Myosin in smooth muscle is not only the motor but is also directly involved in the regulation of contraction. Phosphorylation of the regulatory light chains (LC20) is required to initiate the force generation in smooth muscle contraction. the overall goal of this project is to correlate the structural features of gizzard myosin with its functions. In particular, how the effects of the phosphorylation of LC20 are communicated to the regions that are responsible for myosin activities will be examined. Specifically, the following studies will be carried out: (1) To determine the effects of phosphorylation on the structure of LC20, fluorescence probes and crosslinkers will be attached to the single, indigenous Cys-108 of LC20, and the changes in fluorescence and the intra-LC20 crosslinking pattern due to phosphorylation will be determined with and without the association of heavy chains (HC). (2) To determine the effects of phosphorylation of LC20 on its interaction with HC, the sites on HC that are involved in binding of phosphorylated or unphosphorylated LC20 will be identified by using thiol specific photoactivatable crosslinkers attached to Cys-108 of LC20. (3) To characterize the conformational changes of myosin upon LC20 phosphorylation, the effects of phosphorylation on the intra-heavy chain crosslinking pattern of myosin will be monitored by using crosslinkers attached to the reactive thiols on the head and neck/S2 portion of heavy chains. (4) To examine the effects of LC20 phosphorylation on the actin-myosin interaction, the interfaces between actin and myosin head with phosphorylated or unphosphorylated LC20 will be identified by using a zero length crosslinker. Site-directed mutagenesis will be a major tool to generate variants of LC20. Resonance energy transfer will be used to map the distances between landmarks. Other techniques include circular dichroism measurements, EM, ultracentrifugation, proteolytic susceptibility and fluorescence measurements.
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