TESTING THE CORRELATION BETWEEN DELTA-A AND DELTA-V OF PROTEIN UNFOLDING USING M-VALUE MUTANTS OF STAPHYLOCOCCAL NUCLEASE

The application of hydrostatic pressure to aqueous protein solutions r esults in the unfolding of the protein structure because the protein-s olvent system volume is smaller for the unfolded state. Contributions to this decrease in volume upon unfolding (Delta V-u) derive from alte r-ed interactions of the protein with solvent and are presumed to incl ude electrostriction of charged residues, elimination of packing defec ts, and hydration of hydrophobic surfaces upon unfolding. If the contr ibution of hydrophobic surface area solvation to the observed volume c hange of unfolding were large and negative, as is generally assumed, t hen one would expect to find a correlation between the amount of surfa ce area exposed on unfolding, Delta A(u), and the volume change, Delta V-u. In order to test this correlation. we have determined Delta V-u for two mutants of staphylococcal nuclease, A69T + A90S and H121P, who se unfolding by denaturant is, respectively, either significantly more (28%) or significantly less (28%) cooperative than that observed Tor wild-type (WT). This cooperativity coefficient or In value has been sh own to correlate with Delta A(u). If, in rum, Delta V-u is correlated with Delta A(u), we would expect the m(+) mutant, A69T + A90S, to exhi bit a Delta V-u that is more negative than WT nuclease, while the Delt a V-u for the m(-) mutant, H121P, should be smaller In absolute value. To verify the correlation between m value and Delta A(u) for these mu tants, we determined the xylose concentration dependence of the stabil ity of each mutant al atmospheric pressure and as a function of pressu re. The efficiency of xylose stabilization was found Co be much greate r for the m(+) mutant than for WT, consistent with an increase in Delt a A(u), while that of the m(-) mutant was found to be only slightly gr eater than fur WT, indicating that other factors may contribute to the denaturant m value in this case. Regardless of the denaturant m value or the effect of xylose on stability, the volume changes upon unfoldi ng for both mutants were found to be within error of that observed for WT. Thus, there does not appear to be a correlation between the volum e change and the change in exposed surface area upon unfolding. We hav e previously shown a lack of pH dependence of the volume change, rulin g out electrostriction as a dominant contribution to Delta V-u of nucl ease. These studies implicate either compensation between polar and no npolar hydration or excluded volume effects as the major determinant f or the value of Delta V-u.