Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conforma- tion space from the unfolded state toward the native state and vice versa. Herein. we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions. The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps. indicative of folding through a two-state mechanism. However. for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple un- folding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimen- tal requirements for these to be observed.
展开▼