Determination of intramolecular distance distribution during protein folding on the millisecond timescale

Department of Life Sciences, Bar Ilan University, Ramat-Gan, 52900, Israel.
Journal of Molecular Biology (Impact Factor: 4.33). 07/2000; 299(5):1363-71. DOI: 10.1006/jmbi.2000.3814
Source: PubMed


A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed. The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined. The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument). At that time the width of that distribution was rather large (16+/-2 A). The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10(-7) cm(2)/second. In a second slower phase of the refolding transition, the width was reduced to its native value (6+/-4 A).

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    • "trFRET experiments are ideal for detecting the formation of each closed long loop since it is possible to follow selected distances between two sites that are separated by large number of residues, their distributions, and fast fluctuations (Beechem and Haas 1989; Haas 2005). This led to our efforts towards the development of the trFRET-based " double kinetics " method for the detection of transient intramolecular distance distribution in the rapid collapsed state of globular proteins and during the full folding transition (Ratner et al. 2000; Jacob et al. 2005; Ben Ishay et al. 2012a). The NLI-based model for initiation of folding raises several questions that must be addressed. "
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