Kinetics of fast changing intramolecular distance distributions obtained by combined analysis of FRET efficiency kinetics and time-resolved FRET equilibrium measurements

E. Lerner, T. Orevi, E. Ben Ishay, D. Amir, E. Haas

פרסום מחקרי: פרסום בכתב עתמאמרביקורת עמיתים

תקציר

Detailed studies of the mechanisms of macromolecular conformational transitions such as protein folding are enhanced by analysis of changes of distributions for intramolecular distances during the transitions. Time-resolved Förster resonance energy transfer (FRET) measurements yield such data, but the more readily available kinetics of mean FRET efficiency changes cannot be analyzed in terms of changes in distances because of the sixth-power dependence on the mean distance. To enhance the information obtained from mean FRET efficiency kinetics, we combined the analyses of FRET efficiency kinetics and equilibrium trFRET experiments. The joint analysis enabled determination of transient distance distributions along the folding reaction both in cases where a two-state transition is valid and in some cases consisting of a three-state scenario. The procedure and its limits were tested by simulations. Experimental data obtained from stopped-flow measurements of the refolding of Escherichia coli adenylate kinase were analyzed. The distance distributions between three double-labeled mutants, in the collapsed transient state, were determined and compared to those obtained experimentally using the double-kinetics technique. The proposed method effectively provides information on distance distributions of kinetically accessed intermediates of fast conformational transitions induced by common relaxation methods.

שפה מקוריתאנגלית אמריקאית
עמודים (מ-עד)667-676
מספר עמודים10
כתב עתBiophysical Journal
כרך106
מספר גיליון3
מזהי עצם דיגיטלי (DOIs)
סטטוס פרסוםפורסם - 4 פבר׳ 2014

ASJC Scopus subject areas

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