TROPOMYOSIN-MYOSIN INTERACTION IN MUSCLE

这项提议的广泛的长期目标是阐明 原肌球蛋白在调节横纹肌和平滑肌收缩中的作用。 这需要详细了解它与肌动蛋白和肌动蛋白的相互作用。 肌钙蛋白受肌球蛋白、钙离子和三磷酸腺苷的调节。荧光测量 连接在原肌球蛋白半胱氨酸基上的探针，将用于 监测原肌球蛋白状态的协同变化 肌球蛋白亚段1与细丝结合。具体地说， 测量将用于：显示原肌球蛋白的状态是 与肌球蛋白亚片段1 ATPase活性相关，阐明其作用 肌球蛋白的两个头部，确定心脏和 原肌球蛋白状态下的骨骼肌钙蛋白成分，验证 原肌球蛋白状态，并确定光滑和光滑的行为的差异 横纹肌原肌球蛋白。圆二向色性、荧光性和 将采用化学修饰的方法来研究其作用机理。 α和β原肌球蛋白亚基的组装(其氨基酸 序列略有不同)为二聚体(AlphaAlpha、Alphabeta和 斗鱼)。每个二聚体的构象和调节性质将 以使用原肌球蛋白的展开/组装研究为特征 兔蛙横纹肌、鸡胆和牛主动脉光滑 肌肉，以及突变的条纹和光滑的α-原肌球蛋白 重组DNA技术。氨基酸亚基之间的相互关系 将允许表征正常的遗传变异以及 与遗传缺陷相关的改变。肌球蛋白棒(光滑和 条纹)展开/组装研究将继续进行以澄清 杆状蛋白特定区域的构象变化与 细丝的形成和活性。这些原肌球蛋白和肌球蛋白 构象研究还提供了有关折叠的基本信息 适用于其他蛋白质的中间体和组装机制 类似的盘绕线圈结构。

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.