Functional diversity of myosin VII

肌球蛋白 VII 的功能多样性

• 批准号: 7197443
• 负责人: ENRIQUE M DE LA CRUZ
• 金额: $ 29.69万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7197443
• 关键词: ADP-G-actin; ATP phosphohydrolase; Actins; Actomyosin; Adhesions; Affect; Affinity; Amino Acids; Anisotropy; Behavior; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Brush Border; Cells; Chemicals; Chimera organism; Chimeric Proteins; Class; Coiled-Coil Domain; Data; Dependence; Dimerization; Disease; Disruption; Engineering; Equilibrium; Filament; Functional disorder; Generations; Head; Human; In Vitro; Investigation; Kidney; Kinetics; Labyrinth; Length; Link; MYO7A gene; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Methods; Microfilaments; Molecular; Molecular Conformation; Motor; Motor Activity; Muscle Rigidity; Mutation; Myosin ATPase; Myosin Type V; Nucleotides; Organism; Phenotype; Play; Point Mutation; Population; Property; Protein Isoforms; Proteins; Range; Rate; Reaction; Research Personnel; Resolution; Retina; Role; Side; Slide; Structure; Techniques; Testing; Usher Syndrome; Variant; analytical ultracentrifugation; base; cell motility; cellular microvillus; clinically relevant; deafness; density; design; dimer; gastrointestinal microvillus; hearing impairment; human disease; insight; intestinal epithelium; member; monomer; mutant; myosin VI; particle; phosphorescence; physical property; research study; sedimentation equilibrium; time use; trafficking

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.

描述（申请人提供）：人类有两种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，只是肌球蛋白VIIb在肌动蛋白结合位点具有独特的19个氨基酸插入片段，并且缺乏推定的卷曲螺旋结构域。肌球蛋白VIIa的突变与1b型Usher综合征（人类最常见的致盲性疾病）以及人类的另外两种听力损失疾病DFNB 2和DFNA 11有关。肌球蛋白VIIb被认为是运输和组织膜和膜相关蛋白。尽管VII类肌球蛋白在细胞中发挥着重要作用，但由于缺乏关于肌球蛋白VII化学和物理性质的信息，对肌球蛋白VII功能基础和与肌球蛋白VIIa相关的耳聋表型的理解受到限制。 在这个建议的努力集中在了解肌球蛋白VIIa和VIIb的酶和电机性能。目的1将确定全长肌球蛋白VIIa和VIIb是单体还是二聚体。目标2将提供肌球蛋白VIIa和VIIb ATP酶循环的完整动力学特征，通过突变体和嵌合蛋白的产生确定19个氨基酸插入对ATP酶循环动力学的贡献，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肌球蛋白的结合，并评估应变依赖性头-头协调的机制。目的4将采用高分辨率光谱方法测量高占空比肌球蛋白结合引起的肌动蛋白丝结构动力学的协同变化。 这两个高度保守的亚型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。拟议的反应机制和结构动力学的详细分析将有助于开发可检验的假设，关于肌球蛋白VII的功能和机制，并说明如何在肌球蛋白马达的序列和结构变化定义其酶的特性，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相关性，因此拟议的实验将促进我们对几种人类疾病分子基础的理解。