It is generally accepted that the phosphorylation-dephosphorylation of the 20,000 dalton light chains of myosin primarily regulates the smooth muscle contractile machinery. The phosphorylation of the 20,000 dalton light chain by myosin light chain kinase at the serine 19 residue activates actomyosin ATPase activity which initiates contraction. However, it is not understood how the ATPase activity of actomyosin is regulated by light chain phosphorylation at the molecular level. The experiments described in this proposal will study in detail the molecular mechanism of the regulation of smooth muscle actomyosin. First, we will study the structure-function relationship of the 20,000 dalton regulatory light chain. We recently found that the amino acid residues of the N-terminal side of serine-19 are essential to express the phosphorylation mediated regulation. the structure required for the phosphorylation mediated regulation will be determined using site directed mutagenesis of the recombinant protein. The conformation of the regulatory light chain as well as the interaction with other subunits of the myosin molecule will be monitored using fluorescence probes and chemical cross-linking probes to elucidate the effects of phosphorylation on myosin conformation. Second, we will study the function of the 17,000 dalton light chain. Previous results suggest that the 17,000 dalton light chain may be involved in the catalitic site or regulatory mechanism of myosin ATPase. Using affinity labelling, monoclonal antibodies which influence the myosin function, fluorescence probes, chemical cross-linking and site directed mutagenesis, we will elucidate the role of the 17,000 dalton light chain on the regulation of myosin function. Third, the structural change of myosin molecule will be visualized by means of electron microscopy. The change in the structure during hydrolysis of ATP as well as the effects of phosphorylation will be observed using mica flake quick-freeze deep-etch technique. fourth, the conformation of the actin binding site of smooth muscle myosin will be determined using 2D NMR spectroscopy. Fifth, the characterization of the two heavy chain isozymes of smooth muscle myosin will be performed, and differential intracellular localization of the isoforms will be determined. Sixth, the possible regulation of myosin filament formation in vivo will be studied using microinjection techniques. The proposed studies employing protein biochemistry, molecular biology, biophysics and cell biology are part of a long term effort to determine the molecular mechanism of regulation of smooth muscle contractile machinery and should provide much insight into derangements of smooth muscle function in diseases such as hypertension, asthma and spastic disorders.
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