To quantitate the contributions of the ionizable amino acids to the stability of the native state of staphylococcal nuclease, each of the 23 lysines, 5 arginines, 4 histidines, 12 glutamic acids, and 8 aspartic acids was substituted with both alanine and glycine. This collection of 104 mutant proteins was analyzed by guanidine hydrochloride (GuHCl) denaturation, using intrinsic tryptophan fluorescence to quantitate the equilibrium between native and denatured states. From the analysis of these data, each mutant protein's stability in the absence of denaturant (ΔG(H2O)) and sensitivity to changes in denaturant concentration [m(GuHCl) = d(ΔG)/d[GuHCl]] were obtained. Several general trends in these values suggest that electrostatic interactions make only a minor contribution to the net stability of this protein. For the residue pairs that form ten salt bridges and ten charged hydrogen bonds between side chains, no correlation was observed between the stability losses (ΔΔG) accompanying alanine substitution of each member of the pair. Little or no significant correlation was found between the magnitude of the loss in stability and the local electrostatic potential calculated from the three-dimensional structure by numerical and model dependent solutions of the linearized Poisson-Boltzmann equation. The structural parameters which correlated most strongly with stability loss are measures of the extent of burial of the residue in the native structure, as was previously observed for alanine and glycine substitutions of large hydrophobic residues [Shortle et al. (1990) Biochemistry 29, 8033] and of the polar, uncharged residues [Green et al. (1992) Biochemistry 31, 5717]. These results suggest that the ionizable amino acids contribute to stability predominantly through packing and bonding interactions that do not depend on their electrostatic charge.
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