Dynamic Interactions of Cardiac Troponin and Tropomyosin

描述(申请人提供)：该研究计划的长期目标是通过对细丝的分子水平的研究，获得对心脏控制的深入和临床实用的见解。这将通过研究关键调节蛋白-心肌肌钙蛋白和原肌球蛋白的动态特性来推进，以揭示它们的作用模式和功能。肌钙蛋白-原肌球蛋白复合体通过近乎绝对的钙依赖于肌球蛋白与细丝的生产性相互作用，非常严格地控制横纹肌收缩。这种调节要求肌钙蛋白和原肌球蛋白的重要性质是柔韧性和硬度。它们必须在以临界模式附着在细丝上时移动，并且还必须以其他方式抵抗移动。因此，肌钙蛋白和原肌球蛋白的详细动力学性质对于心脏收缩的调节以及这些蛋白的功能具有普遍的意义。我们用一种非常强大的方法开发了研究这些动力学的方法：氢/氢交换-超高分辨率质谱学(HDX)。这种方法对理解收缩调节有很大的希望，并且在我们的实验室中是唯一可以用于这项工作的方法。我们将使用HDX，当电子显微镜有价值时进行战略性补充，以确定肌钙蛋白调节开关机制的关键方面。在一种方法中，我们将通过描绘肌钙蛋白残基对开启或关闭肌肉收缩最重要的影响来研究这一机制。此外，我们还将通过研究含有靶向突变的肌钙蛋白来研究肌钙蛋白在远距离发挥作用的功能。最后，我们将使用相同的HDX方法来确定和定位对其功能至关重要的原肌球蛋白的可变动力学。这项工作的总体目标是极大地提高我们对直接控制心脏和其他横纹肌收缩的基本分子机制的理解。 与公共健康相关：这项研究将通过研究负责激活和失活每一次收缩的细胞内蛋白质机制，提供关于一般肌肉收缩控制，特别是心脏收缩控制的分子信息。这一课题对于心力衰竭、冠状动脉功能不全、高血压和骨骼肌紊乱的病因和潜在的治疗方法具有重要意义。

DESCRIPTION (provided by applicant): The long term goal of this research program is to achieve deep and clinically practical insights into the control of the heart, by molecular level investigation of the thin filament. This will be advanced by investigating the dynamic properties of the key regulatory proteins, cardiac troponin and tropomyosin, to reveal their modes of action and functions. The troponin- tropomyosin complex very tightly controls striated muscle contraction by conferring near- absolute calcium-dependence upon myosin's productive interactions with the thin filament. This regulation requires both flexibility and stiffness as important properties of troponin and tropomyosin. They must both move while attached to the thin filament in critical patterns, and also resist movement in other ways. Thus, the detailed dynamic properties of troponin and tropomyosin are particularly important for the regulation of cardiac contraction as well as having general significance for the function of these proteins. We have developed the means to study these dynamics with a very powerful approach: hydrogen/deuterium exchange with ultra high resolution mass spectrometry (HDX). The method holds great promise for understanding contractile regulation, and is uniquely available in our laboratory for this work. We will use HDX, strategically supplemented when valuable by electron microscopy, to ascertain critical aspects of troponin's regulatory switch mechanism. In one approach, we will investigate this mechanism by delineation of the effects of the troponin residues most essential for switching muscle contraction either on or off. Also, we will investigate troponin functions involving action at a distance by studies of troponin containing targeted mutations. Finally, we will determine and map the variable dynamics of tropomyosin that are vital for its function, using the same HDX approach. The overall aim of the work is to advance greatly our understandings of the basic molecular mechanism that directly controls the contraction of the heart and other striated muscles. PUBLIC HEALTH RELEVANCE: This research will provide molecular information on the control of muscle contraction generally, and the control of the heart's contraction in particular, by studying the intra-cellular protein machinery responsible for activating and inactivating each contraction. This subject is of fundamental importance for the causes and potential treatments of heart failure, coronary insufficiency, hypertension, and disorders of skeletal muscles.