Stability Mutants of Staphylococcal Nuclease: Large Compensating Enthalpy-Entropy Changes for the Reversible Denaturation Reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant K_appas a function of temperature, the midpoint temperature T_mand apparent enthalpy change ΔH_appon reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a T_mof 53.3 ± 0.2 °C and a ΔH_appof 86.8 ± 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 °C and 96 ± 2 kcal/mol. The heat capacity change on denaturation AC_pwas estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of ΔH_appand AS_appfor a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated AC_pfor the mutant proteins and the d(AG_app)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.