BIOPHYSICS AND ENZYMOLOGY OF KINESIN MOVEMENT

我们正在研究驱动蛋白的机制， 基于微管的细胞内细胞器运输过程。 驱动蛋白 耦合自由能释放化学反应（水解 ATP）的机械步骤的循环，移动酶分子， 沿沿着微管附着细胞器。 我们的长期目标 研究是：1）表征分子事件， 驱动蛋白运动的基本步骤，以及2）确定这些步骤如何 与ATP水解反应相结合。 我们已经开发出一种 光学显微镜数字图像处理技术 驱动蛋白分子产生的运动的纳米级精度。 这项技术提供了一个直接观察 机械酶运动的基本过程。 基于微管的细胞内运动在细胞内运动中起重要作用。 真核细胞生理学。 其功能包括运输 材料、有丝分裂/减数分裂中的染色体和核运动，以及 膜性细胞器的形态发生。 驱动蛋白（或驱动蛋白同系物） 被认为在所有这些类别的细胞过程中起作用。 这项研究是在分子水平上的探索， 功能协调发展的 拟议研究的具体目的是直接观察和 描述由一个单一的催化周转所引起的运动， 单个驱动蛋白分子。 在拟议的实验中，我们将： * 表达并纯化特异性生物素化的驱动蛋白融合蛋白。 以前的研究表明，这个物种应该是充分的， 机械化学功能的。 我们将描述函数 这种重组机械酶的性质，以确保这是 这个案子了 * 合成了一种新型酶标记试剂， 直径的聚苯乙烯珠粒，每个珠粒包含单个位点， 生物素化酶的附着。 * 制备并表征1：1机械酶：珠缀合物。 的 然后将使用共轭物来确认我们可以可靠地观察到 由单个机械酶分子驱动的运动。 我们将 建立一个定量运动分析，将测量 运动的具体活动，通过测量的分数， 能够移动的酶-珠缀合物。 * 通过检查 机械酶的运动：纳米尺度的珠复合物 精度 这些测量将在以下条件下进行： (e.g.,限制ATP），其中离散的“跳跃”对应于 应检测到单次人员流动。 * 描述所观察到的空间和时间特性 跳跃，并确定他们是否有 单次周转运动的特点。

We are studying the mechanism of kinesin, a mechanoenzyme that drives microtubule-based intracellular organelle transport processes. Kinesin couples a free-energy-liberating chemical reaction (the hydrolysis of ATP) to a cycle of mechanical steps that moves the enzyme molecules and attached organelles along microtubules. The long-term goals of our research are to: 1) characterize the molecular events that are the fundamental steps in kinesin movement, and 2) determine how these steps are coupled to the reactions of ATP hydrolysis. We have developed a light microscope digital image processing technique for measuring with nanometer-scale precision the movements generated by kinesin molecules. This technique represents an opportunity to directly observe the fundamental processes of mechanoenzyme movement. Microtubule-based intracellular motility 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. Kinesin (or kinesin homologs) are thought to function in all of these categories of cellular processes. The proposed research is an exploration at the molecular level of these functions. The specific aim of the proposed research is to directly observe and characterize the movement caused by a single catalytic turnover of an individual kinesin molecule. In the proposed experiments, we will: * express and purify a specifically biotinated kinesin fusion protein. Previous studies suggest that this species should be fully mechanochemically functional. We will characterize the functional properties of this recombinant mechanoenzyme to be sure that this is the case. * synthesize a novel enzyme-labelling reagent that consists of 100 nm diameter polystyrene beads each of which contains a single site for the attachment of a biotinated enzyme. * prepare and characterize the 1:1 mechanoenzyme:bead conjugates. The conjugates will then be used to confirm that we can reliably observe movements driven by single mechanoenzyme molecules. We will establish a quantitative motility assay which will measure the specific activity of movement by measuring the fraction of the enzyme-bead conjugates capable of movement. * search for single-turnover movements in the assay by examining the motion of the mechanoenzyme:bead complexes with nanometer-scale precision. These measurements will be conducted under conditions (e.g., limiting ATP) in which discrete "jumps" corresponding to single-turnover movements should be detected. * characterize the spatial and temporal properties of the observed jumps and determine if they have the characteristics of single-turnover movements.