In this Project the structure and function of caldesmon will be investigated. Binding of caldesmon to actin, calmodulin, tropomyosin and myosin, the mechanistic details of the inhibition of actomyosin ATPase activity by caldesmon and the reversal of such an inhibition by calmodulin will be studied. The overall goal is to elucidate the functional role of caldesmon in the thin filament-based regulation in the smooth muscle system. Specifically, we propose to study: (1) The Structure and Function of Various Regions of Caldesmon. This includes the C-terminal region which contains most of the known functional properties such as binding to actin, tropomyosin and calmodulin, and the inhibitory effect, the N-terminal region, which contains the myosin-binding site, and the Central region which appears to be a long, single alpha-helix that is unique for smooth muscle caldesmon. Fluorescence measurements, photo-crosslinking, and resonance energy transfer, in conjunction with electron microscopy, ultracentrifugation and circular dichroism measurements will be used to study the structure-function relationships of caldesmon fragments. Variants of caldesmon fragments prepared by site-directed mutagenesis, and chemically synthesized peptides will also be used. (2) Interactions of Intact Caldesmon with Other Proteins. Similar methodologies will be used to characterize binding of actin, calmodulin, myosin and tropomyosin to the intact caldesmon by specifically labeling of the two thiols with various probes. Information obtained will be compared to that with the fragments and peptides. Possible interaction between caldesmon and calponin under physiological conditions will be investigated. Effects of other protein components, salt, GTP and phosphorylation of individual proteins will also be examined. (3) The Localization and Function of Caldesmon in Organized Smooth Muscle Systems. The location and the mode of binding of caldesmon in smooth muscle cells and isolated native thin filaments will be visualized by light microscopy and electron microscopy with the aid of fluorescently labeled proteins and specific antibodies that do not interfere with the binding of caldesmon. The in situ function of caldesmon will be probed by using fragments with altered amino acids or synthetic peptides that prevent caldesmon from binding to the thin filaments. Antibodies will also be added into the cell to test the postulated function of various parts of the molecule. These studies will enable us to assess the role of caldesmon in the regulatory process of smooth muscle contraction.
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