title: Kinetics of fast changing intramolecular distance distributions obtained by combined analysis of FRET efficiency kinetics and time-resolved FRET equilibrium measurements.
abstract: 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.
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel 52900.
authors: E
authors: E
authors: Lerner
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel 52900.
authors: T
authors: T
authors: Orevi
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel 52900.
authors: E
authors: E
authors: Ben Ishay
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel 52900.
authors: D
authors: D
authors: Amir
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel 52900. Electronic address: elisha.haas@biu.ac.il.
authors: E
authors: E
authors: Haas
mesh_terms: D000263:Adenylate Kinase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D000263:Adenylate Kinase