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 intended 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 six projects, two facility cores and an administrative core. Projects I- A deals with the effect of phosphorylation of the myosin structure and on the interaction between acting and myosin. Project I-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 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 II-C 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. All projects, with the exception of II-C and II, use chemical crosslinking and resonance energy transfer as tools to characterize protein-protein interactions, and practically every project uses-directed mutagenesis to produce protein variants. Projects II-A II-C, in particular, will explore genetic approaches to elucidate the functional significance of some of the regulatory proteins. Other methods include fluorescence and circular dichroism measurements, chemical and enzymatic proteolysis, use of synthetic peptides, analytical ultracentrifugation and 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 ore, 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 continue our investigation on the regulatory mechanisms in the smooth muscle system.
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