Dynamic Interactions of Cardiac Troponin and Tropomyosin

描述（由申请人提供）：本研究计划的长期目标是通过对细丝的分子水平研究，实现对心脏控制的深入和临床实用见解。这将通过调查的关键调控蛋白，心肌肌钙蛋白和原肌球蛋白的动态特性，揭示其作用和功能的模式。肌钙蛋白-原肌球蛋白复合物通过赋予肌球蛋白与细丝的生产性相互作用以接近绝对的钙依赖性来非常紧密地控制横纹肌收缩。这种调节需要作为肌钙蛋白和原肌球蛋白的重要特性的柔韧性和刚度。它们必须在以临界模式附着在细丝上时移动，并且还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对这些蛋白质的功能具有普遍意义是特别重要的。我们已经开发出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）。该方法为理解收缩调节提供了很大的希望，并且在我们的实验室中唯一可用于这项工作。我们将使用HDX，战略性地补充时，有价值的电子显微镜，以确定肌钙蛋白的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的作用来研究这种机制，肌钙蛋白残基对于打开或关闭肌肉收缩是最重要的。此外，我们还将通过对含有靶向突变的肌钙蛋白的研究来研究肌钙蛋白的功能，包括远距离作用。最后，我们将使用相同的HDX方法确定并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.