Mechanistic Analysis of Microtubule-Based Motors

基于微管的电机的机械分析

• 批准号: 6472521
• 负责人: SUSAN P. GILBERT
• 金额: $ 10.19万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6472521
• 关键词: SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; biomechanics; centrifugation; chromosome movement; computer simulation; enzyme activity; intracellular transport; kinesin; microtubules; mitotic spindle apparatus; physical model; protein purification; sedimentation equilibrium; stop flow technique; thermodynamics

DESCRIPTION: The long term goal is to establish the structural and mechanistic basis for force production by kinesin superfamily members. The proposed studies reflect a longstanding interest in intracellular transport and a commitment to biomedical research. The specific aims of this research proposal are to establish the kinetic and thermodynamic basis of force generation of the EgS and Kar3 ATPases in direct comparison to Ncd and conventional kinesin. All use ATP to drive unidirectional microtubule based movements. Ncd, Eg5, and Kar3 are involved in spindle dynamics during meiosis and/or mitosis and therefore are required for proper chromosome segregation. In contrast, kinesin is a neuronal motor that promotes movement of membranous organelles. Kinesins motility is distinctive because of its processivity. Ncd, Kar3 and Eg5 are believed not to be processive. Both Ncd and Kar3 promote minus end directed microtubule movements, yet kinesin and Eg5 promote plus end directed movements. Furthermore, Kar3 as a monomer exhibits unidirectional movement; therefore, Kar3 is an interesting motor to study in direct comparison to the climeric kinesins kinesin, Eg5, Ncd. The results with Ncd and kinesin indicate that both motor domains of the dimer are required for movement. Eg5 is also dimeric, yet evidence to date indicates it is not processive. The studies with EgS, in direct comparison to Ncd and kinesin, are intended to define the mechanistic features required specifically for processivity that may be distinct from those features that drive plus end directed movements. In addition, the experiments with mutant kinesin motors will explore aspects of the ATPase crossbridge cycle that are not accessible by studying the wildtype motor. The proposed experiments will evaluate the mechanistic features that spindle motors have in common, and at the same time address specific questions about energy transduction for dimeric motors in comparison to monomeric motors. A comprehensive analysis of these 4 molecular motors will provide information to begin to understand the structural and mechanistic requirements for the diverse movements occurring during the cell cycle and during neuromuscular development and function where genetic alteration can result in birth defects, degenerative diseases, and cancer.

描述：长期目标是建立结构和机制 驱动蛋白超家族成员产生力的基础。拟议的研究 反映了对细胞内转运的长期兴趣，并致力于 生物医学研究本研究建议的具体目标是 建立了EgS力生成的动力学和热力学基础 和Kar 3 ATP酶与Ncd和常规驱动蛋白的直接比较。都使用 ATP驱动单向微管运动。Ncd、Eg 5和Kar 3是 参与减数分裂和/或有丝分裂期间的纺锤体动力学，因此 这是染色体分离所必需的相比之下，驱动蛋白是一种神经元蛋白， 促进膜状细胞器运动的马达。驱动蛋白运动性是 因为它的持续性而与众不同。Ncd、Kar 3和Eg 5被认为不 要循序渐进。Ncd和Kar 3均促进负末端定向微管 运动，而驱动蛋白和Eg 5促进加结束定向运动。 此外，Kar 3作为单体表现出单向运动;因此， Kar 3是一个有趣的马达，可以直接与climeric 驱动蛋白，Eg 5，Ncd. Ncd和驱动蛋白的结果表明， 二聚体的运动域是运动所必需的。Eg 5也是二聚体，但 迄今为止的证据表明，它不是进行性的。研究EgS，在 与Ncd和驱动蛋白的直接比较，旨在定义 持续合成能力所需的特定特征， 驱动器加上端部定向运动的功能。此外，实验 与突变驱动蛋白马达将探讨方面的ATP酶过桥循环 是无法通过研究野生型马达获得的。拟议 实验将评估主轴电动机的机械特性， 共同的，同时解决有关能源的具体问题 与单体马达相比，二聚体马达的转导。一 对这4种分子马达的综合分析将为 开始了解结构和机械要求的多样性 在细胞周期和神经肌肉发育过程中发生的运动 基因改变可能导致出生缺陷，退行性疾病， 疾病和癌症。