Mechanism of Thin Filament Regulation of Cardiac Actomyosin Hydrolysis

心肌肌动球蛋白水解的细丝调节机制

• 批准号: 7433231
• 负责人: HOWARD D. WHITE
• 金额: $ 30.56万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7433231
• 关键词: ATP Hydrolysis; Acceleration; Actins; Actomyosin; Affect; Affinity; Amino Acid Sequence; Amino Acids; Binding; Binding Sites; Biochemistry; Calcium; Calcium Binding; Cardiac; Cardiac Muscle Contraction; Cardiac Myosins; Complex; Condition; Data; Dependence; Dilated Cardiomyopathy; Disease; Dissociation; Electron Microscopy; Electrons; Enzyme Kinetics; Filament; Freezing; Goals; Hydrolysis; Kinetics; Laboratories; Measurement; Measures; Methods; Modeling; Monitor; Myocardium; Myosin ATPase; Negative Staining; Numbers; Phosphorylation; Phosphorylation Site; Physiology; Positioning Attribute; Range; Rate; Regulation; Research Personnel; Shapes; Skeletal Muscle; Skeletal system; Specimen; Structure; Textbooks; Thin Filament; Time; Tropomyosin; Troponin; Troponin C; Work; genetic regulatory protein; image processing; particle; programs; research study; response

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.

描述（由申请人提供）：我们将使用预稳态动力学方法和电子显微镜来确定细丝调节心脏肌动球蛋白ATP水解的机制。大多数生物化学和生理学教科书描述了细丝调节的“立体阻断”机制，其中原肌凝蛋白阻断了低钙状态下肌动蛋白与肌凝蛋白的结合。然而，现在有令人信服的实验数据与这一机制不一致。钙和肌凝蛋白与快速骨骼肌细丝的结合加速了肌凝蛋白- adp - pi的最大解离速率约200倍，但使肌凝蛋白- adp - pi与细丝结合的亲和力最多增加3倍。这表明空间阻断是调控机制的一个非常小的组成部分。骨骼和心脏肌动蛋白、肌凝蛋白、肌钙蛋白和原肌凝蛋白之间的氨基酸序列存在显著差异，钙调节心脏肌动蛋白ATP水解的机制尚不清楚。心脏肌钙蛋白- C只有一个调节钙结合位点，心脏肌钙蛋白- 1有26个氨基酸延伸，包含一个生理上重要的磷酸化位点。除了为细丝调节机制的研究提供关键信息外，所提出的工作还与许多涉及心肌收缩调节控制的疾病直接相关，例如收缩力不足的扩张型心肌病。多次混合停流将用于测量天然心脏细丝对心肌肌球蛋白-ADP-Pi的产物解离加速（Pi和ADP）对钙和严格肌球蛋白与细丝结合以及肌钙蛋白- 1磷酸化的依赖性。使用阴性染色和冷冻电镜方法的平行电子显微镜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）和肌球蛋白结合在细丝上的分布。结构数据将通过单粒子方法进行分析，该方法已被CoPI实验室（John Trinick）应用于细丝结构。合并后的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性调节机制的理解。