Despite the importance of smooth muscle in health and disease, the details of its operation and regulation remain largely unclear. This renewal application is a continued effort to gain more information in this area. The overall goal is to use molecular and cellular methodologies to gain information on the functional roles of various smooth muscle proteins and to enhance our understanding of the regulatory mechanism of smooth muscle contraction. The program contains five projects, two facility cores and one administrative core. Project I deals with the thick filament regulation by investigating the activation the activation mechanism of myosin light chain kinase by Ca/calmodulin and how myosin is recognized by the enzyme. Projects IIA and IIB study the structure and function of caldesmon and calponin, respectively, in the hope that such studies will lead to the elucidation of their roles in the thin filament- based regulation of smooth muscle contraction. Project IIC searches for the regulatory pathway involving the mitogen activated protein kinases in smooth muscle. 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. Projects I, IIA and IIB all use chemical crosslinking and resonance energy transfer as tools to characterize protein-protein interactions, Projects IIA, IIB and IIC will involve extensive physiological measurements of smooth muscle tissues, and particularly every project uses site-directed mutagenesis to produce variants. Projects IIA and IIC, in particular will explore genetic approaches to elucidate the functional significance of some of these regulatory proteins. Other methods include fluorescence and circular dichroism measurements, rapid kinetics, chemical and enzymatic proteolysis, use of synthetic peptides, analytical ultracentrifugation and immuno-electron microscopy. The Biophysical/Biochemical Core includes Protein Chemistry service, the Analytical Ultracentrifugation service and the Electron Microscopy and Immunocytochemistry service. In the Protein Expression Core, recombinant proteins will be generated by baculovirus expression system in insect cells. Both cores will be used by all projects. Funding of this program will allow us to expand our current investigation on the regulatory mechanism in the smooth muscle system.
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