pH dependence of stability of staphylococcal nuclease: Evidence of substantial electrostatic interactions in the denatured state

The pH dependence of stability of staphylococcal nuclease was studied with two independent equilibrium thermodynamic approaches. First, by measurement of stability in the pH range 9 to 3.5 by fluorescence-monitored denaturati on with urea (DeltaG degrees (urea)), GdnHCl (DeltaG degrees (Gdn)), and he at (DeltaG degrees (T)). Second,by numerical integration of H+ titration cu rves (DeltaG degrees (Deltav)) measured potentiometrically under native (10 0 mM KCl) and unfolding (6.0 M GdnHCl) conditions. The pH dependence of sta bility described by DeltaG degrees (urea), DeltaG degrees (Gdn) and DeltaG degrees (T) was comparable but significantly different from the one describ ed by DeltaG degrees (Deltav). The decrease in DeltaG degrees (Deltav) betw een pH 9 and pH 4 was 4 kcal/mol greater than the decrease in DeltaG degree s (urea), DeltaG degrees (Gdn), and DeltaG degrees (T) in the same pH range . In 6 M GdnHCl, all the ionizable groups titrated with the pK(a) values of model compounds. Therefore, DeltaG degrees (Deltav) represents the free en ergy difference between the native state (N) and an ensemble of unstructure d, or expanded, and highly screened conformations. In contrast, the shallow er pH dependence of stability described by DeltaG degrees (urea) and by Del taG degrees (T) between pH 9 and 5 was consistent with the titration of his tidines with depressed, nativelike pK(a) values in the denatured state (D), These depressed pK(a) values likely reflect long-range electrostatic inter actions with the other 29 basic groups and are a consequence of the compact character of the D state. The steep change in DeltaG degrees (urea) at and DeltaG degrees (T) at pH < 5 suggests that near pH 5 the structural and th ermodynamic character of the D state shifts toward a state in which acidic residues titrate with normal pK(a) values, presumably because the electrost atic interactions with basic residues are lost, maybe as a consequence of a n expansion.