Protein Exchange to Study Muscle Function and Disease

描述（由申请人提供）：肌钙蛋白T或I亚基的点突变或缺失可能导致家族性肥厚性心肌病和线状性肌病等疾病。我们的数据表明，肌钙蛋白的突变倾向于改变具有不同活性的肌动蛋白-原肌球蛋白-肌钙蛋白状态之间的分布。我们建议通过检查这些肌钙蛋白亚基的几个突变来检验这一假设。我们将通过在溶液和动物肌纤维模型中使用表达的人肌钙蛋白亚基来避免获得人体组织的困难。溶液中的生化研究将确定定义调控反应的关键参数的变化。我们将检验突变倾向于改变肌动蛋白状态分布或这些状态之间转换速率的假设。重点将放在肌凝蛋白S1对稳态ATP水解的协同激活，S1与肌动蛋白-原肌凝蛋白-肌钙蛋白（调节肌动蛋白）的协同平衡结合，以及S1与调节肌动蛋白结合的速率。我们将研究肌钙蛋白T和I亚基的几种突变，并对一组对调节有不同程度影响的突变进行详细测量。如果有证据表明在多个步骤中发生了变化，我们将确认这些变化。肌钙蛋白突变的影响将在骨骼肌和心肌纤维制剂中进行检查。使用我们的交换方案，纤维中的天然肌钙蛋白将被突变肌钙蛋白和/或荧光标记肌钙蛋白取代。我们的交换方法是这个提议的重要组成部分，因为这个过程本身并不影响纤维的机械性能。我们将观察由突变引起的纤维力学性能的变化。通过测量探针在肌钙蛋白上的荧光，我们可以将性能变化与溶液中测量的生化变化联系起来。我们将利用数学模型来测试调节模型，将解决结果与力学研究结果进行比较，并预测可以测试的其他行为。最重要的是，详细的数学分析可能使我们能够预测具体的干预措施，以限制这些突变对患病个体的影响。

DESCRIPTION (provided by applicant): Point mutations or deletions in the T or I subunits of troponin may result in disorders such as Familial Hypertrophic Cardiomyopathy and Nemaline myopathy. Our data indicate that mutations of troponin tend to alter the distribution between states of actin-tropomyosin-troponin having different activities. We propose to test this hypothesis by examining several mutations of these troponin subunits. We will avoid the difficulty of obtaining human tissues by using expressed human troponin subunits in solution and animal muscle fiber models. Biochemical studies in solution will determine changes in the key parameters that define the regulatory response. We will test the hypothesis that mutations tend to alter the distribution of actin states or the rate of transition among these states. Emphasis will be placed on the cooperative activation of steady-state ATP hydrolysis by myosin S1, the cooperative equilibrium binding of S1 to actin-tropomyosin-troponin (regulated actin), and on the rate of S1 binding to regulated actin. We will examine several mutations of the T and I subunits of troponin and make detailed measurements on a set of mutants with varying degrees of effect on regulation. If there is evidence of changes in multiple steps we will confirm those changes. The effect of the troponin mutations will be examined in both skeletal and cardiac muscle fiber preparations. The native troponin in the fibers will be replaced with mutant troponin and/or fluorescently labeled troponin using our exchange protocol. Our method of exchange is an important part of this proposal because the procedure itself does not affect the mechanical properties of the fiber. We will observe changes in the mechanical properties of fibers caused by the mutations. By measuring the fluorescence of a probe on the troponin we can correlate changes in performance with biochemical changes that are measured in solution. We will utilize mathematical modeling to test models of regulation, to compare the solution results with the results from mechanical studies and to predict other behaviors that can be tested. Most importantly, the detailed mathematical analyses may allow us to predict specific interventions to limit the effects of these mutations in afflicted individuals.