A Mutagenic Study of Yeast Actin Conformational Changes

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DESCRIPTION (provided by applicant): Actin is involved in generation of contractile force, cell shape determination, cytokinesis, and determination of cell polarity. Proper actin fuction, essential for cell viability, depends on a certain degree of filament stability and conformational flexibility. The dynamic nature of the actin monomer, both free and within the actin filament, is required for allosteric regulation of actin, both internally, and imposed upon it by an array of actin regulatory proteins. A manifestation of this regulation is the difference in functional behavior of yeast vs. muscle actin. They are 87% identical and have virtually superimposable structures. Yet they differ in parameters such as speed of polymerization, propensity of filaments to fragment, rate of nucleotide exchange, rate of phosphate release following bound nucleotide hydrolysis (a determinant of filament dynamics), ability to activate myosin, and response to actin binding proteins such as cofilin and Arp2/3 complex. To understand at the molecular level, the ways in which actin filament dynamics are regulated, one must first understand the way in which conformational changes are transmitted within the protein and how differences in these communication networks result in the behavioral differences of actin isoforms. We will use a series of yeast/muscle hybrid actins, manufactured by site-directed mutagenesis, to better understand the roles of actin's two domains in controlling the behavior of the actin monomer in the G-form and in the filament. With yeast as a model system, we will extend our initial work with such hybrid proteins using a combination of cell biological and protein chemical approaches such as polymerization kinetics, covalent crosslinking, and hydrogen/deuterium exchange to address the following aims. We will delineate the role of a subdomain 1 column of amino acids in regulation of domain/domain interactions. We will assess the effect of filament fragmentation on cytoskeletal function and how the balance between inherent filament stability and cofilin severing affects cytoskeletal behavior. We will examine the importance of two proposed intermonomer ionic interactions on filament stability, and we will assess the role of two critical subdomain 4 residues on the behavior of subdomains 1 and 2 in their interaction with actin binding proteins. Health Significance: This work will provide new insight into the molecular basis of actin regulation which, when abnormal, leads to pathological states such as cancer, muscle disease, and hearing loss.