Functional diversity of myosin VII

描述(申请人提供)：人类有两个VII类肌球蛋白，肌球蛋白VIIa和VIIb，它们在结构域结构上非常相似，不同的是肌球蛋白VIIb在肌动蛋白结合部位有一个独特的19个氨基酸插入，并且缺乏一个假定的卷曲结构域。肌球蛋白VIIa的突变与1b型Usher综合征有关，这是人类最常见的聋盲疾病，以及人类的另外两种听力损失疾病，DFNB2和DFNA11。肌球蛋白VIIB被认为运输和组织膜和膜相关蛋白。尽管第VII类肌球蛋白在细胞中扮演着重要的角色，但由于缺乏有关肌球蛋白VII的化学和物理性质的信息，对肌球蛋白VII功能的基础和与肌球蛋白VIIa相关的耳聋表型的了解一直受到限制。 本提案中的工作重点是了解肌球蛋白VIIa和VIIb的酶和运动特性。目标1将确定全长肌球蛋白VIIa和VIIb是单体还是二聚体。目的2将提供肌球蛋白VIIa和VIIb ATPase循环的完整动力学特征，通过产生突变体和嵌合蛋白来确定19个氨基酸插入对ATPase循环动力学的贡献，并确定导致耳聋的突变如何影响肌球蛋白VIIa运动活性。目的3将测量外部负荷如何调节ADP和肌动蛋白与高占空比的肌球蛋白结合，并评估依赖应变的头-头协调的机制。目标4将使用高分辨率光谱方法来测量高占空比肌球蛋白结合引起的肌动蛋白细丝结构动力学的协同变化。 这两种高度保守的异构体提供了一个独特的机会来测试肌动蛋白结合环和二聚化对肌球蛋白功能的结构和功能影响。对反应机制和结构动力学的详细分析将有助于开发关于肌球蛋白VII的功能和机制的可检验的假说，并阐明肌球蛋白马达的序列和结构变化如何定义其酶特性，最终决定其生物学功能。由于肌球蛋白VIIa功能的突变和破坏具有临床意义，拟议的实验将促进我们对几种人类疾病的分子基础的理解。

DESCRIPTION (provided by applicant): Humans have two class VII myosins, myosin VIIa and VIIb, which are very similar in domain structure except that myosin VIIb possesses a unique 19 amino acid insert at the actin-binding site and lacks a putative coiled-coil domain. Mutations in myosin VIIa have been linked to type 1b Usher syndrome, the most common deafness-blindness disorder in humans, and two other hearing loss disorders in humans, DFNB2 and DFNA11. Myosin VIIb is believed to traffic and organize membranes and membrane-associated proteins. Despite the critical roles class VII myosins play in cells, understanding the basis of myosin VII function and the deafness phenotype associated with myosin VIIa has been limited by a lack of information regarding the chemical and physical properties of myosin VII. Efforts in this proposal focus on understanding the enzymatic and motor properties of myosin VIIa and VIIb. Aim 1 will determine if full-length myosin VIIa and VIIb are monomers or dimers. Aim 2 will provide a complete kinetic characterization of the myosin VIIa and VIIb ATPase cycles, determine the contributions of the 19 amino acid insertion to the ATPase cycle kinetics through the generation of mutants and chimeric proteins, and identify how mutations that generate deafness affect myosin VIIa motor activity. Aim 3 will measure how external loads modulate ADP and actin binding to high duty ratio myosins and evaluate mechanisms of strain-dependent head-head coordination. Aim 4 will employ high-resolution spectroscopic methods to measure cooperative changes in the structural dynamics of actin filaments induced by high duty ratio myosin binding. The two highly conserved isoforms provide a unique opportunity to test the structural and functional effects of the actin-binding loop and of dimerization on myosin function. The proposed detailed analysis of reaction mechanisms and structural dynamics will help develop testable hypotheses regarding the function and mechanism of myosin VII, and illustrate how sequence and structural variations in myosin motors define their enzymatic properties, which ultimately dictates their biological functions. Because mutations and disruption of myosin VIIa function are of clinical relevance, the proposed experiments will advance our understanding of the molecular basis of several human diseases.