Significance of local electrostatic interactions in staphylococcal nuclease studied by site-directed mutagenesis

In this paper, we show that amino acids Glu(73) and Asp(77) of staphylococc al nuclease cooperate unequally with Glu(75) to stabilize its structure loc ated between the C-terminal helix and beta -barrel of the protein. Amino ac id substitutions E73G and D77G cause losses of the catalytic efficiency of 24 and 16% and cause thermal stability losses of 22 and 26%, respectively, in comparison with the wild type (WT) protein. However, these changes do no t significantly change global and local secondary structures, based on meas urements of fluorescence and CD222nm. Furthermore, x-ray diffraction analys is of the E75G protein shows that the overall structure of mutant and WT pr oteins is similar. However, this mutation does cause a loss of essential hy drogen bonding and charge interactions between Glu(75) and Lyg(9), Tyr(93), and HiS(121). In experiments using double point mutations, E73G/ D77G, E73 G/E75G, and E75G/D77G, significant changes are seen in all mutants in compa rison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47, 59, and 58%, for E73G/ D77G, E73G/E75G, and E75G/D77G, respectively. The triple mutant, E73G/E75G/D77G, results in fluorescence intensity and CD222nm close to those of the denatured state a nd in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic intera ctions result in the formation of a locally stable structure in staphylococ cal nuclease.