Pressure perturbation calorimetic studies of the solvation properties and the thermal unfolding of staphylococcal nuclease

Abstract

A rather new technique, pressure perturbation calorimetry (PPC), was applied to study volumetric and solvation properties of staphylococcal nuclease (Snase) in its native and unfolded state with high precision. Furthermore, the effects of various chaotropic and cosmotropic co-solvents on the solvation and unfolding behaviour of Snase was investigated in detail. In PPC, the apparent coefficient of thermal expansion of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent at the protein–solvent interface. In the native state, the protein shows a very strong thermal expansion of 1.0×10−3 K−1 at 10°C, which decreases steeply to 0.65×10−3 K−1 at 40°C. This behaviour is discussed in terms of a continuous release of condensed water from the protein surface. Upon unfolding, the volume decreases by about 19 mL×mol−1. Solutions of the cosmotropic and chaotropic compounds glycerol, sorbitol, K2SO4 and urea, respectively, show characteristic deviations from the thermal expansion and volumetric properties of the pure buffer solution. The solvent contribution to the apparent coefficient of thermal expansion of the protein, α, is enhanced considerably when the protein is immersed in a solvent known to be more structured than H2O (even the more structured D2O has a drastic effect) and nearly eliminated in a solvent in which “normal” water is largely absent (e.g., in 1.5 M urea). Similarly to D2O, a continuous increase in solvation was observed with increase in glycerol or sorbitol content in the buffer, which leads to an increase in protein stability, as is verified by the increasing Tm and ΔH values obtained by microcalorimetric measurements (DSC). In this regard, sorbitol is the more efficient agent. The reduction of ΔV in the presence of these stabilisers can in part be attributed to the formation of a partial unfolded state of the protein, in part it is due to the temperature dependence of ΔV. The preferential binding of urea reduces the hydration level, also in the native state, causing the protein to approach a more disordered state at high urea concentration. The increase in ΔV and the decrease in ΔH with increasing urea concentration support these conclusions. A stabilising effect, even though there is a reduction in solvation around the protein is observed for 0.5 M K2SO4 as co-solvent. In this case, surprisingly, ΔV is found to be positive which is an indication of the formation of a swollen, molten globule kind of unfolded state at the transition. Finally, ΔV values for the temperature-induced unfolding are compared with corresponding data for the pressure-induced unfolding of Snase.