NANOSECOND ROTATIONAL DYNAMICS OF THIN FILAMENT PROTEINS

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The long term goal of this project is to determine the molecular mechanism of force generation and its regulation in the actomyosin contractile system of skeletal muscle. The project seeks to achieve this goal through measurements of the nanosecond rotational motions of individual proteins of the actomyosin contractile apparatus during ATP hydrolysis in a reconstituted model system. The rotational dynamics of individually labeled proteins of the thin filament complex will be measured using time-resolved polarization anisotropy of fluorescence from probes covalently attached to specific sulfhydryls in the proteins actin, tropomyosin, troponin C, and troponin I. (Appropriate probes include IAEDANS and pyrene). The nanosecond rotational dynamics of each of these proteins will be monitored in the thin filament complex (F-actin + Tropomyosin + Troponin) and in complexes with myosin and its proteolytic fragments (myosin heads) under conditions that mimic the physiologically relevant states of rigor (-Ca2+, -ATP), relaxation (-Ca2+,+ATP), and contraction (+Ca2+,+ATP). These studies will compliment our ongoing studies of the rotational dynamics of these proteins on the microsecond time scale using phosphorescence emission anisotropy.