Molecular mechanics of mutant cardiac myosin

摘要 家族性肥厚所致心肌肥大、肌原纤维紊乱及猝死 心肌病(FHC)是由肌节蛋白的常染色体显性突变引起的。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，是一个六角体。 由两条重链组成的蛋白质。每个重链结合两个轻链，一个是必需的(ELC)，另一个是必需的 监管(RLC)。轻链结合(颈部/杠杆)结构域放大依赖于ATP的构象 起源于肌球蛋白活性部位的变化产生力量和运动。考虑到光的重要性 在肌球蛋白的链结合(颈部/杠杆)结构域的产生中，难怪有几个FHC突变 已在RLC中确定。这项建议的目标是为患者的FHC提供分子基础 与RLC的突变有关。由于肌球蛋白分子生物化学与产生构象的力有关 变化，预计肌球蛋白生化循环的几个步骤是依赖于菌株的。因此，我们 将研究外力通过肌球蛋白颈部区域向肌球蛋白活性部位的传递。自.以来 临床表现取决于特定的突变，这些研究是必要的先兆 制定治疗方案。 我们将验证肌球蛋白RLC突变降低肌球蛋白颈部能力的假设 结构域充当应变传感器，它改变力到活动部位的传递，导致改变应变 依赖于动力学和功率输出。选择用于研究的突变位于可磷酸化的附近 丝氨酸和RLC分子的EF-Hand(A13T、N47K、R58Q和D166V)。在历史上，这些地区 已被证明对肌球蛋白功能很重要；因此，我们的实验不仅提供了一个分子基础 ，但也将阐述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动力测试来评估RLC突变对功率的影响 产量(目标1)以及在整体(多分子)水平上依赖应变的肌球蛋白动力学(目标2)。 将在单个肌球蛋白分子水平上进一步研究整体应变依赖性的任何变化，以 确定受突变影响的特定潜在菌株依赖的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的磷酸化已被提出以抑制 肥大有助于增强收缩性能和效率。因此，我们将确定是否 RLC的磷酸化挽救了RLC-FHC表型(目标4)。我们的方法测量的是机械 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 肌动球蛋白。了解RLC突变如何影响肌球蛋白的固有功能将使 较高功能单位的改变，如心肌纤维，或心脏本身与 原发性收缩缺陷症。

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.