Molecular Analysis of Flagellar Dynein Function

鞭毛动力蛋白功能的分子分析

• 批准号: 7393704
• 负责人: Stephen M King
• 金额: $ 27.36万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7393704
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Active Sites; Address; Area; Arginine; Binding; Binding Sites; Biochemical; Biochemistry; Bioinformatics; Biological Models; C-terminal; Calmodulin; Cell division; Cell physiology; Chlamydomonas; Complex; Consensus; Docking; Dynein ATPase; Electron Microscopy; Family; Fingers; Flagella; Frequencies; Glycine; Goals; Head; Homologous Gene; Hydrolysis; In Vitro; Investigation; Isoenzymes; Length; Light; Mechanics; Methods; Microtubules; Modeling; Molecular; Molecular Analysis; Molecular Genetics; Molecular Motors; Motor; Motor Activity; Movement; Mutagenesis; Mutation; N-terminal; Nuclear Envelope; Nucleotides; Oxidation-Reduction; Pathway interactions; Phenotype; Physiological; Play; Power stroke; Proteins; Regulation; Role; Rotation; Series; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Structural Models; Structure; Testing; Thioredoxin; Upper arm; Variant; Vesicle; base; design; in vivo; insight; leucine-rich repeat protein; member; mutant; oxidation; particle; response; rho; rho GTP-Binding Proteins; sensor; stem; trafficking

DESCRIPTION (provided by applicant): Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicle trafficking, nuclear envelope breakdown, ciliary/flagellar motility and cell division. The 1.9 MDa outer dynein arm from flagella of Chlamydomonas offers an excellent model system in which to study dynein structure, function and regulation as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetics and can be purified in large amounts for biochemical analysis. Dynein is a member of the AAA+ family of ATPases, however, the mechanisms by which ATP hydrolysis is converted to mechanical movement and how that motor activity is regulated at the molecular level remain almost completely unknown. This application proposes four specific areas of investigation. 1) We will use site-directed mutagenesis and in vitro biochemistry to address which domains bind and/or hydrolyze nucleotide. We will also test whether inter-domain interactions regulate ATP/ADP binding and whether the microtubule-binding stalk plays an active role in transmitting ATP-driven conformational change. 2) We will use electron microscopy and mutagenesis to test our model for subdomain assignments within the dynein motor unit. Further, we will insert a glycine linker between AAA1 and the N-terminal domain to test whether the power stroke occurs between these two segments as has been recently proposed. 3) We will examine whether thioredoxin light chains and the redox-sensitive docking complex protein (DCS) are involved in regulating dynein motor function in response to alterations in cellular redox poise. This will involve analysis of mutant strains expressing altered versions of DC3 and mutagenesis (Cys to Ser) of the thioredoxin redox-active sites combined with both in vitro and in vivo analysis of motor function. 4) We will use site-directed mutagenesis to test the hypothesis (based on our NMR structural studies) that the C-terminal domain of the LC1 protein controls gamma heavy chain ATPase in a manner similar to that observed with the GAPs that activate Ras/Rho GTPases. Finally, we will also examine whether the y heavy chain-associated Ca2+-binding LC4 protein acts as the Ca2+ sensor responsible for modulating ATP-dependent dyneinmicrotubule interactions. This project will provide detailed information on the fundamental motor mechanism of dynein and enable us to define the molecular pathways by which motor function is controlled.

描述（由申请人提供）：动力蛋白是基于微管的分子电机，涉及各种必需的细胞功能，包括逆行囊泡运输，核包膜崩溃，纤毛/鞭毛运动和细胞分裂。 来自衣原体鞭毛的1.9 MDA外动力蛋白臂提供了一个出色的模型系统，可以研究与细胞质同工酶中与细胞质同工酶密切相关的组件，可研究动力蛋白的结构，功能和调节，可与经典/分子遗传学相融合，并且可以在大量的生物化学分析中净化。 Dynein是AAA+ ATPases家族的成员，但是，ATP水解转化为机械运动的机制以及在分子水平上如何调节运动活性几乎完全未知。该申请提出了四个特定的调查领域。 1）我们将使用位置定向的诱变和体外生物化学来解决哪些结构和/或水解核苷酸的结合。我们还将测试域间相互作用是否调节ATP/ADP结合以及微管结合茎是否在传输ATP驱动构象变化中起积极作用。 2）我们将使用电子显微镜和诱变来测试我们的模型在动力蛋白运动单元中的子域分配。此外，我们将在AAA1和N末端结构域之间插入一个甘氨酸接头，以测试像最近提出的这两个片段之间是否发生功率中风。 3）我们将检查硫氧还蛋白的光链和对氧化还原敏感的对接复合物蛋白（DC）是否参与调节动力蛋白运动功能，以应对细胞氧化还原的改变。这将涉及对表达变化版本的DC3和诱变（CYS至SER）的突变菌株的分析，并结合体外和体内运动功能分析。 4）我们将使用位置定向的诱变来检验假设（基于我们的NMR结构研究），即LC1蛋白的C末端结构域以类似于激活RAS/Rho GTPases的间隙的方式以类似的方式控制γ重链ATPase。最后，我们还将研究Y重链相关的Ca2+结合LC4蛋白是否充当CA2+传感器，负责调节ATP依赖性的Dyneinmicroutule相互作用。该项目将提供有关动力蛋白基本运动机制的详细信息，并使我们能够定义控制运动功能的分子途径。