(230e) Mutation Effects On Protein Folding Over Small Barriers

Small, ultra-fast folding proteins are attractive for studies that directly compare simulation to experiment, and thus make it possible to obtain detailed information on protein-folding pathways. A crucial, but poorly understood issue pertaining to these proteins is the interpretation of mutation effects on folding kinetics. For two-state proteins with high barriers mutation effects are measured using phi-value analysis. However, for proteins with small barriers where positions along the reaction coordinate other than the transition state are kinetically relevant, this approach is not valid. Furthermore, this issue is particularly important to comparisons between computations and experiment, as phi values are frequently used as points of comparison between the two approaches. To study this issue, we measured the thermodynamics and kinetics of the ultra-fast folding chicken-villin heapiece subdomain (villin) and seven mutants over a large-temperature range. This protein is particularly relevant, as it has been the subject of numerous papers involving high-resolution molecular dynamics simulations that can, in principle, reveal microscopic details of the folding pathway. Our experimental data show that while several mutations have a notable effect on thermodynamics, all mutations have only marginal effects on the kinetics. We are now analyzing this data using an Ising-like analytical model for protein folding that has been successful in describing folding kinetics and thermodynamics for a large number of proteins. Our analysis will allow us to make novel statements pertaining to the interpretation of mutation effects on folding kinetics over small barriers, and allow us to verify a previously-held hypothesis pertaining to the folding of villin. More specifically, we will verify that its transition state moves to more folded structures as solution conditions become more favorable to unfolding.