Pressure-Jump Small-Angle X-Ray Scattering Detected Kinetics of Staphylococcal Nuclease Folding

Abstract

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the 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 formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique.

Cooperative water filling of a nonpolar protein cavity observed by high-pressure crystallography and simulation

“…At higher pressures, cavity filling should become so favorable that new cavities will grow to accommodate water and the protein will partially unfold. Even at lower pressures, our work supports the idea that collapsed intermediate states in protein folding may be hydrated internally (4,28,29) and that dehydration may be a rate-limiting step in folding (4,(29)(30)(31).…”

Random-coil behavior and the dimensions of chemically unfolded proteins

“…Differences in the magnitude of the residual denatured state structure from one unfolded protein to another would thus be expected to produce significant scatter around any underlying power-law relationship across a diverse set of unfolded proteins. Although proteins unfolded in water by means of mutation under pressure or at low pH are sometimes relatively compact (9)(10)(11), the dimensions of most urea-or GuHCl-denatured proteins obey the theoretically expected random-coil scaling. Studies of this issue date from the 1960s, when Tanford et al (12) used intrinsic viscosity measurements to determine that, for a set of 12 proteins unfolded in 5-6 M GuHCl, ϭ 0.67 Ϯ 0.09 (95% confidence interval).…”

Amyloid Features and Neuronal Toxicity of Mature Prion Fibrils Are Highly Sensitive to High Pressure

“…To prove this hypothesis, a high pressure and particularly the pressure-jump technique (43)(44)(45)(46) was applied to study the activation energy parameters and structural changes involved in mature amyloid fibril disassembly by using the recombinant prion protein (PrP) 3 increase in pressure forced PrP amyloid fibrils to change irreversibly to a new, less cytotoxic state that was still partly fibrillar but lacked the typical structural features of amyloids. The analysis of the relaxation kinetics toward this new state gave information about the reaction mechanism and the transition state ensemble.…”