Regulatory Mechanisms of Cardiac Muscle

描述（由申请人提供）：心肌收缩是由钙与TnC结合引起的，TnC是异三聚体肌钙蛋白复合物（TnC, Tnl, TnT）的一员。这种结合导致构成肌肉细丝的几种蛋白质发生结构变化，并改变这些蛋白质之间的相互作用。这些分子变化决定了肌肉从不活跃状态到活跃状态的转变。在激活/失活期间，钙开关控制这些事件。作为反馈控制调节心脏功能的机制涉及蛋白激酶A （PKA）对位于Tnl独特n端延伸的2个相邻丝氨酸残基的磷酸化。钙结合和磷酸化程度的改变可导致心功能异常。分子开关位于两个亚基Tnl和TnC之间的界面上。这种开关如何在结构改变和蛋白质-蛋白质相互作用的变化方面运作仍有待阐明。该计划的长期目标是使用荧光光谱/福斯特共振能量转移（FRET）和快速动力学作为主要工具，定义这些分子事件和这些事件的变化与正常心脏功能相关。该系统是由肌钙蛋白复合物、原肌凝蛋白和肌动蛋白组成的合成细丝。大多数这些蛋白质将是重组蛋白与特定的氨基酸取代设计的FRET研究。另一个长期目标与PKA磷酸化Tnl的信号传递到分子的远处部分和其他2个肌钙蛋白亚基的机制有关。第三和第四个目标是构建肌钙蛋白复合物的分子模型，利用大量的FRET距离来研究钙结合反应的结构变化，特别是在已知的功能重要的复合物区域，但没有高分辨率的结构信息。阐明这些机制将有助于了解心脏病变状态的结构基础。

DESCRIPTION (provided by applicant): Contraction of cardiac muscle is initiated by calcium binding to TnC, which is a member of the heterotrimeric troponin complex (TnC, Tnl, TnT). This binding results in structural changes that occur in several proteins that make up the muscle thin filament and alterations of the interactions among these proteins. These molecular changes define the transition of muscle from the inactive to the active state. A calcium switch controls these events during activation/deactivation. A mechanism that regulates cardiac function as a feed back control involves phosphorylation by protein kinase A (PKA) of 2 adjacent serine residues located in the unique N-terminal extension of Tnl. Alteration of the extent of calcium binding and phosphorylation can lead to abnormal cardiac functions. The molecular switch is located in the interface between the 2 subunits Tnl and TnC. How this switch operates in terms of structural alterations and changes in protein-protein interactions remains to be elucidated. A long-term goal of this program is to define these molecular events and changes of these events as related to normal cardiac function, using fluorescence spectroscopy/Forster resonance energy transfer (FRET) and rapid kinetics as major tools. The system to be used is the synthetic thin filament consisting of the troponin complex, tropomyosin and actin. Most of these proteins will be recombinant proteins with specific amino acids substitutions designed for FRET studies. Another long-term goal relates to the mechanisms by which the signal of PKA phosphorylation of Tnl is transmitted to distant parts of the molecule and to the other 2 troponin subunits. The third goal and fourth goals are construction of molecular models for the troponin complex using a large number FRET distances to study structural changes in response to calcium binding, particularly in regions of the complex known to be functionally important, but for which no high-resolution structural information is available. Elucidation of these mechanisms will contribute to the understanding of the structural basis of the diseased state of the heart.