Despite the importance of smooth muscle in health and disease, relatively little is known about many details of its operation compared with striated muscle. This application is a coordinated effort intended to gain more information in this area. The overall goal is to apply the biophysical and biochemical methodologies that have been successfully used in the striated muscle system to the smooth muscle system, and to enhance our understanding of the process that regulates smooth muscle contraction. The program contains six projects, a biophysical and biochemical core and an administrative core. Project I-A deals with the effect of phosphorylation on the myosin structure and on the interaction between actin and myosin. Project II-B is concerned with the mechanism by which myosin light chain kinase is activated by Ca/calmodulin and that by which myosin light chain is recognized by the enzyme. Projects II-A and II-B study the structure- function relationships of caldesmon and calponin, respectively, in the hope that such studies will lead to the elucidation of their functional roles in the thin filament-based regulation of smooth muscle contraction. Project II-C uses X-ray crystallography to gain information on the 3D structures of the thin filament regulatory proteins: tropomyosin, caldesmon and calponin. In Project III the integrated system of the thick and the thin filaments will be examined, and the possible role of tropomyosin in the cooperative properties of smooth muscle thin filament will be explored. All projects except II-C and III, use chemical crosslinking and resonance energy transfer as major tools to characterize protein-protein interactions, and practically every project uses site-directed mutagenesis to produce protein variants. Other methods include fluorescence and circular dichroism measurements, chemical and enzymatic proteolysis, use of synthetic peptides, analytical ultracentrifugation and electron microscopy. The in vitro motility assay featured in Project III will also be available for other projects. In Biophysical/Biochemical Core, the Protein Chemistry service will be used by every project; the Analytical Ultracentrifugation service will support Projects I-A, I-B, II-A and II-B; and the Electron Microscopy and Immunocytochemistry service is anticipated to be used by Projects I-A, II-A, II-B and possibly III. Funding of this program will allow us to launch a major investigation on the regulatory mechanisms in the smooth muscle system.
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