Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding

Staphylococcal nuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of protein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in thermal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lower ΔH_cal values than the wild-type protein. Furthermore, two mutants, E75Q and E129Q, were created and the results imply that substitution of the Gly residue has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability. Two local stable areas formed by the charge-charge interactions around E75 and E129 with particular positively charged amino acids are thus identified as being significant in maintenance of the three-dimensional structure of the protein.