Conformational equilibration time of unfolded protein chains and the folding speed limit.
The speed with which the conformers of unfolded protein chains interconvert is a fundamental question in the study of protein folding. Kinetic evidence is presented here for the time constant for interconversion of disparate unfolded chain conformations of a small globular protein, cytochrome c, in the presence of guanidine hydrochloride denaturant. The axial binding reactions of histidine and methionine residues with the Fe(II) heme cofactor were monitored with time-resolved magnetic circular dichroism spectroscopy after photodissociation of the CO complexes of unfolded protein obtained from horse and tuna and from several histidine mutants of the horse protein. A kinetic model fitting both the reaction rate constants and spectra of the intermediates was used to obtain a quantitative estimate of the conformational diffusion time. The latter parameter was approximated as a first-order time constant for exchange between conformational subensembles presenting either a methionine or a histidine residue to the heme iron for facile binding. The mean diffusional time constant of the wild type and variants was 3 +/- 2 mus, close to the folding "speed limit". The implications of the relatively rapid conformational equilibration time observed are discussed in terms of the energy landscape and classical pathway time regimes of folding, for which the conformational diffusion time can be considered a pivot point.
Abel
Christina J
CJ
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Goldbeck
Robert A
RA
Latypov
Ramil F
RF
Roder
Heinrich
H
Kliger
David S
DS
D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics; D004058:Diffusion; D006736:Horses; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013997:Time Factors; D014413:Tuna
D045304:Cytochromes c