Molecular mechanics of mutant cardiac myosin

摘要 家族性肥厚性心肌病致心肌肥厚、肌原纤维紊乱与猝死 心肌病（FHC）由肌节蛋白的常染色体显性突变引起。肌球蛋白 肌节分子马达与肌动蛋白相互作用，为心肌收缩提供动力，是一种六聚体 由两条重链组成的蛋白质。每条重链结合两条轻链，一条是必需的（ELC），一条是必需的（ELC）。 监管（RLC）。轻链结合（颈/杠杆）结构域放大ATP依赖性构象 源自肌球蛋白活性部位的变化，以产生力和运动。鉴于光线的重要性 肌球蛋白的链结合（颈/杠杆）结构域在部队的生产，这是不足为奇的，几个FHC突变 在RLC中被发现。本提案的目的是为FHC患者提供分子基础 RLC中的突变。由于肌球蛋白分子生物化学与力产生构象有关， 变化，预期肌球蛋白生化循环中的几个步骤是菌株依赖性的。所以我们 将研究外力通过肌球蛋白颈部区域传递到肌球蛋白活性部位。以来 临床表现取决于特定的突变，这些研究是必要的前兆， 制定治疗方案。 我们将检验肌球蛋白RLC突变降低肌球蛋白颈的能力的假设， 结构域作为应变传感器，其改变力到活性位点的传递，导致应变改变 依赖动力学和动力输出。选择用于研究的突变位于可磷酸化的 丝氨酸和RLC分子的EF-手（A13 T、N47 K、R58 Q和D166 V）。这些地区在历史上 已被证明是重要的肌球蛋白的功能，因此，我们的实验将不仅提供了分子基础 但也将解决RLC功能的基本方面和肌球蛋白运动的分子基础 一代我们的方法将利用体外运动试验来评估RLC突变对功率的影响。 输出（目标1）以及应变依赖性肌球蛋白动力学在合奏（多分子）水平（目标2）。 任何整体应变依赖性的改变将在单个肌球蛋白分子水平上进一步研究， 确定受突变影响的特定潜在应变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，已提出RLC磷酸化抑制 通过促进增强的收缩性能和效率来抑制肥大。因此，我们将确定， RLC的磷酸化挽救了RLC-FHC表型（目的4）。我们的方法测量机械 分离的收缩蛋白的性质。因此，我们将确定FHC突变的直接影响， 关于肌动球蛋白了解RLC突变如何影响肌球蛋白的固有功能，将有助于了解 更高功能单位的改变，如心肌纤维，或心脏本身与心脏病相关。 原发性收缩缺陷

Abstract 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.