Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules (original) (raw)
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Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer
Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.
Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments
Biochemistry, 2017
Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor-acceptor pairs during solution-based smFRET. We use this "caged FRET" methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV r...
Single-Molecule Fluorescence Resonance Energy Transfer
Springer eBooks, 2018
Fluorescence resonance energy transfer (FRET); Single-molecule Förster resonance energy transfer; Single-pair fluorescence resonance energy transfer Definition Single-molecule fluorescence resonance energy transfer (smFRET) is a technique used to measure nanometer-scale distances between specific sites on an individual molecule, usually as a function of time.
A practical guide to single-molecule FRET
Nature Methods, 2008
Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.
Journal of Physical Chemistry B, 2011
Inter-or intramolecular distances of biomolecules can be studied by F€ orster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1À10 nm, and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensitybased methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleed-through, thereby tolerating a high excess (up to 100-fold) of donoronly labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50% in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores, and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in vitro and in vivo FRET-based spectroscopy and imaging. 13361 dx.
Small, 2006
The development of nanobiotechnological devices requires the ability to build various components with nanometer accuracy. DNA is a well-established nanoscale building block that self assembles due to specific interactions that are encoded in its sequence. Recently, it has become possible to couple proteins to DNA, thereby expanding the capabilities of DNA for use with molecular photonics and bioelectronics. Here, we present the design and characterization of a supramolecular Fçrster resonance energy transfer (FRET) system by using a fluorescent protein bound to single-stranded DNA (ssDNA), a fluorophore attached to a second ssDNA molecule, and a complementary strand for hybridizing the two fluorophores together. The FRET efficiency was studied by using both ensemble and single-pair FRET measurements. The distance between the two fluorophores was determined from the single-pair FRET efficiency and could be described by a simple cylindrical model for the DNA. Hence, DNA can be used as a scaffold for positioning fluorescent proteins, as well as traditional fluorophores, with nanometer accuracy and shows great potential for use in the future of nanobiotechnology.
FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET
Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete softwa...
Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble. Advanced microscopy methods have become powerful tools for structural studies of biomolecules. These methods can complement classical biochemical and biophysical techniques 1,2 , but most importantly emerged as key player in understanding structural dynamics 3,4. The underlying biophysical concept is straight forward: Construct a one-dimensional molecular ruler, in which the biochemical state of the system can be read out as a distance-related measure. Such a molecular ruler often uses a photophysical property such as fluorophore brightness or fluorescence lifetime to provide information on the structure of biomolecules in real time 5. A classic example for such a molecular ruler is Förster-type resonance energy transfer (FRET) 5 , which allows achieving structural information with a spatial resolution in the nanometre range and (sub)millisecond temporal resolution 6–10. However, other photophysical effects such as photo-induced electron transfer (PET) 11–14 or protein-induced fluorescence enhancement (PIFE) 15–32 can be used for similar purposes. Since the fluorescent signal can be read out with high time-resolution, even fast conformational changes 33–39 , as well as interactions between biomolecules, can be mapped in physiologically relevant environments in vitro 40,41 and in vivo 42,43 with a sensitivity allowing to address individual molecules. These molecular rulers suffer from limitations such as their restricted distance ranges and the need for labelling with fluorescent dyes. Most importantly, in the assessment of a three-dimensional (dynamic) structure, the largest limitation is embedded in the information accessible to these methods, which at best follow a single distance to capture a complex structural state.
Proceedings of the National Academy of Sciences of the United States of America, 1999
Photon bursts from single diffusing donoracceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of f luorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.
PLOS Computational Biology, 2020
Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit so...