9603807 Frieden The goal of this project is to examine the effect of site directed mutations on interactions between similar units within a filamentous protein. The model system to be used is actin. The crystal structure of monomeric muscle actin is known but it is not necessarily clear what the important contacts between actin monomers within the filament are. Thus, within the actin filament the interactions to be examined are those that occur between actin monomers. For this study it is planned to use mutants of yeast actin, this protein being approximately 90% identical to muscle actin. Yeast (Saccharomyces cerevisiae) contains a single actin gene obviating the need for separating different actin isoforms. A number of yeast strains expressing mutant actins are already available while others are under construction in this laboratory. Yeast actin can be obtained in reasonable yields from both wild-type and mutant strains. The first goal of this project is to examine the effect of site directed mutations, based on the known monomeric crystal structure, on the polymerization of actin itself to determine whether the mutation affects the formation of actin nuclei leading to filament formation or the elongation of the filament or both processes. Determination of the mechanism of actin polymerization can be made using sophisticated kinetic studies. Such studies are to be carried out following the polymerization process either by using a fluorescent label covalently attached to the actin or the intrinsic fluorescence of actin. Both these fluorescent methods are applicable because the fluorescence change is known to follow the incorporation of monomer into polymeric actin. Actin, a protein that is ubiquitous in all cells from yeast to humans, is a critical component in metabolic function. In muscle cells it interacts with the protein myosin to give rise to muscle contraction. In non-muscle cells, either alone or in combination with other proteins, actin i s responsible for maintaining the structure of the cell and for many cellular functions. The polymerization and depolymerization of actin controls such processes as cell movement, uptake of other molecules and response to extracellular signals such as hormones. The actin molecule is made up of 375 amino acid residues which fold together to give a specific three dimensional structure. Specific residues within this structure play an important role in the interaction of actin molecules with themselves to form the polymeric species while others are important for interacting with other protein molecules which control cellular processes. In this project site-directed mutagenesis,will be used to change specific residues in the actin molecule. The effect of these changes on the properties of the actin molecule itself and on its interactions with other proteins will be determined. The results will help to clarify the general role of certain amino acid residues in the interactions between proteins.
