BIOPHYSICS AND ENZYMOLOGY OF KINESIN MOVEMENT

驱动蛋白运动的生物物理学和酶学

• 批准号: 2181983
• 负责人: JEFF GELLES
• 金额: $ 24.37万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181983
• 关键词: biomechanics; chemical conjugate; conformation; enzyme activity; enzyme structure; intracellular transport; kinesin; microtubules; protein structure function; protoplasm motility

Kinesin is a mechanoenzyme that drives microtubule-based intracellular organelles transport processes. Kinesin couples a free-energy-liberating chemical reaction (the hydrolysis of ATP) to a cycle of mechanical processes that move the enzyme molecules and attached organelles along microtubules. We want to characterize the cycle of mechanical processes by which kinesin moves and to determine how these processes are coupled to the reactions of ATP hydrolysis. We have developed a novel experimental system that allows us to directly monitor mechanical processes and chemical steps in single kinesin molecules specifically conjugated to microscopic polystyrene beads. The system makes it possible to quantitatively compare mechanical and chemical reaction rates under identical conditions, thereby allowing direct studies of mechanochemical coupling. Intracellular organelle transport by kinesin and kinesin homologs plays an essential role in the physiology of eukaryotic cells. Its functions include transport of materials, chromosome and nuclear movements in mitosis/meiosis, and morphogenesis of membranous organelles. To explore these functions at the molecular level, we will: 1) measure the distance moved by kinesin per ATP hydrolyzed. We will measure the ATPase Vmax for bead-conjugated single kinesin molecules and compare this to the movement velocity of the conjugates under the same conditions. This study will test the validity of models in which one ATP is hydrolyzed per mechanical step. 2) measure the rate of ADP-induced release of microtubule-bound kinesin heads. This study will test the hypothesis that head release is an essential step in the kinesin movement cycle. Knowing the kinetics of head release will help us understand how single two-headed kinesin molecules remain associated with the microtubule while moving along it. 3) derive the structure of a two-headed kinesin derivative from two- dimensional molecular crystals. Structural data will help reveal the molecular conformational changes that drive kinesin movement and the nature of interactions between kinesin heads. 4) prepare one-headed kinesin derivatives and characterize their functional properties. By characterizing the steady-state ATPase, microtubule release kinetics, and single-molecule motility, this study will help reveal the role of head-head interactions in two-headed kinesin function.

驱动蛋白是一种机械酶，其驱动基于微管的细胞内 细胞器运输过程。 驱动蛋白结合了一种释放自由能的 化学反应（ATP的水解）到机械循环 使酶分子和附着的细胞器沿着运动的过程 微管 我们想描述机械过程的循环 并确定这些过程是如何耦合的 ATP水解的反应。 我们已经开发出一种新颖 实验系统，使我们能够直接监测机械 单个驱动蛋白分子中的特定过程和化学步骤 与微观聚苯乙烯珠结合。 系统让它 可以定量比较机械和化学反应速率 在相同的条件下，从而允许直接研究 机械化学耦合 驱动蛋白及其同源物参与的细胞内细胞器转运 在真核细胞的生理学中起着重要作用。 其职能 包括物质运输、染色体和细胞核运动， 有丝分裂/减数分裂和膜细胞器的形态发生。 探讨 这些功能在分子水平上，我们将： 1)测量每水解一次ATP驱动蛋白移动的距离。 我们将 测量珠缀合的单个驱动蛋白分子的ATP酶Vmax， 将其与相同条件下共轭物的运动速度进行比较， 条件 本研究将测试模型的有效性，其中一个ATP 在每个机械步骤中水解。 2)测量ADP诱导的微管结合驱动蛋白的释放速率 构造标准部件. 这项研究将测试的假设，头部释放是一个 驱动蛋白运动周期中的重要步骤。 了解了 头部释放将帮助我们理解单个双头驱动蛋白 分子在沿微管沿着移动时保持与微管结合。 3)从两个- 三维分子晶体 结构数据将有助于揭示 驱动蛋白运动的分子构象变化， 驱动蛋白头部之间相互作用的性质。 4)制备单头驱动蛋白衍生物并表征其 功能特性 通过表征稳态ATP酶， 微管释放动力学和单分子运动，这项研究 将有助于揭示头-头相互作用在双头驱动蛋白中的作用 功能