The thermodynamic stability of staphylococcal nuclease was studied against the variation of both temperature and pressure by utilizing (1)H NMR spectroscopy at 750 MHz in 20 mM Mes buffer containing 99.9 % (2)H(2)O, pH 5.3. Equilibrium fractions of folded and unfolded protein species were evaluated with the proton signals of two histidine residues as monitor in the pressure range of 30-3300 bar and in the temperature range of 1.5 degrees C-35 degrees C. From the multi-parameter fit of the experimental data to the Gibbs energy equation expressed as a simultaneous function of pressure and temperature, we determined the compressibility change (Deltabeta), the volume change at 1 bar (DeltaV degrees ) and the expansivity change (Deltaalpha) upon unfolding among other thermodynamic parameters: Deltabeta=0.02(+/-0.003) ml mol(-1) bar(-1); Deltaalpha=1.33(+/-0.2) ml mol(-1) K(-1); DeltaV degrees =-41.9(+/-6. 3) ml mol(-1) (at 24 degrees C); DeltaG degrees =13.18(+/-2) kJ mol(-1) (at 24 degrees C); DeltaC(p)=13.12(+/-2) kJ mol(-1) K(-1); DeltaS degrees =0.32(+/-0.05) kJ mol(-1) K(-1 )(at 24 degrees C). The result yields a three-dimensional free energy surface, i.e. the free energy-landscape of staphylococcal nuclease on the P-T plane. The significantly positive Deltabeta and Deltaalpha values suggest that, in the pressure-denatured state, staphylococcal nuclease forms a loosely packed and fluctuating structure. The slight but statistically significant difference between the unfolding transitions of the His8 and His124 environments is considered to reflect local fluctuations in the native state, leading to pre-melting of the His124 environment prior to the cooperative unfolding of the major part of the protein.
Copyright 2000 Academic Press.