ELECTROSTATIC AND PACKING MUTANTS IN STAPH NUCLEASE

点击翻译按钮获取中文摘要

The long range goals of our research program are to better understand the principles of protein folding and association. This fundamental problem is relevant to a better understanding of a wide range of biological systems and problems. We are attacking the problems through four approaches. All these studies are designed to be carried out by undergraduate workers. First, we making six polar mutations at a buried site in a more stable variant of our model protein staphylococcal nuclease. The variation of the stability of these mutant proteins with pH will be determined. This data will allow us to determine the microscopic dielectric constant of this site in the protein interior. An accurate experimental determination of the dielectric constant would increase the accuracy of modeling of electrostatic interactions in proteins. Second, we plan to make a large number of single packing mutants of the aliphatic residues of nuclease. We will substitute every leucine with isoleucine and valine, every isoleucine with leucine and valine, and every valine with leucine and isoleucine. The thermodynamic stability of these mutants will be determined. This data will allow us to examine the effects of packing in stabilizing this protein structure and provide a basic dataset to complement the data generated f rom our third approach. This third approach is to select two subsets of four aliphatic residues in nuclease's core and make all 81 possible mutant arrangement of isoleucine, leucine and valine at these four sites. We will also sample the possible mutants at six sites in the core. A library of some 210-220 different multiple mutant arrangements in the nuclease core will thus be generated. The thermodynamics stability of the proteins will be determined. This dataset will be the largest set of multiple mutants with complete thermodynamic characterization. Together with the data from the single mutations at every aliphatic site in the molecule we will have a splendid set of test data for modeling the packing of protein interiors. Our last initiative will be to clone, express, and purify two recently discovered proteins that are homologous to staphylococcal nuclease. The characterization of the structure and function of these protein relative to nuclease will provide useful information to complement the large number of existing studies of nuclease's structure and function.