The control of muscle contraction by myosin is studied at the molecular level in molluscan muscles. We have previously shown that in these muscles calcium triggers contraction by binding directly to myosin and that the regulatory components are the regulatory and essential light chains of myosin which maintain the resting state in the absence of calcium. The mechanism of this subunit regulation is most conveniently studied on scallop myosin since the regulatory light chains can be fully and reversibly dissociated from the heavy chains without denaturation. Removal of the RLCs and immobilization of the light chains at a site where the two myosin heads join locks muscle in the """"""""on"""""""" state, losing its ability to relax. The interactions between the light chains and between the heavy and light chains are studied with the aid of photolabile, heterobifunctional cross-linkers, and with chemical and genetic modifications. We plan to identify the cross-linked products formed with various foreign light chains in rest and rigor. Since cross-linkers introduced into the various foreign light chains are located differently in the sequence, the experiments will help to ascertain the polarity, colinearity and relative position of the light chains in myosin in its different functional states. Cross-linkers introduced via the fast reacting heavy chain thiols will relate the heavy chains to light chain movement. Fragmentation of the heavy chain with proteolytic enzymes may identify regions within the molecule responsible for particular functions. Light chains modified by site-directed mutagenesis will help to identify residues responsible for binding to the heavy chain, calcium binding and calcium regulation of contractile functions. Introduction of thiol groups in predetermined positions of the sequence will allow for a more precise localization of the light chains in myosin, and a more accurate description of their rearrangement. The findings will clarify the basic mechanisms of thick-filament regulation They may also be extended to vertebrate smooth and non-muscle myosins, which are also regulated by light chains but triggered by phosphorylation instead of calcium binding.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Brandeis University
Schools of Arts and Sciences
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