(386a) Ultra-Fast Folding Of The Villin Subdomain: Nanosecond T-Jump Measurements In Denaturant

Over the past 10-15 years much effort has been devoted by biophysical scientists towards understanding the mechanisms of protein folding. Rapid advancement of computer power has facilitated efforts to visualize the folding of small proteins in molecular dynamics trajectories. Experiments on small, fast-folding proteins are required to validate the findings from these revolutionary simulations. We are investigating the folding of the villin headpiece subdomain, a 35 residue mini-protein consisting of 3 helices surrounding a hydrophobic core in a non-trivial topology. This protein has already been the subject of numerous high-profile theoretical and computational studies. Using nanosecond-laser temperature jump measurements, we show that the denaturant guanidinium chloride (GdmCl) exerts almost no effect on villin's unfolding/refolding relaxation rate at concentrations up to 4 M at 40C. For conventional two-state proteins, this relaxation rate often varies by several orders of magnitude over this concentration range, while the villin relaxation rate only varies from 5-8 microseconds. In order to obtain a quantitative estimate of the free energy barrier height, both the equilibrium and kinetic data are being fit with an Ising-like statistical mechanical model that has been extraordinarily successful in predicting the relative rates of folding for small, single domain proteins. We also present data on the GdmCl dependence of villin's ~100 nanoseconds phase. We find that this ?fast? phase disappears at high GdmCl concentrations. Circular dichroism measurements show a decrease in the helical structure of the native state over the same concentration range, supporting the interpretation that the fast phase arises from helix fraying. Further studies are aimed at further stabilizing the protein to unequivocally produce a downhill folder, thereby allowing direct interrogation by spectroscopy of the evolution of the folded state all along the reaction coordinate.