Investigation of the conformational equilibration time of unfolded cytochrome c chains using time-resolved magnetic circular dichroism

The speed with which unfolded protein chains reach conformational equilibrium is a fundamental question in the study of protein folding. If unfolded chains reach equilibration on a time scale slower than protein folding, then it is possible to experimentally distinguish between a landscape folding mechanism and a classical folding mechanism. However, if unfolded chains reach equilibrium on a time scale faster than protein folding, folding proceeds from equilibrated conformations over a thermally activated barrier. Kinetic evidence is presented here for the first quantitative measurement of the rate of conformational equilibration from a folding-capable protein. The protein investigated in this work is the small globular protein, cytochrome c (cyt c), studied in the presence of guanidine hydrochloride (GndHCl) denaturant. The axial binding reactions of reduced cyt c's heme cofactor with histidine and methionine residues were monitored with time-resolved magnetic circular dichroism (TRMCD) 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 interpreted as a first-order time constant for exchange between conformational sub-ensembles presenting either a methionine or histidine residue to the heme iron for facile binding. The mean diffusional tau of the wild type and variants was 3 +/- 2 mus, close to the folding "speed limit" described recently in the literature. 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 pivotal point.