Mechanism of Thin Filament Regulation of Cardiac Actomyosin Hydrolysis

描述（由申请人提供）：我们将使用稳态动力学方法和电子显微镜来确定心脏肌动球蛋白ATP水解的细丝调节机制。大多数生物化学和生理学教科书描述了一种“空间阻断”机制，即原肌球蛋白在低钙时阻断肌球蛋白与肌动蛋白的结合。然而，现在有令人信服的实验数据与这种机制不一致。钙和僵硬的肌球蛋白结合到快速骨骼肌细丝上，使肌球蛋白-ADP-Pi的产物解离的最大速率加速约200倍，但使肌球蛋白-ADP-Pi结合到细丝上的亲和力最多增加3倍。这表明空间阻断是调节机制的一个非常小的组成部分。骨骼肌和心脏肌动蛋白、肌球蛋白、肌钙蛋白和原肌球蛋白之间的氨基酸序列存在显著差异，并且心脏肌动球蛋白ATP水解的钙调节机制还不太清楚。心肌肌钙蛋白- C仅具有单一调节性钙结合位点，心肌肌钙蛋白-I具有26个氨基酸的延伸，其包含生理上显著的磷酸化位点。除了在细纤维调节机制的研究中提供关键信息外，所提出的工作与涉及控制心肌收缩调节的许多疾病直接相关，例如收缩力不足的扩张型心肌病。将使用多重混合停流来测量天然心脏细丝从心脏肌球蛋白-ADP-Pi中解离产物（Pi和ADP）的加速对钙和僵硬肌球蛋白与细丝结合以及肌钙蛋白磷酸化的依赖性-l。使用负染色和冷冻EM方法的平行电子显微镜检查将用于确定心脏细丝的结构（在钙和结合肌球蛋白的各种激活状态下）以及与细丝结合的肌球蛋白的分布。结构数据将通过CoPI实验室（John Trinick）采用的适用于细丝结构的单粒子方法进行分析。结合数据将用于确定心脏细丝的调节机制，并增加我们对心脏收缩力调节机制的理解。

DESCRIPTION (provided by applicant): We will use presteady state kinetic methods and electron microscopy to determine the mechanism of thin filament regulation of cardiac actomyosin ATP hydrolysis. Most biochemistry and physiology textbooks depict a "steric blocking" mechanism of thin filament regulation in which tropomyosin blocks the binding of myosin to actin at low calcium. However, there is now compelling experimental data that are inconsistent with this mechanism. Calcium and rigor myosin binding to fast skeletal muscle thin filaments accelerate the maximum rate of the product dissociation from myosin-ADP-Pi approximately 200 times but produce at most a 3 fold increase in the affinity of myosin-ADP-Pi binding to the thin filament. This indicates that steric blocking is a very small component of the regulatory mechanism. There are significant differences in the amino acid sequences between skeletal and cardiac actin, myosin, troponin, and tropomyosin and the mechanism of calcium regulation of cardiac actomyosin ATP hydrolysis is much less well characterized. Cardiac troponin- C has only a single regulatory calcium binding site and cardiac troponin-l has a 26 amino acid extension that contains a physiologically significant phosphorylation site. In addition to providing key information in the study of the mechanism of thin filament regulation, the proposed work is directly relevant to a number of diseases involving control of the regulation of cardiac muscle contraction, such as dilated cardiomyopathy, in which the force of the contraction is inadequate. Multi-mixing stopped-flow will be used to measure the dependence of the acceleration of product dissociation (Pi and ADP) from cardiac myosin-ADP-Pi by native cardiac thin filaments upon calcium and rigor myosin binding to the thin filament, and from phosphorylation of troponin-l. Parallel electron microscopy using negative stain and cryo-EM methods will be used to determine the structure of cardiac thin filaments (in various states of activation by calcium and bound myosin) and the distribution of the myosin bound to thin filaments. The structural data will be analyzed by single particle methods that have been adapted to filament structures by the laboratory of the CoPI (John Trinick). The combined data will be used to determine the mechanism of regulation by cardiac thin filaments and increase our understanding of mechanism of regulation of cardiac contractility.