BIOPHYSICS AND ENZYMOLOGY OF KINESIN MOVEMENT

动蛋白是一种机械酶，可以驱动基于微管的细胞内 细胞器的运输过程。Kinesin配对释放自由能量 化学反应(三磷酸腺苷的水解)形成一个机械循环 移动酶分子和附着的细胞器的过程 微管。我们想要描述机械过程的循环 并确定这些过程是如何耦合的 三磷酸腺苷的水解反应。我们开发了一部小说 允许我们直接监控机械设备的实验系统 单个动蛋白分子中的过程和化学步骤 结合在聚苯乙烯微珠上。这个系统让它 可以定量比较机械反应和化学反应的速度 在相同的条件下，从而允许直接研究 机械力化学耦合。 动蛋白及其同系物在细胞器内的转运作用 在真核细胞的生理学中起着重要作用。它的功能 包括物质的运输、染色体和核的运动 有丝分裂/减数分裂和膜细胞器的形态发生。去探索 这些在分子水平上的功能，我们将： 1)测量肌动蛋白每降解一次ATP的移动距离。我们会 测量微珠结合的单一动蛋白分子的ATPase Vmax和 将此与相同条件下共轭分子的移动速度进行比较 条件。这项研究将测试模型的有效性，在该模型中，一个ATP 每一个机械步骤都会被水解。 2)测定ADP诱导的微管结合运动蛋白的释放速率 头朝上。这项研究将检验这样一种假设，即释放头部是一种 在动蛋白运动周期中必不可少的一步。了解其动力学过程 头部释放将帮助我们了解单双头运动是如何 当分子沿着微管移动时，它仍然与微管相联系。 3)从两个基团推导出双头激动素衍生物的结构。 多维分子晶体。结构性数据将有助于揭示 驱动动蛋白运动的分子构象变化和 头部运动蛋白相互作用的性质。 4)制备单头肌动蛋白衍生物，并对其结构进行表征 功能属性。通过描述稳定状态的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-头交互在双头运动中的作用 功能。

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.