PRESSURE DENATURATION OF PROTEINS - EVALUATION OF COMPRESSIBILITY EFFECTS

One of the key pieces of information from pressure denaturation experi ments is the standard volume change for unfolding (Delta V-o). The pre ssure dependence of the volume change, the standard compressibility ch ange (Delta K-T(o)), is typically assumed to be zero in the analysis o f these experiments. We show here that this assumption can be incorrec t and that the neglect of compressibility differences can skew the int erpretation of experimental results. Analysis of experimental, variabl e-pressure NMR data for bovine pancreatic ribonuclease A in (H2O)-H-2 at pH 2.0 and 295 K yielded the following statistically significant, non-zero values: Delta K-T(o) = 0.015 +/- 0.002 mL mol(-1) bar(-1), De lta V-o = -21 +/- 2 mL mol(-1), and Delta G(o) = 2.8 +/- 0.3 kcal mol( -1). The experimental protein stability is in good agreement with one (Delta G(o) = 2.5 kcal mol(-1)) determined independently for the same protein by calorimetry at atmospheric pressure under equivalent condit ions [Makhatadze, G. L., Clore, G. M., and Gronenborn, A. M. (1995) Na t. Struct. Biol. 2, 852-855]. The positive value for Delta K-T(o) indi cates that the denatured form of ribonuclease A is more compressible t han the native form; this is explained in terms of an interplay betwee n the intrinsic compressibility of the protein and solvation effects. When the same data were fitted to a model that assumes a zero compress ibility change, the Delta G(o) value of 4.0 +/- 0.1 kcal mol(-1) retur ned by the model no longer agreed with the independent measurement, an d the Delta V-o returned by the model was a Very different -59 +/- 1 m L mol(-1). By contrast, it was not possible to carry out a similar the rmodynamic analysis of fluorescence spectroscopic data for the denatur ation of staphylococcal nuclease to yield well-defined values of Delta G(o), Delta V-o, and Delta K-T(o). This limitation was shown by evalu ation of synthetic data to be intrinsic to spectroscopic data whose an alysis requires fitting of the plateaus at either side of the transiti on. Because NMR data do not have this requirement, they can be analyse d more rigorously.