Protoplasma (2014) 251:317–332 DOI 10.1007/s00709-013-0596-6
SPECIAL ISSUE: NEW/EMERGING TECHNIQUES IN BIOLOGICAL MICROSCOPY
Studying DNA–protein interactions with single-molecule Förster resonance energy transfer Shazia Farooq & Carel Fijen & Johannes Hohlbein
Received: 6 December 2013 / Accepted: 9 December 2013 / Published online: 28 December 2013 # Springer-Verlag Wien 2013
Abstract Single-molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and precise characterisation of dynamic and rarely occurring events, which are otherwise averaged out in ensemble-based experiments. In this review, we will highlight some recent developments that provide new means of studying complex biological systems either by combining smFRET with force-based techniques or by using data obtained from smFRET experiments as constrains for computer-aided modelling. Keywords Single-molecule Förster resonance energy transfer . Fluorescence . Enzymes . DNA . DNA–protein interactions Abbreviations AFM Atomic force microscopy ALEX Alternating-laser excitation ATP Adenosine tri-phosphate BVA Burst variance analysis bp Base pair CAP Catabolite activator protein DNA Deoxyribonucleic acid dsDNA Double-stranded deoxyribonucleic acid dNTP Deoxyribonucleoside tri-phosphate FRET Förster resonance energy transfer Handling Editor: J.W. Borst S. Farooq and C. Fijen contributed equally to this study. S. Farooq : C. Fijen : J. Hohlbein (*) Laboratory of Biophysics, Wageningen UR, Wageningen, The Netherlands e-mail: [email protected]
FPS MFD NMR NPS PDA PIFE Pol quFRET RNA RNAP rNTP smFRET SSB ssDNA TIRF TBP TF
FRET-restrained positioning and screening Multiparameter fluorescence detection Nuclear magnetic resonance Nano-positioning system Probability distribution analysis Protein-induced fluorescence enhancement Polymerase Quenchable Förster resonance energy transfer Ribonucleic acid RNA polymerase Ribonucleoside tri-phosphate Single-molecule Förster resonance energy transfer Single-stranded DNA binding protein Single-stranded deoxyribonucleic acid Total internal reflection fluorescence TATA box binding protein Transcription factor
Introduction and theoretical background In order to understand the structure and function of biomolecular systems despite their often breath-taking complexity, scientists have been developing an ever-growing arsenal of sophisticated instrumentation and analytical methods. Nuclear magnetic resonance (NMR) spectroscopy (Wüthrich 2001; Foster et al. 2007) and X-ray crystallography (Ilari and Savino 2008), for example, provide structural information with atomic resolution, but both methods ultimately fall short of resolving dynamic interactions within and especially between biomolecular complexes under physiologically relevant conditions. A major limitation of conventional biochemical analysis originates from ensemble- and time-averaging effects. In other words, the analysis reports on averaged properties of a population rather than the properties of individual species forming this population. With the
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advent of single-molecule techniques, researchers gained new exciting possibilities to study time-dependent sample distributions, conformational dynamics (Fig. 1a), reaction pathways, intermediate states, and asynchronous reactions (Kapanidis and Strick 2009). In this review, we will focus on an important member of the class of fluorescence based methods namely the singlemolecule Förster resonance energy transfer (smFRET). This methodology allows detecting (relative) changes of distances between two fluorophores in the 2 to 10 nm range thus operating in a range comparable to the size of biomolecules such as proteins, lipids and nucleic acids. We will further limit our review to smFRET-based applications to study structure, dynamics and functions of DNA and DNA/protein interactions. We will also briefly discuss the development of techniques combining smFRET with force-based techniques such as optical and magnetic tweezers. For more general reviews about single-molecule techniques and smFRET, the interested reader is referred to (Moerner 2007; Deniz et al. 2008; Roy et al. 2008; Walter et al. 2008; Hohlbein et al. 2010; Preus and Wilhelmsson 2012; Kim and Ha 2013). Single-molecule Förster resonance energy transfer FRET describes the distance-dependent and non-radiative energy transfer from a donor fluorophore to an acceptor chromophore via a dipole–dipole interaction and was first reported by Theodor Förster more than 60 years ago (Förster 1946, 1948). Three basic conditions need to be fulfilled for FRET to occur: (1) the spectra for donor emission and acceptor absorption must overlap, (2) donor and acceptor must be in close proximity (
SPECIAL ISSUE: NEW/EMERGING TECHNIQUES IN BIOLOGICAL MICROSCOPY
Studying DNA–protein interactions with single-molecule Förster resonance energy transfer Shazia Farooq & Carel Fijen & Johannes Hohlbein
Received: 6 December 2013 / Accepted: 9 December 2013 / Published online: 28 December 2013 # Springer-Verlag Wien 2013
Abstract Single-molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and precise characterisation of dynamic and rarely occurring events, which are otherwise averaged out in ensemble-based experiments. In this review, we will highlight some recent developments that provide new means of studying complex biological systems either by combining smFRET with force-based techniques or by using data obtained from smFRET experiments as constrains for computer-aided modelling. Keywords Single-molecule Förster resonance energy transfer . Fluorescence . Enzymes . DNA . DNA–protein interactions Abbreviations AFM Atomic force microscopy ALEX Alternating-laser excitation ATP Adenosine tri-phosphate BVA Burst variance analysis bp Base pair CAP Catabolite activator protein DNA Deoxyribonucleic acid dsDNA Double-stranded deoxyribonucleic acid dNTP Deoxyribonucleoside tri-phosphate FRET Förster resonance energy transfer Handling Editor: J.W. Borst S. Farooq and C. Fijen contributed equally to this study. S. Farooq : C. Fijen : J. Hohlbein (*) Laboratory of Biophysics, Wageningen UR, Wageningen, The Netherlands e-mail: [email protected]
FPS MFD NMR NPS PDA PIFE Pol quFRET RNA RNAP rNTP smFRET SSB ssDNA TIRF TBP TF
FRET-restrained positioning and screening Multiparameter fluorescence detection Nuclear magnetic resonance Nano-positioning system Probability distribution analysis Protein-induced fluorescence enhancement Polymerase Quenchable Förster resonance energy transfer Ribonucleic acid RNA polymerase Ribonucleoside tri-phosphate Single-molecule Förster resonance energy transfer Single-stranded DNA binding protein Single-stranded deoxyribonucleic acid Total internal reflection fluorescence TATA box binding protein Transcription factor
Introduction and theoretical background In order to understand the structure and function of biomolecular systems despite their often breath-taking complexity, scientists have been developing an ever-growing arsenal of sophisticated instrumentation and analytical methods. Nuclear magnetic resonance (NMR) spectroscopy (Wüthrich 2001; Foster et al. 2007) and X-ray crystallography (Ilari and Savino 2008), for example, provide structural information with atomic resolution, but both methods ultimately fall short of resolving dynamic interactions within and especially between biomolecular complexes under physiologically relevant conditions. A major limitation of conventional biochemical analysis originates from ensemble- and time-averaging effects. In other words, the analysis reports on averaged properties of a population rather than the properties of individual species forming this population. With the
318
S. Farooq et al.
advent of single-molecule techniques, researchers gained new exciting possibilities to study time-dependent sample distributions, conformational dynamics (Fig. 1a), reaction pathways, intermediate states, and asynchronous reactions (Kapanidis and Strick 2009). In this review, we will focus on an important member of the class of fluorescence based methods namely the singlemolecule Förster resonance energy transfer (smFRET). This methodology allows detecting (relative) changes of distances between two fluorophores in the 2 to 10 nm range thus operating in a range comparable to the size of biomolecules such as proteins, lipids and nucleic acids. We will further limit our review to smFRET-based applications to study structure, dynamics and functions of DNA and DNA/protein interactions. We will also briefly discuss the development of techniques combining smFRET with force-based techniques such as optical and magnetic tweezers. For more general reviews about single-molecule techniques and smFRET, the interested reader is referred to (Moerner 2007; Deniz et al. 2008; Roy et al. 2008; Walter et al. 2008; Hohlbein et al. 2010; Preus and Wilhelmsson 2012; Kim and Ha 2013). Single-molecule Förster resonance energy transfer FRET describes the distance-dependent and non-radiative energy transfer from a donor fluorophore to an acceptor chromophore via a dipole–dipole interaction and was first reported by Theodor Förster more than 60 years ago (Förster 1946, 1948). Three basic conditions need to be fulfilled for FRET to occur: (1) the spectra for donor emission and acceptor absorption must overlap, (2) donor and acceptor must be in close proximity (
