Determination of intramolecular distance distribution during protein folding on the millisecond timescale.
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).
Ratner
V
V
Department of Life Sciences, Bar Ilan University, Ramat-Gan, 52900, Israel.
Sinev
M
M
Haas
E
E
D000263:Adenylate Kinase / Q000737:chemistry* / Q000378:metabolism*; D004058:Diffusion; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D019791:Guanidine / Q000494:pharmacology; D007546:Isoquinolines / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D007546:Isoquinolines; D015335:Molecular Probes; C017475:lucifer yellow; D000263:Adenylate Kinase; D019791:Guanidine