Substructure and Communication in Myosin and Actin

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9904599 Reisler Myosin and actin, the two major muscle proteins, interact with each other in a cyclic fashion and use the chemical fuel of cells, adenosine triphosphate (ATP), to power muscle contraction and many motile and transport processes in non-muscle cells. The cycle of actomyosin interaction involves the formation of weakly and strongly bound complexes of these proteins. Force and motion are generated during, or after, transition between the weakly and strongly bound actomyosin states. The first goal of this project is to determine the role and contribution of specific sites on actin to such a transition. To this end, mutations will be engineered in yeast actin, at its N-terminus, and loops 21-29 and 92-103, to alter their charge and to introduce cysteine residues for attachment of fluorescent probes. The mutant and labeled mutant actins will be used in the in vitro motility assays, spectroscopic studies, and biochemical assays of actomyosin interactions to clarify the structural determinants of the transition between weakly and strongly bound actomyosin states. The second goal of this project is to clarify the role of structural dynamics of actin filaments in the contractile function of actomyosin. This goal will be pursued through specific chemical cross-linking reactions between actin monomers in the filament. These reactions will immobilize the interface between subdomain 1 and 2 on adjacent actins, and thus allow the testing of functional significance of actin dynamics in the in vitro motility and other assays. Mutant yeast actins will be prepared with cysteine substitutions to enable such cross-linking reactions and to introduce probes for monitoring the conformational states of actin. The results of this work will provide a description of the changes which occur in the areas of contact between myosin and actin at different steps of the force generation process by actomyosin. This work will also clarify the role of actin and its dynamic properties in the biological function of actomyosin. Such information is necessary for a detailed understanding of the basic biological process of motion and force generation by this complex of proteins. General principles of actin and myosin interactions and the regulation of their interactions, which will be elucidated in this work, should help in the understanding of how other protein complexes carry out similar force and motion generation tasks in living cells. The work on these projects will provide broad training for postdoctoral, graduate, and undergraduate students in the areas of molecular biology, biochemistry, and biophysics and their applications in the studies on the structure and function of proteins.