MOLECULAR ANALYSIS OF FHC MISSENSE MUTATIONS

肌球蛋白如何移动肌动蛋白并产生力的结构模型 马达结构域和轻链（LC）之间的界面 结合颈区域是两个域弯曲的枢轴点 相对于彼此。许多点突变与家族性 肥大性心肌病（FHC）聚集在β-肌球蛋白重 在必需的LC（ELC）结合界面上以及LC中， 自己在FHC中发现的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 第一目标。重链突变将被工程化到平滑肌中 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都至关重要。并发 目的是增加表达的心脏HMM的产量， 可以对表达的心脏突变体进行实验。心肌肌球蛋白 这些FHC突变的一个子集也将从转基因动物中分离出来。 小鼠（获自Core C），并类似地分析。这样的比较 研究应该有助于理解基本的机械性能 所有肌球蛋白，以及提供一个分子基础的影响， 选择FHC突变。第二个目标是关注FHC突变如何在 心脏调节和必需LC影响心肌肌球蛋白的酶促和 力学性能由于心房ELC在心室中积聚， 不同形式的人类心室肥大，动力学和 β-心肌肌球蛋白/心房ELC嵌合体的机械特性将 也要坚决。在这两种情况下，细菌表达的心脏LC将 用LC缺陷型β-心肌肌球蛋白重链重建 从组织中制备，并通过 第一个目标描述的技术。提案的总体目标 是阐明FHC中涉及的突变如何影响机械的 肌球蛋白的性能。

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.