Identifying molecular dynamics in single-molecule FRET experiments with burst variance analysis.

Abstract:

:Histograms of single-molecule Förster resonance energy transfer (FRET) efficiency are often used to study the structures of biomolecules and relate these structures to function. Methods like probability distribution analysis analyze FRET histograms to detect heterogeneities in molecular structure, but they cannot determine whether this heterogeneity arises from dynamic processes or from the coexistence of several static structures. To this end, we introduce burst variance analysis (BVA), a method that detects dynamics by comparing the standard deviation of FRET from individual molecules over time to that expected from theory. Both simulations and experiments on DNA hairpins show that BVA can distinguish between static and dynamic sources of heterogeneity in single-molecule FRET histograms and can test models of dynamics against the observed standard deviation information. Using BVA, we analyzed the fingers-closing transition in the Klenow fragment of Escherichia coli DNA polymerase I and identified substantial dynamics in polymerase complexes formed prior to nucleotide incorporation; these dynamics may be important for the fidelity of DNA synthesis. We expect BVA to be broadly applicable to single-molecule FRET studies of molecular structure and to complement approaches such as probability distribution analysis and fluorescence correlation spectroscopy in studying molecular dynamics.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Torella JP,Holden SJ,Santoso Y,Hohlbein J,Kapanidis AN

doi

10.1016/j.bpj.2011.01.066

subject

Has Abstract

pub_date

2011-03-16 00:00:00

pages

1568-77

issue

6

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(11)00185-8

journal_volume

100

pub_type

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