Chemical denaturants such as guanidine hydrochloride and urea are currently the primary tools used to perturb landscapes, but their mechanism of action is poorly understood. Existing models describing denaturant interactions with proteins are inadequate for accurately extrapolating thermodynamic and kinetic measurements at high denaturant to the physiologically relevant conditions. None of the models treat the experimental observation in several protein systems of curvature in denaturant dependence of unfolding free energies and activation free energies in a physically interpretable manner. Furthermore, most models treat only the effect of denaturant on solvent interactions by modeling the hydrophobic effect. However, there are also well-documented interactions of denaturants with the unfolded and folded polypeptide, which are more likely due to electrostatic forces. The ability to accurately deconvolute the hydrophobic and electrostatic components underlying denaturant-protein interactions would lead to more accurate extrapolated values for theoretical predictions of protein behavior, and reveal insights into the nature of unfolded transition states and intermediates.
We have exploited the unique kinetic stability of αLP to measure unfolding kinetics over the widest denaturant and temperature range so far reported in the protein folding field (Jaswal, Journal of Molecular Biology 347, 355-66, 2005). We are using the data to develop a general and physically based model that can fit denaturant dependent unfolding data accurately regardless of the degree of curvature observed.