The long term objective of the proposed research is to understand the structural and conformational events which occur in myosin during smooth muscle contraction. Toward this end, the dynamic conformational changes which chicken gizzard myosin undergoes in vitro will be probed. Two inter-related projects, one focusing on the myosin rod and other on the influence of actin, will probe how myosin filaments may be stablized in smooth muscle. A third project concentrates on conformational changes of the myosin head and swivel region, in an effort to understand on a molecular level how phosphorylation of the myosin 20-kDa light chain (LC- 20) regulates the actin-activated MgATPase.
The specific aims of proposed studies of the myosin rod are (i) to monitor the disposition of portions of the rod with respect to the filament backbone, under the influence of factors which affect myosin filament stability; (ii) to examine effects of these factors on myosin rod polymerization; (iii) to probe for regions of the rod whose conformation is dramatically changed in the """"""""10S"""""""" monomeric conformer of smooth muscle myosin. A vareity of methods will be used including proteolysis, cross-linking techniques, electron microscopy, analytical centrifugation and light scattering techniques.
The specific aims of studies examining influence of actin on myosin filament stability are (i) to probe in vitro the effects of actin on the dynamic equilibrium between filamentous myosin and the monomeric 10S conformer in the presence of MgATP; (ii) to examne the actin binding characteristics of the 10S conformer of myosin. Airfuge sedimentation, chemical cross-linking techniques, fluorescence quenching methods will be used.
The specific aims of work focusing on conformational changes of the myosin heads are (i) to examine the synergistic effects of actin binding and LC-20 phosphorylation on the myosin """"""""swivel"""""""" region; (ii) to probe for the interaction of actin with essential light chain of smooth muscle myosin; (iii) to clarify the functional role of the 50-kDa region of smooth muscle myosin; (iv) to introduce a specific fluorescent label into the essential light chain for subsequent probing by fluorescent techniques. A variety of biochemical methods will be employed, including proteolytic rate methods, cross-linking techniques and chemical modifications.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Biophysical Chemistry Study Section (BBCB)
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Mount Sinai School of Medicine
Schools of Medicine
New York
United States
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