Mechanism of Thin Filament Regulation of Cardiac Actomyosin Hydrolysis

描述(申请人提供)：我们将使用稳态前动力学方法和电子显微镜来确定心肌肌动球蛋白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“立体阻断”的细丝调节机制，即原肌球蛋白在低钙条件下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一机制不一致。钙和僵直的肌球蛋白与快速骨骼肌细丝结合可加快肌球蛋白-ADP-PI的最大解离速率约200倍，但最多使肌球蛋白-ADP-PI与肌球蛋白-ADP-PI结合的亲和力增加3倍。这表明空间位阻在调节机制中只是一个非常小的组成部分。骨骼肌动蛋白和心脏肌动蛋白、肌球蛋白、肌钙蛋白和原肌球蛋白的氨基酸序列存在显著差异，而心肌肌动球蛋白ATP水解的钙调节机制尚不清楚。心肌肌钙蛋白-C只有一个调节钙结合位点，而心肌肌钙蛋白-L有一个26个氨基酸的延伸，包含一个生理上有意义的磷酸化位点。除了为细丝调节机制的研究提供关键信息外，拟议的工作还与一些涉及心肌收缩调节控制的疾病直接相关，例如扩张型心肌病，在这些疾病中，收缩的力量不足。多次混合停流将被用来测量天然心脏细丝从心肌肌球蛋白-腺苷二磷酸-PI解离产物的速度(等电点和二磷酸腺苷)对钙离子和肌球蛋白结合的依赖性，以及肌钙蛋白-L的磷酸化的依赖性。使用负染和冷冻-EM方法的平行电子显微镜将被用来确定心脏细丝(在钙和结合的肌球蛋白激活的不同状态下)的结构以及结合在细丝上的肌球蛋白的分布。结构数据将通过单粒子方法进行分析，这种方法已经被COPI的实验室改装成灯丝结构(约翰·特里尼克)。这些数据将被用来确定心脏细丝的调节机制，并加深我们对心脏收缩能力调节机制的理解。

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.