Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding

The kinetics of chain disruption and collapse of staphylococcal nuclease af ter positive or negative pressure jumps was monitored by real-time small-an gle x-ray scattering under pressure. We used this method to probe the overa ll conformation of the protein by measuring its radius of gyration and pair -distance-distribution function p(r) which are sensitive to the spatial ext ent and shape of the particle. At all pressures and temperatures tested, th e relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure for mation. Whereas refolding at low pressures occurred in a few seconds, at hi gh pressures the relaxation was quite slow, similar to1 h, due to a large p ositive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydr ation of the transition state and a high degree of similarity in terms of t he packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-in duced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obt ained from a combination of small-angle x-ray scattering using high intensi ty synchrotron radiation with the high pressure perturbation technique.