Since a protein's function depends on its structure, basic research in protein structure facilitates the solution of many practical problems, such as the synthesis of more effective medicines. With this larger goal in sight, the purpose of this research project is to understand better the chemical principles that underlie protein structure and stability. Disulfide bonds are a potentially stabilizing feature of many proteins. They may form between cysteine residues in close proximity to one another if the orientation is favorable. Often found in proteins produced by organisms that grow at high temperatures, disulfide bonds may anchor side chains together, making a protein resistant to thermal or chemical denaturation. In order to provide a better understanding of the stabilizing effects of disulfide bonds, disulfides are artificially introduced into the protein staphylococcal nuclease to create mutant versions of the protein. Wild-type S. nuclease has no cysteine residues, so disulfide bonds must be engineered by substituting cysteines for pairs of amino acid residues in the wild-type protein. To synthesize these double mutants, successive rounds of site-directed mutagenesis are performed on bacteriophage DNA using the Kunkel method. After transformation with the modified DNA, E. coli bacteria are used to synthesize the mutant proteins for analysis. Biophysical techniques such as solvent and thermal denaturation provide essential thermodynamic data for characterizing the stabilities of the mutants. On the basis of the data obtained from the S. nuclease mutants, generalized predictions about protein structure and stability can be established.
Terry, A. (2000). Double Cysteine Mutations in Staphylococcal Nuclease: The effect of Artificially Introduced Disulfide Bonds on Protein Structure and Stability. Inquiry: The University of Arkansas Undergraduate Research Journal, 1(1). Retrieved from https://scholarworks.uark.edu/inquiry/vol1/iss1/9