Tropomyosin and the Regulation of Muscle Contraction

描述(由申请人提供)：广泛的、长期的目标是通过确定步兵和钙离子和肌球蛋白如何调节重要的原肌球蛋白-肌动蛋白相互作用来阐明横纹肌收缩调节的分子细节。与健康相关：了解与某些肌肉疾病相关的功能障碍，如TM、TNT、TNL和肌球蛋白突变引起的家族性肥厚性心肌病(FHC)，需要了解每个组成部分的作用和它们之间的相互作用。在与肌动蛋白的相互作用中，TM在决定肌肉细丝的3种状态之间的平衡中起着关键作用，即阻塞、闭合和开放(收缩发生在开放状态)。肌钙蛋白(在钙离子的辅助下)和肌球蛋白头部通过与肌动蛋白和TM相互作用，改变状态之间的平衡，从而开启和关闭收缩。具体目的：(A)确定TM在肌动蛋白上的灵活性与激活的肌动蛋白亚基数量(合作单位大小)之间的关系；确定FHC-TM突变体的调节特性改变；(B)确定Tn复合体的Tn1和TnT组分在肌动蛋白Tm丝上的位置以及在活性状态之间发生的变化；(C)确定有组织的肌节中细丝成分的相互作用的变化。方法：除了用ATPase、结合和动力学研究表征溶液中的3种状态外，还将广泛使用高分辨率的距离测量方法，利用溶液中时间分辨荧光检测能量转移(FRET)来测量组分之间的距离，以及在交换成单一肌原纤维的组分的肌节区域。这些特定标记氨基酸残基之间的测量将提供有关调控过程中涉及的成分之间变化的动态信息。

DESCRIPTION (provided by applicant): The broad, long-term objective is to elucidate the molecular details of the regulation of striated muscle contraction by determining how trooping and Ca2+ and myosin, regulate the important tropomyosin-actin interaction. Health Relatedness: The role of each component and their interactions are required to understand the malfunctions associated with certain muscle diseases, e.g., familial hypertrophic cardiomyopathy (FHC) resulting from mutations in Tm, TnT, Tnl and myosin. Tm, in its interaction with actin, plays a key role in determining the equilibria between the 3 states of the muscle thin filament, Blocked, Closed and Open (contraction takes place in the Open state). By interacting with actin and Tm, troponin (assisted by Ca2+) and myosin heads change the equilibrium between the states, thereby turning contraction on and off. Specific Aims: (a) to determine the relationship between the flexibility of Tm on actin and the number of actin subunits activated (the cooperative unit size); to determine the altered regulatory properties of the FHC Tm mutants; (b) to determine the location of the Tnl and TnT components of the Tn complex on the actinTm filament and the changes that take place in shifting between activity states; (c) to determine the changing interactions of the thin filament components in the organized sarcomere. Methods: In addition to characterization of the 3 states in solution with ATPase, binding and kinetic studies, extensive use will be made of high resolution distance measurements between components with time-resolved fluorescence-detected energy transfer (FRET) in solution and in regions of the sarcomere of components exchanged into single myofibrils. These measurements between specific labeled amino acid residues will provide information about the dynamics of the changes between the components involved in the regulatory process.