Substructure and Communication in Myosin and Actin

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9630997 Reisler The contraction of skeletal, smooth, and cardiac muscles is fueled by the hydrolysis of ATP which is coupled to cyclic interactions between the two main muscle proteins, myosin and actin. These proteins, along with other motor proteins, are involved also in diverse functions of non-muscle cells including their locomotion, division, organelle transport, etc. According to prevalent views, motion and force are generated by myosin and actin as a result of cyclic changes in their interaction and structures during the different steps of ATP hydrolysis by myosin. A major goal of our research is to clarify the molecular basis of these events. To achieve this goal we will map those sites on actin which bind myosin in the presence of ATP and will analyze their role in specific steps of ATP hydrolysis and force and motion generation. The experimental materials that will be used in the mapping of the dynamic changes in myosin binding to actin during ATP hydrolysis include engineered actin mutants and chemically modified actins. These materials will be used in the in vitro measurements of actin filament motion and the force generated with myosin and ATP, and in biochemical, biophysical, and kinetic assays of actomyosin interactions. In another part of this program, the role of actin filament structure in actomyosin-based cellular functions will be clarified. The specific aim of this project is to determine the relationship between the functional interactions of actin with myosin and the dynamic changes in the organization of actin filaments. Proteolytically cleaved actin, chemically crosslinked actin, and actin mutants will be studied in this project by functional and structural analysis methods, by assays of actomyosin interactions, and by spectroscopic methods. The results of the proposed work will provide a structural description of the evolution of actomyosin interactions during force and motion generation by these proteins. This information will lead to the understanding of the basic molecular mechanisms by which chemical energy is converted into mechanical events in biological systems. On a broader scope, such an understanding will be instrumental in the design of biomimetic devices that will generate force or motion. %%% Muscle contraction involves the interaction between actin and myosin in a process that is dependent upon energy in the form of ATP hydrolysis. This project will investigate actin-myosin interactions and the mechanism by which these interactions contribute to the generation of force and motion in muscle as well as non-muscle cells. A variety of methods will be used including in vitro motility assays, force measurements, fluorescence spectroscopy, and biochemical characterization of the actomyosin interactions. These studies will improve the understanding of muscle contraction and the transduction of chemical energy into work in biological systems. *** .. . __,