Regulatory Mechanisms of Cardiac Muscle

说明(申请人提供)：心肌收缩是由钙结合TNC启动的，TNC是异源三聚体肌钙蛋白复合体(TNC，TnL，TnT)的一员。这种结合导致组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。这些分子变化定义了肌肉从不活跃状态到活跃状态的转变。钙开关在激活/去激活过程中控制这些事件。一种调节心脏功能的反馈控制机制涉及蛋白激酶A(PKA)对位于TnL独特的N-末端延伸的两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脏功能异常。分子开关位于两个亚基Tn1和Tnc之间的界面上。这种转换如何在结构改变和蛋白质-蛋白质相互作用的变化方面发挥作用仍有待阐明。该计划的一个长期目标是使用荧光光谱/Forster共振能量转移(FRET)和快速动力学作为主要工具，将这些分子事件及其变化定义为与正常心功能相关的。所使用的系统是由肌钙蛋白复合体、原肌球蛋白和肌动蛋白组成的合成细丝。这些蛋白质中的大多数将是为FRET研究设计的带有特定氨基酸替代的重组蛋白质。另一个长期目标涉及TnL的PKA磷酸化信号传递到分子的远端部分和其他2个肌钙蛋白亚基的机制。第三个目标和第四个目标是使用大量的FRET距离来构建肌钙蛋白复合体的分子模型，以研究钙结合引起的结构变化，特别是在已知具有重要功能但没有高分辨率结构信息的复合体区域。阐明这些机制将有助于理解心脏疾病状态的结构基础。

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.