CONTRIBUTIONS OF THE IONIZABLE AMINO-ACIDS TO THE STABILITY OF STAPHYLOCOCCAL NUCLEASE

To quantitate the contributions of the ionizable amino acids to the st ability 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 t he equilibrium between native and denatured states. From the analysis of these data, each mutant protein's stability in the absence of denat urant (Delta G(H2O)) and sensitivity to changes in denaturant concentr ation [m(GuHCl) = d(Delta G)/d[GuHCl]] were obtained. Several general trends in these values suggest that electrostatic interactions make on ly a minor contribution to the net stability of this protein. For the residue pairs that form ten salt bridges and ten charged hydrogen bond s between side chains, no correlation was observed between the stabili ty losses (Delta Delta G) accompanying alanine substitution of each me mber of the pair. Little or no significant correlation was found betwe en 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 equ ation. The structural parameters which correlated most strongly with s tability loss are measures of the extent of burial of the residue in t he native structure, as was previously observed for alanine and glycin e substitutions of large hydrophobic residues [Shortle et al. (1990) B iochemistry 29, 8033] and of the polar, uncharged residues [Green et a l. (1992) Biochemistry 31, 5717]. These results suggest that the ioniz able amino acids contribute to stability predominantly through packing and bonding interactions that do not depend on their electrostatic ch arge.