The goal of the proposed research is to quantify the individual effects of filament structure and phosphorylation in smooth and skeletal muscle myosins, and to examine their roles in modifying the mechanical behavior of single myosin molecules. Smooth and skeletal muscle myosins polymerize to form two very distinct filament structures. The functional consequence of this structural difference is unknown. Furthermore, there is tremendous disagreement in the literature as to the """"""""step displacement"""""""" of myosin, with estimates ranging from 5 - 20 nm. The value obtained appears to correspond to the structural state of the myosin being used in each laboratory. Taken as a whole, these data suggest that the level of structural organization is critical to the overall performance of myosin, and that myosin molecules within a filament may cooperate to generate force and motion. However, intermolecular cooperativity at the level of myosin filaments has never been systematically investigated, and is thus the first major thrust of the proposed research. We will use a laser trap force transducer to measure and compare the single steps in force and displacement generated by smooth and skeletal muscle myosins both in their monomeric and filamentous states. The objective is to understand if the constituent monomers of myosin filaments act cooperatively, and whether smooth and skeletal muscle filaments behave differently in this respect. Phosphorylation of the regulatory light chain of myosin alters the structures of myosin filaments in both smooth and skeletal muscles. Because of this, we are obligated to examine both phosphorylation and filament structure in order to understand either. The structural change that occurs upon phosphorylation appears to be an extension of the myosin heads away from the myosin filament and toward actin. This conformational change may underlie the well-established regulation of contraction in smooth muscle by phosphorylation, as well as the enhancement of force generation in skeletal muscle. However, the mechanical consequences of phosphorylation have yet to be examined at the level of the individual myosin heads. Thus, the second major thrust of the proposed research is to examine the effects of phosphorylation on the forces and displacements generated by these myosins in their monomeric and filamentous states. These data are critical to our understanding of how myosin functions at the molecular level in whole cells and tissues.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Lymn, Richard W
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University of Virginia
Biomedical Engineering
Schools of Medicine
United States
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