Three dimensional structures have recently been determined for both actin and myosin, leading to an atomic level model for force production. In this model, the sequential opening and closing of two deep clefts in the myosin structure has been proposed to drive the rotation of a long lever arm known as the neck region. One cleft forms the nucleotide binding site (known as the pocket), the other spans the actin binding site. While this model is plausible, direct evidence for the motion of any one of the three of these elements is lacking. We propose to use both fluorescent and paramagnetic probes to monitor the opening and closing of the two clefts. We will also attach paramagnetic probes to myosin light chains and use them to measure the orientation of the neck region of the myosin head in muscle fibers to determine if this region rotates during force generation. These experiments will be carried out in muscle fibers, and in isolated myosin heads that have been genetically altered, to arrest them at different points in the acto-myosin cycle. In a separate but related set of experiments, we will extend these approaches to a study of microtubular based motors. The data will be analyzed in terms of molecular models of actin, myosin S1 and of models of their complexes. Docking of analogs into the ATP binding site of myosin will aid in designing and interpreting experiments.
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