Evidence for barrier-limited protein folding kinetics on the microsecond time scale.

Although important structural events in protein folding are known to occur on the submillisecond time scale, the limited time resolution of conventional kinetic methods has precluded direct observation of the initial 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 cytochrome c from approximately 5-10(-5)-10(2) s, including the previously unresolved quenching of Trp 59 fluorescence (burst phase) indicative of the formation of compact states. The kinetics of folding exhibits a major exponential process with a time constant of approximately 50 micros, independent 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 accumulates prior to the rate-limiting formation of specific tertiary interactions, confirming previous indications that folding involves at least two distinct stages.