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Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein-DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software., (© 2023. Springer Nature Limited.)

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology., (© 2023. The Author(s).)

Membrane proteins fulfil a plethora of vital functions, are major drug targets, and are implicated in many diseases. Their importance, however, is in no way paralleled by our current understanding of the dynamic processes by which these proteins fold into and function within cellular membranes. This is mainly due to fundamental challenges in resolving the structural dynamics of proteins embedded within lipid-bilayer membranes or membrane-mimetic environments. Single-molecule spectroscopy bears great potential for dissecting this complexity. Particularly, single-molecule Förster resonance energy transfer (smFRET), owing to its sensitivity and versatility, has emerged as a new tool for accessing the spatial, temporal, and energetic features of membrane-protein folding reactions, providing unique insights into protein subpopulations and their associated dynamics on timescales ranging from nanoseconds to hours. Here, we review recent advances in the application of smFRET to the structural dynamics of membrane-protein folding and discuss the benefits that this new toolset affords to provide a molecular-level description of the dynamics governing this physiologically and therapeutically eminent class of proteins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

An increasing number of membrane proteins in different membrane-mimetic systems have become accessible to reversible unfolding experiments monitored by well-established ensemble techniques. However, only little information is available about kinetic processes during membrane-protein folding, mainly because of experimental challenges and a lack of methods suitable for observing highly dynamic membrane proteins. Here, we present single-molecule Förster resonance energy transfer (smFRET) confocal spectroscopy as a powerful tool in kinetic studies of membrane-protein folding in membrane-mimetic environments. We have developed a rigorous workflow demonstrating how to identify and quantify such dynamic processes using a set of qualitative, semiquantitative, and quantitative analytical tools. Using this workflow, we analyzed urea-induced folding and unfolding experiments on the α-helical membrane protein Mistic in the presence of the zwitterionic detergent n-dodecylphosphocholine (DPC). We identified two-state interconversion dynamics on the millisecond time scale of a protein folding into and out of detergent micelles. Our results demonstrate that smFRET is a promising tool for probing the chemical physics of membrane-protein structure and dynamics in the complex and anisotropic environment of a hydrophilic/hydrophobic interface, providing insights into protein interconversion dynamics without the need and challenges of synchronization.

Thermodynamic properties of single molecules including enthalpic and entropic contributions are often determined from experiments by a direct control and precise measurement of the local temperature. However, common temperature monitoring techniques using, for example, ultrafine temperature probes can lead to uncertainties as the probe cannot be placed in the vicinity of the molecule of interest. Here, we devised an approach to measure the local temperature in freely diffusing confocal single-molecule Förster Resonance Energy Transfer (smFRET) experiments in situ by directly adding the temperature-sensitive fluorescent dye Rhodamine B, whose fluorescence lifetime serves as a probe of the local temperature in the confocal volume. We demonstrate that the temperature and FRET efficiencies of static and dynamic molecules can be extracted within one measurement simultaneously, without the need of a reference chamber. We anticipate this technique to be particularly useful in the physicochemical analyses of temperature-dependent biomolecular processes from single-molecule measurements. [ABSTRACT FROM AUTHOR]