MOLECULAR ANALYSIS OF FHC MISSENSE MUTATIONS

肌球蛋白如何移动肌动蛋白并产生作用力假说的结构模型 马达结构域和轻链(LC)之间的界面 结合颈区是两个结构域弯曲的支点 相互之间的关系。许多点突变与家族性 肥厚性心肌病(FHC)聚集在β-肌球蛋白重度 基本LC(ELC)结合界面的链，以及LCS中的链 他们自己。FHC重链突变对肌球蛋白的影响 酶和机械性能(稳态和瞬时动力学， 速度、单位力和单位步长)将在 第一个目标。重链突变将被改造成平滑的肌肉 肌球蛋白并在杆状病毒/昆虫细胞系统中表达，以确定 哪些突变对所有肌球蛋白的功能至关重要。一个并发的 目标是提高表达的心脏HMM的产量，以便具有可比性 可以在表达的心脏突变体上进行实验。心肌肌球蛋白 有了这些FHC突变的子集，也将从转基因 小鼠(从Core C获得)，并进行类似的分析。这种可比性 研究应该有助于理解基本的力学性能 并为肌球蛋白的作用提供了分子基础 精选的FHC突变。第二个目标集中在FHC突变是如何在 心脏调节和必要的LC影响心肌肌球蛋白的酶和 机械性能。由于心房ELC积聚在 不同形式的人类心室肥厚，运动性和 β-心肌肌球蛋白/心房ELC嵌合体的力学性质 也要有决心。在这两种情况下，细菌表达的心脏LCS将 与缺乏LC的β-心肌肌球蛋白重链重组 从组织中制备，并通过 为第一个目的而描述的技术。该提案的总体目标是 是为了阐明FHC中涉及的突变如何影响机械性 肌球蛋白的表现。

Structural models of how myosin moves actin and produces force hypothesize that the interface between the motor domains and the light-chain (LC) binding neck region is a pivot point for bending of the two domains relative to each other. Many point mutations implicated in familial hypertrophic cardiomyopathies (FHC) are clustered in the beta-myosin heavy chain at the essential LC (ELC) binding interface, as well as in the LCs themselves. The effect of heavy chain mutations found in FHC on myosin's enzymatic and mechanical properties (steady-state and transient kinetics, velocity, unitary force, and unitary step size) will be analyzed in the first aim. Heavy chain mutations will be engineered into smooth muscle myosin and expressed in the baculovirus/insect cell system, to determine which mutations are critical for the function of all myosins. A concurrent goal is to increase the yield of expressed cardiac HMM so that comparable experiments can be performed on expressed cardiac mutants. Cardiac myosin with a subset of these FHC mutations will also be isolated from transgenic mice (obtained from Core C), and analyzed similarly. Such comparative studies should contribute to understanding the basic mechanical properties of all myosins, as well as providing a molecular basis for the effect of selected FHC mutations. The second aim focuses on how FHC mutations in the cardiac regulatory and essential LCs affect cardiac myosin's enzymatic and mechanical properties. Since atrial ELC accumulates in the ventricle in different forms of human ventricular hypertrophies, the kinetic and mechanical properties of a beta-cardiac myosin/atrial ELC chimera will also be determined. In both cases, bacterially expressed cardiac LCs will be reconstituted with LC-deficient beta-cardiac myosin heavy chain prepared from tissue, and assayed for functional properties by the techniques described for the first aim. The overall goal of the proposal is to elucidate how mutations implicated in FHC affect the mechanical performance of myosin.