Molecular Mechanics of Mutant Cardiac Myosin

描述（由申请人提供）：家族性肥厚性心肌病（FHC）引起的心脏肥大、肌纤维紊乱和猝死是由肌体蛋白常染色体显性突变引起的。肌凝蛋白是一种由两条重链组成的六聚体蛋白，是一种与肌动蛋白相互作用以促进心肌收缩的肌分子马达。每条重链结合两条轻链，一条基本链（ELC）和一条调节链（RLC）。轻链结合（颈/杠杆）结构域放大源自肌球蛋白活性位点的ATP依赖构象变化，以产生力和运动。考虑到肌球蛋白轻链结合（颈/杠杆）结构域在力量产生中的重要性，在RLC中发现几个FHC突变并不奇怪。本提案的目的是为RLC突变患者的FHC提供分子基础。由于肌凝蛋白分子生物化学与产生构象变化的力有关，因此预计肌凝蛋白生物化学循环中的几个步骤依赖于菌株。因此，我们将研究外力通过肌凝蛋白颈区传递到肌凝蛋白活性部位的过程。由于临床表现取决于特定的突变，这些研究是制定治疗方案的必要前提。我们将测试肌凝蛋白RLC突变降低肌凝蛋白颈域作为应变传感器的能力的假设，这改变了向活性部位的力传递，导致应变依赖动力学和功率输出的改变。研究中选择的突变位于RLC分子（A13T, N47K， R58Q和D166V）的可磷酸化丝氨酸和af -hand附近。这些区域在历史上被证明对肌凝蛋白的功能很重要；因此，我们的实验不仅将为FHC提供分子基础，而且将解决RLC功能的基本方面和肌球蛋白运动产生的分子基础。我们的方法将利用体外运动分析来评估RLC突变对功率输出的影响（目的1），以及在集合（多分子）水平上的菌株依赖肌球蛋白动力学（目的2）。将在单个肌凝蛋白分子水平上进一步研究整体菌株依赖性的任何改变，以确定受突变影响的特定潜在菌株依赖性肌动球蛋白动力学转变（目的3）。此外，与我们的初步数据一致，RLC磷酸化已被提出通过促进收缩性能和效率的提高来抑制肥大。因此，我们将确定RLC的磷酸化是否能挽救RLC- fhc表型（Aim 4）。我们的方法测量了分离的可收缩蛋白的机械特性。因此，我们将确定FHC突变对肌动球蛋白的直接影响。了解RLC突变如何影响肌球蛋白的固有功能，将允许更高功能单位的改变程度，如心肌纤维或心脏本身与原发性收缩缺陷相关。

DESCRIPTION (provided by applicant): The cardiac hypertrophy, myofibrillar disarray and sudden death caused by familial hypertrophic cardiomyopathy (FHC) results from autosomal dominant mutations in sarcomeric proteins. Myosin, the sarcomeric molecular motor that interacts with actin to power cardiac muscle contraction, is a hexameric protein consisting of two heavy chains. Each heavy chain binds two light chains, one essential (ELC) and one regulatory (RLC). The light chain binding (neck/lever) domain amplifies ATP dependent conformational changes originating in the myosin active site to generate force and motion. Given the importance of the light chain binding (neck/lever) domain of myosin in force production, it is not surprising that several FHC mutations have been identified in the RLC. The goal of this proposal is to provide a molecular basis for FHC in patients with mutations in the RLC. Since myosin molecule biochemistry is linked to force producing conformational changes, it is expected that several steps in the myosin biochemical cycle are strain dependent. Therefore, we will study the transmission of external forces to the myosin active site via the myosin neck region, . Since the clinical presentation depends on the specific mutation, these studies are a necessary precursor to development of therapeutic protocols. We will test the hypothesis that mutations in the myosin RLC decrease the ability of the myosin neck domain to act as a strain sensor, which alters the delivery of force to the active site, leading to altered strain dependent kinetics and power output. The mutations chosen for study are localized near the phosphorylatable serine and the EF-hand of the RLC molecule (A13T, N47K, R58Q and D166V). These regions have historically been shown to be important for myosin function; thus our experiments will not only provide a molecular basis for FHC but will also address fundamental aspects of RLC function and the molecular basis of myosin motion generation. Our approach will utilize in vitro motility assays to assess the effects of RLC mutations on power output (Aim 1) as well as strain dependent myosin kinetics at the ensemble (multiple molecule) level (Aim 2). Any alterations in ensemble strain dependence will be further pursued at the single myosin molecule level to determine the specific underlying strain dependent actomyosin kinetic transitions affected by the mutations (Aim 3). Furthermore, consistent with our preliminary data, RLC phosphorylation has been proposed to inhibit hypertrophy by contributing to enhanced contractile performance and efficiency. Therefore, we will determine if phosphorylation of the RLC rescues the RLC-FHC phenotypes (Aim 4). Our approach measures the mechanical properties of isolated contractile proteins. Therefore, we will determine the direct effects of the FHC mutations on actomyosin. Knowledge of how the RLC mutations affect myosin's inherent function will allow the degree of alteration of higher functional units, such as the cardiac muscle fiber, or the heart itself to be correlated with a primary contractile defect. PUBLIC HEALTH RELEVANCE: Familial hypertrophic cardiomyopathy (FHC) is a genetic heart disease that is caused by mutations in the molecular machinery that allows the heart to contract. This study will examine how FHC mutations in one of the proteins of the heart (the myosin regulatory light chain), alters the ability of the heart to generate force and power at the molecular level.