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.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2-or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the pausedbacktracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.T ranscription in Escherichia coli comprises three stages: initiation, elongation and termination. Initiation, which is typically the rate-limiting and the most regulated stage of transcription, is by itself a complex, multistep process consisting of the following successive steps (1, 2): (i) association of RNA polymerase (RNAP) core enzyme (subunit composition α2ββ′ω) with the promoter specificity factor σ (such as σ70 for transcription of housekeeping genes) to form RNAP holoenzyme; (ii) binding of holoenzyme to the −10 and −35 DNA elements in the promoter recognition sequence (PRS) upstream to the transcription start site (TSS) to form closed promoter complex (RPc); (iii) isomerization of RPc through multiple intermediates into an open promoter complex (RPo), in which a ∼12-bp DNA stretch (bases at registers −10 to +2) is melted to form a transcription bubble, the template DNA strand is inserted into RNAP major cleft positioning the base at register +1 of TSS at the active site, the nontemplate strand is tightly bound by σ70 and the downstream DNA duplex (bases +3 up to +20) is loaded into RNAP β′ DNA-binding clamp; (iv) an abortive initiation step (AI), where binding of nucleoside triphosphate (NTPs) and the start of RNA synthesis leads to formation of an initial transcribing complex (RP ITC ) followed by RNAP cycling through multiple polymerization trials via a DNA scrunching mechanism (3, 4), release of short "abortive transcripts" and repositioning itself in RP O for a new synthesis trial (5-7); and finally, (v) RNAP promoter escape, when enough strain is built in the enzyme, the σ70 undergoes structural transition to relieve blockage of ...

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66 -86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.natively disordered ͉ domain-domain interaction ͉ quaternary structure ͉ FRET ͉ time-resolved T he tumor suppressor p53 is a tetrameric, multidomain transcription factor that plays key roles in maintaining the integrity of the human genome and in DNA repair machinery (1, 2). p53 is a partly intrinsically disordered protein, containing two folded domains: the DNA-binding core domain (CD; residues 94-294) and the tetramerization domain (TetD; residues 323-360) (3, 4). The intrinsically disordered N-terminal domain (NTD; residues 1-94) and C-terminal domain (CTD; residues 360-393) (5, 6) mediate interactions with several proteins such as p300/CBP, MDM2, 14-3-3, and S100 family that in turn regulate the activity of p53. Moreover, the NTD and CTD are the target sites of numerous posttranslational modificiations that modulate the activity of p53.High-resolution structures of the CD and the TetD have been solved by using X-ray crystallography and NMR spectroscopy (3,4,7,8). But, the intrinsic instability and the presence of highly disordered regions in p53 have impeded the application of conventional structural studies on full-length p53 (flp53). A combination of NMR spectroscopy and small-angle X-ray scattering (SAXS) in solution with electron microscopy on immobilized samples was recently used to solve the quaternary structures of a mutationally stabilized human flp53 and its DNA complex (...

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.

Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein–DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

The B domain of protein A (BDPA), a three-helix bundle of 60 residues, folds via a nucleation-condensation mechanism in apparent two-state kinetics. We have applied a time-resolved FRET (tr-FRET) approach to characterize the ensembles of BDPA during chemical denaturation. The distribution of the distance between residues 22 and 55, which are close and separated by helices 2 and 3 in the native state, was determined by global analysis of the time-resolved fluorescence decay curves of the probes. Narrow distributions were observed when the protein was equilibrated in guanidinium chloride (GdmCl) concentrations below 1.5 M (native state, N) and above the transition zone at 2.6-3.0 M GdmCl (denatured state, D). Considerably broader distributions were found around the transition point (2.0 M GdmCl) or much higher GdmCl concentrations (>3.0 M). Comparative global analysis of the tr-FRET data showed a compact denatured state of the protein, characterized by narrow distribution and relatively small mean distance between residues 22 and 55 that was observed at mild denaturing conditions (<3 M GdmCl). This experiment supports the two-state folding mechanism of BDPA and indicates the existence of effective nonlocal, probably hydrophobic, intramolecular interactions that stabilize a pretty uniform ensemble of compact denatured molecules at intermediate denaturing conditions.

The structural heterogeneity of a-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds Graphical abstract Highlights d trFRET-guided DMD simulations are used to study a-synuclein monomer conformations d Multifunctions of a-synuclein are explained by its monomeric structures d Millisecond conformational dynamics of a-synuclein monomer is discovered by smPIFE