Dynamic Interactions of Cardiac Troponin and Tropomyosin

描述（由申请人提供）：本研究项目的长期目标是通过细丝的分子水平研究，对心脏的控制获得深入的临床实践见解。这将通过研究关键调节蛋白，心脏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-原肌凝蛋白复合物通过赋予肌凝蛋白与细丝的生产相互作用近乎绝对的钙依赖性，非常严格地控制横纹肌收缩。这种调节需要肌钙蛋白和原肌凝蛋白的柔韧性和刚度这两个重要特性。当它们以临界模式附着在细丝上时，它们必须既移动，又以其他方式抵抗运动。因此，肌钙蛋白和原肌球蛋白的详细动态特性对心脏收缩的调节尤为重要，对这些蛋白的功能也具有普遍意义。我们已经开发了一种非常强大的方法来研究这些动力学：氢/氘交换与超高分辨率质谱（HDX）。该方法对理解收缩调节具有很大的希望，并且在我们的实验室中对这项工作是唯一可用的。我们将使用HDX，在有价值时通过电子显微镜进行战略性补充，以确定肌钙蛋白调节开关机制的关键方面。在一种方法中，我们将通过描述肌钙蛋白残基对打开或关闭肌肉收缩最重要的影响来研究这种机制。此外，我们将通过研究含有靶向突变的肌钙蛋白来研究肌钙蛋白涉及远距离作用的功能。最后，我们将使用相同的HDX方法确定和绘制原肌球蛋白的可变动力学，这对其功能至关重要。这项工作的总体目标是大大推进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。

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.