Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-48192
Hoffmann, A; Nettels, D; Clark, J; Borgia, A; Radford, S E; Clarke, J; Schuler, B (2011). Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP). Physical Chemistry Chemical Physics (PCCP), 13(5):1857-1871.
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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.
|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||04 Faculty of Medicine > Department of Biochemistry|
07 Faculty of Science > Department of Biochemistry
|DDC:||570 Life sciences; biology|
|Deposited On:||01 Jun 2011 12:57|
|Last Modified:||27 Nov 2013 18:55|
|Publisher:||Royal Society of Chemistry|
|Additional Information:||Persons who receive the PDF must not make it further available or distribute it|
|Citations:||Web of Science®. Times Cited: 19|
Scopus®. Citation Count: 19
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