Protein folding and unfolding on a complex energy landscape

Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation spate from the unfolded state toward the native state and vice verse. Herein, we introduc e a new type of kinetics experiment that shows how different unfolding path ways can be selected by varying the initial reaction conditions. The relaxa tion kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small tem perature jumps, indicative of folding through a two-state mechanism. Howeve r, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can oc cur simultaneously in the same ensemble and provide the experimental requir ements for these to be observed.