Nanosecond laser T-jump was used to measure the viscosity dependence of the folding kinetics of the villin subdomain under conditions where the viscogen has no effect on its equilibrium properties. The dependence of the unfolding/refolding relaxation time on solvent viscosity indicates a major contribution to the dynamics from internal friction. The internal friction increases with increasing temperature, suggesting a shift in the transition state along the reaction coordinate toward the native state, and therefore toward more compact structures with a smaller diffusion coefficient due to increased landscape roughness. Fitting the data with an Ising-like model yields a relatively small position-dependence for the diffusion coefficient. This finding is consistent with the excellent correlation found previously between experimental and calculated folding rates based on free energy barrier heights with the same diffusion coefficient for every protein. To further investigate the folding mechanism of the villin subdomian, we have studied the equilibrium and kinetics using laser temperature jump of 8 additional mutants in which the amino replacement contains a simple methyl group deletion. In most cases this results in a non-natural amino acid, but the protein is made by peptide synthesis so this does not pose a problem. Instead of calculating phi values, the equilibrium curves are being fit with the Ising-like model, and the new relaxation rate is calculated on the perturbed free energy surface to compare with the measured relaxation rate. Collaboration with group at University of Parma, Italy, on hemoglobin allostery: Following an extended period of steady state laser illumination of the carbon monoxide complex of hemoglobin in the T quaternary structure to maintain the hemes in the deoxy state, the kinetics of carbon monoxide rebinding after turning off the laser switch from biphasic to monophasic, indicating that all subunits are in the same conformation as deoxyhemoglobin in the absence of light or carbon monoxide. These experiments dramatically confirm our interpretation of gel-encapsulated rebinding kinetics following nanosecond photodissociation of carbon monoxide in the T quaternary structure as arising from an equilibrium population of liganded subunits in the same conformation as found in the R quaternary structure.
| Cellmer, Troy; Buscaglia, Marco; Henry, Eric R et al. (2011) Making connections between ultrafast protein folding kinetics and molecular dynamics simulations. Proc Natl Acad Sci U S A 108:6103-8 |
| Cellmer, Troy; Henry, Eric R; Hofrichter, James et al. (2008) Measuring internal friction of an ultrafast-folding protein. Proc Natl Acad Sci U S A 105:18320-5 |
| Kubelka, Jan; Henry, Eric R; Cellmer, Troy et al. (2008) Chemical, physical, and theoretical kinetics of an ultrafast folding protein. Proc Natl Acad Sci U S A 105:18655-62 |
