Two-state expansion and collapse of a polypeptide

The initial phase of folding for many proteins is presumed to be the collap se of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a t wo-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers and random heteropolymers is continuous and multi- phasic. A new rapid-mixing flow technique has been used to resolve the late stages of polypeptide collapse,at time scales greater than or equal to 45 mu s. We have used a laser temperature-jump with fluorescence spectroscopy to resolve the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential i n time and thermally activated, with an apparent activation energy similar to 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because o f the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse. (C) 2000 Academic Press.