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，可驱动基于微管的细胞内 细胞器运输过程。 动力学夫妻伴侣 化学反应（ATP的水解）至机械循环 移动酶分子和附着的细胞器的过程 微管。 我们想表征机械过程的周期 运动蛋白移动并确定如何耦合这些过程 对ATP水解的反应。 我们已经开发了一本小说 实验系统允许我们直接监视机械 单驱蛋白分子中的过程和化学步骤特别 与微观聚苯乙烯珠共轭。 系统使它成为 可以定量比较机械和化学反应速率 在相同的条件下，从而直接研究 机械化学耦合。 驱动蛋白和动力蛋白同源物的细胞内细胞器转运 在真核细胞生理学中的重要作用。 它的功能 包括材料的运输，染色体和核运动 有丝分裂/减数症以及膜细胞器的形态发生。 探索 这些功能在分子水平上，我们将： 1）测量每个ATP水解动力蛋白移动的距离。 我们将 测量珠偶联的单驱动蛋白分子的ATPase Vmax和 将此与在同一偶联物的运动速度进行比较 状况。 这项研究将测试一个ATP的模型的有效性 根据机械步骤被水解。 2）测量ADP诱导的微管结合驱动蛋白的释放速率 头。 这项研究将检验以下假设：头部释放是 运动蛋白运动周期中的重要步骤。 知道动力学 头部释放将帮助我们了解单个双头运动 沿着微管移动时，分子仍然与微管相关。 3）从两个 - 尺寸分子晶体。 结构数据将有助于揭示 分子构象变化，驱动动力素运动和 驱动蛋白头之间相互作用的性质。 4）准备单头运动衍生物并描述其特征 功能性能。 通过表征稳态ATPase， 微管释放动力学和单分子运动，这项研究 将有助于揭示头头互动在两头动力蛋白中的作用 功能。