Smooth and non-muscle myosin IIs are unique in that their activity is regulated by light chain phosphorylation. The goal of the first specific aim is to determine how regulatory light chain phosphorylation controls smooth muscle myosin's motor activity at the molecular level. A mutational analysis will be used to deduce the interactions that are essential for obtaining the off state from which phosphate release is blocked. The hypothesis is that multiple interactions between unphosphorylated myosin heads, as well as between the heads and specific sequences in the rod, are required for complete inhibition. To complement the functional/mutational studies, structural information about smooth muscle myosin will be obtained from crystallographic studies of expressed proteins in specific aim two. Structural studies will provide a framework for understanding regulation as well as for interpreting the effect of mutations that alter motor function. These studies will also provide insight into some of smooth muscle myosin's unique properties, such as its slow ATPase activity, and tail movement upon ADP binding. The investigation of regulation of motor activity will be extended to one of the classes of unconventional myosins in specific aim three, since similar strategies are probably exploited to control all myosin motor activity. the goal is to determine how calcium binding regulates the activity of unconbentional myosin V via multiple calmodulin molecules bound to IQ motifs in the neck region.
