TOPOGRAPHICAL STUDIES OF SMOOTH MUSCLE MYOSIN

荧光技术将用于探测光滑的拓扑结构

Fluorescence techniques will be used to probe the topology of smooth muscle myosin. The major goal of these experiments is to answer questions about the molecular mechanism of regulation of smooth muscle activity. Fluorescent and/or chromophoric compounds will be used to specifically label the myosin active site, various positions within the heavy chain, the two different kinds of light chains, and a specific site on the rod portion of the molecule. A novel new method of attaching probes specifically to serines or threonines that are phosphorylated by kinases is presented. This labeling method has potential to be generally useful to study the structural aspects of phosphorylated sites in other systems specifically phosphorylated by kinases. Fluorescence energy transfer techniques will be used as a spectroscopic ruler to probe conformational transitions which may be important to the mechanism of smooth muscle myosin regulation by light chain phosphorylation. The effects of phosphorylation of the regulatory light chains by either myosin light chain kinase or protein kinase C upon these distances will be examined. Using these approaches, the hypothesis that the head-tail junction of the myosin molecule plays a key role in the mechanism of regulation of smooth muscle myosin will be tested. This information will be useful to determine how the phosphorylation dependent conformational changes observed in vitro relate to physiologically important structural changes within myosin filaments in vivo.