TOPOGRAPHICAL STUDIES OF SMOOTH MUSCLE MYOSIN

荧光技术将被用来探测光滑的拓扑结构 肌球蛋白 这些实验的主要目的是回答 平滑肌活动调节的分子机制。 荧光和/或发色化合物将用于特异性地 标记肌球蛋白活性位点，重链内的各个位置， 两种不同的轻链，以及杆上的特定位点， 分子的一部分。 一种新的探针附着方法 特别是被激酶磷酸化的丝氨酸或苏氨酸 给出 这种标记方法具有普遍适用的潜力 研究其他系统中磷酸化位点的结构方面 被激酶特异性磷酸化。 荧光能量转移 技术将被用作光谱尺来探测构象 可能对平滑肌的机制很重要的转变 通过轻链磷酸化调节肌球蛋白。 的影响 调节轻链被肌球蛋白轻链磷酸化， 链激酶或蛋白激酶C对这些距离的影响。 使用这些方法，假设头尾连接的 肌球蛋白分子在平滑肌细胞的调节机制中起着关键作用， 将测试肌肉肌球蛋白。 这些信息将有助于 确定磷酸化依赖的构象变化 在体外观察到的与生理学上重要的结构变化有关 在体内肌球蛋白丝内。

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.