Document Detail


Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).
MedLine Citation:
PMID:  21218223     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Armin Hoffmann; Daniel Nettels; Jennifer Clark; Alessandro Borgia; Sheena E Radford; Jane Clarke; Benjamin Schuler
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-01-07
Journal Detail:
Title:  Physical chemistry chemical physics : PCCP     Volume:  13     ISSN:  1463-9084     ISO Abbreviation:  Phys Chem Chem Phys     Publication Date:  2011 Feb 
Date Detail:
Created Date:  2011-01-19     Completed Date:  2011-07-14     Revised Date:  2013-07-02    
Medline Journal Info:
Nlm Unique ID:  100888160     Medline TA:  Phys Chem Chem Phys     Country:  England    
Other Details:
Languages:  eng     Pagination:  1857-71     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland.
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MeSH Terms
Descriptor/Qualifier:
Diffusion
Fluorescence Resonance Energy Transfer / methods*
Kinetics
Molecular Conformation*
Probability
Protein Structure, Tertiary
Staphylococcal Protein A / chemistry
Grant Support
ID/Acronym/Agency:
064417//Wellcome Trust; 064417//Wellcome Trust; //Biotechnology and Biological Sciences Research Council
Chemical
Reg. No./Substance:
0/Staphylococcal Protein A
Comments/Corrections

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