TROPOMYOSIN AND MYOSIN INTERACTION IN MUSCLE

这项建议的广泛的长期目标是阐明 原肌球蛋白在横纹肌和平滑肌收缩调节中的作用。 这需要详细了解它与肌动蛋白的相互作用， 肌钙蛋白由肌球蛋白、Ca 2+和ATP调节。 荧光测量 的探针连接到原肌球蛋白的半胱氨酸基团，将用于 监测原肌球蛋白状态的协同变化， 肌球蛋白亚片段1与细丝结合。 具体而言是 测量将用于：显示原肌球蛋白状态是 与肌动球蛋白亚片段1 ATP酶活性相关，阐明其作用 的两个头的肌球蛋白，确定心脏的影响， 骨骼肌钙蛋白成分对原肌球蛋白状态的影响，验证 原肌球蛋白状态，并确定光滑和光滑肌球蛋白行为的差异 横纹肌原肌球蛋白 圆二色性、荧光性和 采用化学改性的方法，研究其改性机理， α和β原肌球蛋白亚基（其氨基酸 序列略有不同）成二聚体（α α，α β和 betabeta）。 每个二聚体的构象和调节特性将 通过使用来自以下的原肌球蛋白的解折叠/组装研究来表征： 兔、蛙横纹肌、鸡胗、牛主动脉平滑肌 肌肉，和突变的条纹和光滑的α α原肌球蛋白， 重组DNA技术。 亚基氨基酸之间的关系 将允许表征正常的遗传变异以及 与遗传缺陷有关的改变。 肌球蛋白杆（光滑， 条纹）展开/组装研究将继续澄清 在特定区域的杆相关的构象变化 纤维形成和活性。 这些原肌球蛋白和肌球蛋白 构象研究也提供了关于折叠的基本信息 中间体和组装机制适用于其他蛋白质， 类似的螺旋线圈结构。

The broad long-term objective of this proposal is to elucidate the function of tropomyosin in the regulation of striated and smooth muscle contraction. This requires a detailed knowledge of its interactions with actin and troponin as modulated by myosin, Ca2+, and ATP. Fluorescence measurements of probes attached to cysteine groups of tropomyosin, will be used to monitor the cooperative change in state of tropomyosin associated with myosin subfragment 1 binding to the thin filament. Specifically, the measurements will be used to: show that the tropomyosin state is correlated with acto-myosin subfragment 1 ATPase activity, clarify the role of the two heads of myosin, determine the influence of the cardiac and skeletal troponin components on the tropomyosin state, verify that tropomyosin states, and determine the differences in behavior of smooth and striated muscle tropomyosins. Circular dichroism, fluorescence and chemical modification methods will be used to study the mechanism of assembly of the alpha and beta tropomyosin subunits (whose amino acid sequences slightly differ) into dimers (alphaalpha, alphabeta and betabeta). The conformational and regulatory properties of each dimer will be characterized by unfolding/assembly studies using tropomyosin from rabbit and frog striated muscle, chicken gizzard and bovine aorta smooth muscle, and mutated striated and smooth alphaalpha tropomyosin prepared by recombinant DNA techniques. The relationships between subunit amino acid will allow the characterization of normal genetic variation as well as alterations associated with genetic defects. Myosin rod (smooth and striated) unfolding/assembly studies will be continued to clarify conformational changes in specific regions of the rod associated with filament formation and activity. These tropomyosin and myosin conformational studies also provide basic information about folding intermediates and assembly mechanisms applicable to other proteins with similar coiled-coil structures.