Dataset Information

Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).

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 ?s 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.

SUBMITTER: Hoffmann A

PROVIDER: S-EPMC3378030 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).

Hoffmann Armin A Nettels Daniel D Clark Jennifer J Borgia Alessandro A Radford Sheena E SE Clarke Jane J Schuler Benjamin B

Physical chemistry chemical physics : PCCP 20110107 5

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 ...[more]

PMID: 21218223

Similar Datasets

Project description: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 software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.

Dataset Information

Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).

Publications

Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets