EVIDENCE FOR BARRIER-LIMITED PROTEIN-FOLDING KINETICS ON THE MICROSECOND TIME-SCALE

Although important structural events in protein folding are known to o ccur on the submillisecond time scale, the limited time resolution of conventional kinetic methods has precluded direct observation of the i nitial collapse of the polypeptide chain. A continuous-flow capillary mixing method recently developed by us made it possible to account for the entire fluorescence change associated with refolding of cytochrom e c from similar to 5-10(-5)-10(2) s, including the previously unresol ved quenching of Trp 59 fluorescence (burst phase) indicative of the f ormation of compact states. The kinetics of folding exhibits a major e xponential process with a time constant of similar to 50 mu s, indepen dent of initial conditions and heme ligation state, indicating that a common free energy barrier is encountered during the initial collapse of the polypeptide chain. The resulting loosely packed intermediate ac cumulates prior to the rate-limiting formation of specific tertiary in teractions, confirming previous indications that folding involves at l east two distinct stages.