The long term objective of this research is to understand the regulation and function of myosin in smooth muscle cells. It is well accepted that the motor activity of smooth muscle myosin is regulated by phosphorylation of the regulatory light chain (RLC) at Ser19. However, it is not understood how the phosphorylation of RLC regulates the myosin motor activity. Our hypothesis is that phosphorylation of RLC changes a conformation that attenuates an inter-head interaction at RLC binding domain, and this change is transmitted to the motor domain via a long -helix shaft of the C-terminal domain of the head. We will verify this hypothesis by producing and expressing various engineered myosins and characterizing their motor function by enzymatic and mechanical assays. It is known that smooth muscle contraction is not proportional to the level of myosin phosphorylation. This can be, in part, due to the cooperative nature of the two heads of myosin; however, how the motor activity of myosin phosphorylated at a single head affects or is affected by cooperativity is not understood. Thus a major question is whether or not one head influences the motor activity of the other head. This question is best answered (and may in fact only be answerable) by using a single molecule assay system which enables us to determine ATP turnover and mechanical events simultaneously. This will be done in the proposed study. Smooth muscle cells express various myosin isoforms that show distinct function in vitro. However, the physiological significance of these isoforms for smooth muscle contraction is obscure. The critical questions are whether or not these isoforms form heterodimers, and whether or not these isoforms produce co- filaments in cells. These questions will be addressed by the use of advanced 3D fluorescence microscopy and digital imaging systems for precise colocalization analysis, the use of electron microscopy to measure filament structure and composition, and the use of immunochemistry/biochemistry/molecular biology for the detection of heterodimers. Finally, smooth muscle myosin forms a unique filament structure called "side polar" which may be critical for the contractile characteristics of smooth muscle. We will identify the molecular basis of this filament structure. The itemized specific aims are: 1) To define the structure of the 20,000 dalton light chain critical for the phosphorylation- induced activation of the myosin motor; 2) To define the heavy chain structure responsible for the regulation and function of smooth muscle myosin motor activity; 3) To define the cooperativity between the two heads of smooth muscle myosin; 4) To define whether or not myosin forms heterodimers with different isoforms; 5) To define the localization of various myosin isoforms expressed in smooth muscle cells; 6) To define the molecular basis of side polar myosin filament structure of smooth muscle myosin.