| Abstract
| - The application of hydrostatic pressure to aqueousprotein solutions results in the unfoldingof the protein structure because the protein−solvent system volume issmaller for the unfolded state.Contributions to this decrease in volume upon unfolding(ΔVu) derive from altered interactions oftheprotein with solvent and are presumed to include electrostriction ofcharged residues, elimination of packingdefects, and hydration of hydrophobic surfaces upon unfolding. Ifthe contribution of hydrophobic surfacearea solvation to the observed volume change of unfolding were largeand negative, as is generally assumed,then one would expect to find a correlation between the amount ofsurface area exposed on unfolding,ΔAu, and the volume change,ΔVu. In order to test this correlation,we have determined ΔVu for twomutants of staphylococcal nuclease, A69T + A90S and H121P, whoseunfolding by denaturant is,respectively, either significantly more (28%) or significantly less(28%) cooperative than that observedfor wild-type (WT). This cooperativity coefficient or mvalue has been shown to correlate with ΔAu.If,in turn, ΔVu is correlated withΔAu, we would expect them+ mutant, A69T + A90S, toexhibit a ΔVu thatis more negative than WT nuclease, while theΔVu for them- mutant, H121P, should be smallerin absolutevalue. To verify the correlation between m value andΔAu for these mutants, we determined thexyloseconcentration dependence of the stability of each mutant at atmosphericpressure and as a function ofpressure. The efficiency of xylose stabilization was found to bemuch greater for the m+ mutant thanforWT, consistent with an increase in ΔAu, whilethat of the m- mutant was found tobe only slightly greaterthan for WT, indicating that other factors may contribute to thedenaturant m value in this case.Regardlessof the denaturant m value or the effect of xylose onstability, the volume changes upon unfolding for bothmutants were found to be within error of that observed for WT.Thus, there does not appear to be acorrelation between the volume change and the change in exposed surfacearea upon unfolding. Wehave previously shown a lack of pH dependence of the volume change,ruling out electrostriction as adominant contribution to ΔVu of nuclease.These studies implicate either compensation betweenpolarand nonpolar hydration or excluded volume effects as the majordeterminant for the value of ΔVu.
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