Time-resolved absorption and fluorescence spectroscopy are used to study the dynamics of protein structural changes subsequent to rapid mixing or excitation with short laser pulses. Kinetic models are used to fit and interpret the measured data. Our recent efforts have focussed on understanding the folding of the villin subdomain. The 35-residue villin subdomain consists of three helices which interact to form a hydrophobic core. It is the smallest naturally occurring polypeptide that folds autonomously without disulfide bonds or ligand binding, and has been the object of extensive simulation studies. ? ? To probe the folding mechanism of villin we previously carried out structural and physical-chemical studies of molecules with point mutations and other sequence modifications. These studies have included determination of the structures of some of the folded states by X-ray diffraction carried out in collaboration with Dr. Thang Chiu and David Davies in the LMB/NIDDK. Conservative mutations had no significant effects on the folding rates, suggesting that the transition state for folding of villin is close to the denatured ensemble of conformations. On the other hand, the K24Nle and K29Nle ? mutations both stabilize the folded structure and accelerate folding by factors of 2.5, and the double mutant, with a folding time of 700 ns, ranks as the fastest folding protein studied to this time.? ? To further explore the folding of villin we have examined the dependence of the folding equilibruim and kinetics on the denaturant, guanidinium chloride (GdmCl). We observe a nearly denaturant- independent, unfolding/refolding relaxation rate. Both Munoz and coworkers and Fersht and coworkers have argued that this behavior results from the absence of a barrier separating folded and unfolded states. Using an Ising-like model, we find that this behavior can be produced by a large movement of the major free energy barrier, together with a denaturant- and reaction coordinate- dependent diffusion coefficient. The denaturant-independence of the kinetics of this ultrafast folding protein can be explained with a model that yields approximately two-state thermodynamic and kinetic behavior. ? ? See the annual report by William A. Eaton for additional details regarding this project.

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