. Such an alignment enables the measurement of various RDCs using solution NMR spectroscopy. The two flagella with which the algal cell swims are flexible rodlike structures, B500 nm in diameter and 5–15 mm long. These flagella are cellular membrane protrusions encompassed within a circle of nine doublet microtubules surrounding the two central microtubules, which stabilize them.32 Each of these microtubules is made up of a and b-tubulin monomers.33 These tubulins form different periodic units inside microtubule doublets and cause an uneven surface charge distribution on the outside of the doublets. Chem. Commun., 2013, 49, 11403--11405
11403
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Communication Furthermore, the electric dipole moment of the a and b-tubulin monomers and the dimer (ab) were reported to be |pa| = 552 D, |pb| = 1193 D and |pab| = 1740 D, respectively.34 Thus the size and organization of the flagella lead to a net intrinsic dipole moment to align them in an external field.35 The flagella thus cause alignment of molecules by a combination of both electrostatic and steric effects. We have detached flagella from C. reinhardtii cells by adopting a low-pH sucrose method36,37 and purified it for our study. These cells deflagellate after an acid-induced flagellar abscission, which provides a robust method to obtain the flagella, free of cells. This essentially involved subjecting the concentrated cell suspension at pH 7.5 to a brief shock with a low pH of 4.5 for 45–60 s, during which all the cells in the cell culture undergo deflagellation without losing any cell-viability. The cells were brought back to neutral pH and centrifuged to separate the cell-free flagella. The pelleted deflagellated cells, upon resuspension in the neutral pH medium, exhibit efficient regeneration of their flagella (Fig. 1), thereby rendering the cellular system ready for additional rounds of flagella preparation. This deflagellation procedure can be repeated at least three times with the same culture. In the present study, however, we used one round of deflagellation and needed 4 liters cell culture with inexpensive media components to prepare one NMR sample. A step-wise procedure adapted from Witman’s recent protocol is given in the ESI† (Fig. S1).37 The flagella thus isolated and purified appear intact and mostly rod-like, though flexible (ESI,† Fig. S2). To demonstrate the utility of the flagella as an alignment medium we have recorded 1D 2H spectra at 25 1C, without and with different concentrations of flagella taken in an admixture of 90% H2O and 10% 2H2O with 50 mM sodium acetate buffer (pH 5.0) containing 100 mM NaCl. As expected we observed the quadrupolar splitting of the 2H NMR signal arising from the solvent. The splitting increased with increasing flagellar concentration (Fig. 2), indicating that the flagella indeed induce ordering effect, which could be tuned by adjusting their concentration. In the presence of flagella the solution becomes mildly opaque white (ESI,† Fig. S3). Furthermore, a suspension of 12 mg mL 1 of flagella and 200 mM of uniformly 15N-labeled ubiquitin was prepared by dissolving it in a final concentration of 50 mM sodium acetate buffer (pH 5.0) and 100 mM NaCl to measure RDCs arising from the weak alignment of the protein. It is worth mentioning here that the minimum flagella concentration required is 0.6% w/v, which is considerably less compared to the minimum concentrations required by media reported earlier. For example, the minimum concentrations required by bicelles and polyacrylamide gel are
Fig. 1 Bright field images showing flagellar regeneration at different time points following pH shock.
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Chem. Commun., 2013, 49, 11403--11405
ChemComm
Fig. 2 1D 2H spectra recorded without and with different concentrations of flagella taken in an admixture of 90% H2O and 10% 2H2O with 50 mM sodium acetate buffer (pH 5.0) and 100 mM NaCl. These spectra were recorded at 25 1C with four scans and a sweep width of 300 Hz on a Bruker Avance 800 MHz NMR spectrometer.
2.5% w/v and 3.0% w/v, respectively. Panels (A) and (B) in Fig. 3 show illustrative examples of the 2D [15N–1H]-IPAP-HSQC spectra for the u-15N labeled ubiquitin protein sample in the absence and presence of 12 mg mL 1 of flagella, respectively. As illustrated in panels (A) and (B) of Fig. 3, we could measure individual RDCs for almost all the 15N–1H amide pairs, and they ranged from 11.0 to + 11.0 Hz. Panels (C) and (D) show the measured DNH values for ubiquitin and a correlation plot between the measured and calculated DNH values based on its reported structure (pdb id: 1ubq)38 respectively. The calculated alignment tensor (Da) and rhombicity (R) for ubiquitin in the presence of flagella were 7.38 Hz and 0.127, respectively. Euler angles a, b and g, which define the orientation of the alignment frame with respect to the pdb frame of ubiquitin (PDB ID: 1UBQ) were 43.691, 54.391 and 28.571, respectively. These alignment tensors calculated for ubiquitin in the presence of flagella were found to be quite comparable to the alignment tensors (a = 49.801, b = 33.431, g = 39.441, Da = 9.15 Hz and R = 0.17) derived for ubiquitin using the DMPC–DHPC mixture39 as an alignment medium (see ESI,† Table S4). Furthermore, flagella as an alignment medium showed good pH, temperature and detergent tolerance (ESI,† Fig. S5 and S6). It is worth noting here that the mixture of flagella (12.0 mg mL 1) and the protein was quite stable at low pH (4.0) or in a 100 mM dodecylphosphocholine (DPC) solution for more than seven days. No changes were noticed in HSQC recorded during this period. Unaltered 2H splitting or changes in the stability or lack of precipitation over seven days suggests the stability of this system. The highly organized arrangement of nine doublet microtubules surrounding the two central single microtubules, enclosed by membranes, render the flagella, a biological product, as a novel medium for the measurement of RDCs over a wide range of temperature, pH, high ionic strength or in the presence of detergents. No variation was observed in different This journal is
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The Royal Society of Chemistry 2013
View Article Online
Published on 10 October 2013. Downloaded by University of Illinois at Chicago on 25/10/2014 11:02:09.
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Fig. 3 Selected regions of 2D [15N–1H]-IPAP-HSQC spectra for 200 mM of uniformly 15N labeled ubiquitin in 50 mM sodium acetate buffer (pH 5.0), 100 mM NaCl without (A) and with (B) 12 mg mL 1 flagella isolated from C. reinhardtii. (A) JNH splittings seen for the isotropic sample and (B) (J + D)NH splittings seen in the presence of flagella along the 15N axis are shown with dotted vertical lines. (C) Range of measured DNH values for ubiquitin. (D) A correlation plot between the measured DNH values for ubiquitin in a sample containing 12 mg mL 1 flagella and those calculated based on its reported structure (pdb id: 1ubq). Deviations in the correlation plot correspond to the flexible C-terminal of ubiquitin.
flagellar preparations. Thus, it provides a robust, cost-effective and reliable alternative to align biomolecules in an external magnetic field and measure RDCs, for structure determination, refinement and validation. The facilities provided by the National Facility for High Field NMR, supported by the Department of Science and Technology, New Delhi, Department of Biotechnology, New Delhi, Council of Scientific and Industrial Research, New Delhi, and Tata Institute of Fundamental Research, Mumbai, India, are gratefully acknowledged. KVRC and BJ thank DST for their JC Bose Fellowships of Department of Science and Technology, New Delhi, India. This manuscript is dedicated to Late Professor V. S. R. Rao.
Notes and references ¨nger, P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, 1 A. T. Bru R. W. Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges and N. S. Pannu, Acta Crystallogr., Sect. D, 1998, 54, 905–921. 2 A. Bax, G. Kontaxis and N. Tjandra, in Methods in Enzymology, ed. V. D. Thomas L. James and S. Uli, Academic Press, 2001, vol. 339, pp. 127–174.
This journal is
c
The Royal Society of Chemistry 2013
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