Un- and refolding kinetics of WT staphylococcal nuclease

Globular proteins in solution are only marginally stable and represent complex macromolecular systems whose structure and function is determined by the sum of hydrophobic, electrostatic, and hydrogen bond interactions. Because folding in a natural environment is very complex and often happens too fast, the experimental approaches for studying the responsible forces have focused mainly on the unand refolding of small proteins or enzymes using rapid-mixing, temperature-jump, and (only recently developed) pressurejump techniques. The advantage of using pressure jumps as a perturbation technique is guided by the ability to provide fast and bidirectional unfolding as well as refolding environments for the polypeptide in solution. Pressure propagates homogeneously through the system and usually causes no disruption of intramolecular covalent bonds below 20 kbar. In general, pressure is a rather mild perturbation technique for studying biomolecular phase transformations. In the case of protein unand refolding studies, pressure perturbation often has the additional advantage of significantly slowing down the reactions due to positive activation volumes. In this study, we present pressure-jump kinetic studies of the tertiary structural evolution in the folding/unfolding reactions of WT staphylococcal nuclease (SNase) using time-resolved synchrotron small-angle x-ray scattering (SAXS) techniques.