ArticlePhysical chemistry chemical physics : PCCP2011
Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).
Article in Physical chemistry chemical physics : PCCP, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 58 papers.
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58 citing papers in PubMed.
- Benchmarking dynamic analysis methods in diffusion-based single-molecule FRET.Cell reports methods · 2026Article
- ABEL-FRET bridges the timescale gap in single-molecule measurements of the structural dynamics in the ACommunications chemistry · 2026Article
- Disentangling Timescales of Molecular Kinetics with spFRET using ALEX-FCS.Journal of fluorescence · 2025Article
- Quantitative analysis methods for free diffusion single-molecule FRET experiments.Current opinion in structural biology · 2025Review
- Model-free photon analysis of diffusion-based single-molecule FRET experiments.Nature communications · 2025Article
- Disordered regions of human eIF4B orchestrate a dynamic self-association landscape.Nature communications · 2024Article
- Conformational dynamics of the hepatitis C virus 3'X RNA.RNA (New York, N.Y.) · 2024Article
- Quantifying Microsecond Solution-Phase Conformational Dynamics of a DNA Hairpin at the Single-Molecule Level.ACS physical chemistry Au · 2024Article
- Pulsed-Interleaved-Excitation Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.The journal of physical chemistry. B · 2024Article
- An optofluidic antenna for enhancing the sensitivity of single-emitter measurements.Nature communications · 2024Article
- An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics.Nature communications · 2023Article
- Nuclear magnetic resonance/single molecule fluorescence combinations to study dynamic protein systems.Current opinion in structural biology · 2023Review
- Time-resolved burst variance analysis.Biophysical reports · 2023Article
- Analysis of photon trajectories from diffusing single molecules.The Journal of chemical physics · 2023Article
- Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.Nature methods · 2023Article
- The biophysics of disordered proteins from the point of view of single-molecule fluorescence spectroscopy.Essays in biochemistry · 2022Article
- Identification and quantification of within-burst dynamics in singly labeled single-molecule fluorescence lifetime experiments.Biophysical reports · 2022Article
- Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates.Proceedings of the National Academy of Sciences of the United States of America · 2022Article
- Theory and Analysis of Single-Molecule FRET Experiments.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Synergies of Single Molecule Fluorescence and NMR for the Study of Intrinsically Disordered Proteins.Biomolecules · 2021Review
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7 authors.
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Abstract
Single molecule Förster resonance energy transfer (FRET) experiments are a versatile method for investigating the conformational distributions and dynamics of biological macromolecules. In a common type of experiment, the fluorescence bursts from individual molecules freely diffusing in solution are detected as they pass through the observation volume of a confocal microscope. Correlation analysis of the fluorescence bursts shows that under typical experimental conditions, for time scales up to several tens of milliseconds, the probability for a molecule to return to the confocal volume is greater than the probability of a new molecule being detected. Here we present RASP (recurrence analysis of single particles), a method that is based on this recurrence behavior and allows us to significantly extend the information that can be extracted from single molecule FRET experiments. The number and peak shapes of subpopulations within the sample can be identified essentially in a model-free way by constructing recurrence FRET efficiency histograms. These are obtained by first selecting photon bursts from a small transfer efficiency range (initial bursts), and then building the FRET efficiency histogram only from bursts detected within a short time (the recurrence interval) after the initial bursts. Systematic variation of the recurrence interval allows the kinetics of interconversion between subpopulations to be determined on time scales from ~50 μs up to ~100 ms from equilibrium measurements. We demonstrate the applicability of the method on measurements of several peptides and proteins with different degrees of conformational heterogeneity and folding dynamics. The concepts presented here can be extended to other observables available from single molecule fluorescence experiments.
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