BIOPHYSICS AND ENZYMOLOGY OF KINESIN MOVEMENT

驱动蛋白运动的生物物理学和酶学

• 批准号: 3302443
• 负责人: JEFF GELLES
• 金额: $ 8.33万
• 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 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302443
• 关键词: adenylate kinase; affinity chromatography; chimeric proteins; enzyme structure; eukaryote; genetic manipulation; image processing; intracellular transport; kinesin; organelles; protein structure function; transfection; transport proteins

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.

我们正在研究激动素的机制，这是一种推动 基于微管的细胞内细胞器运输过程。动蛋白 耦合释放自由能的化学反应(水解物 ATP)到一个机械步骤的循环，该循环移动酶分子和 沿微管附着的细胞器。我们的长期目标是 研究的目的是：1)描述分子事件是 运动蛋白运动的基本步骤，以及2)决定这些步骤如何 都与三磷酸腺苷的水解反应相耦合。我们已经开发出一种 光学显微镜数字图像处理技术在测量中的应用 运动蛋白分子产生的运动的纳米级精确度。 这项技术提供了一个直接观察 机械酶运动的基本过程。 以微管为基础的细胞内运动在 真核细胞生理学。它的功能包括运输 有丝分裂/减数分裂中的物质、染色体和核运动，以及 膜细胞器的形态发生。Kinesin(或Kinesin同系物) 被认为在所有这些类别的细胞过程中起作用。 提出的研究是在分子水平上的探索。 功能。 拟议研究的具体目的是直接观察和 描述由一次催化周转所引起的运动 个体动蛋白分子。在建议的试验计划中，我们会： *表达和纯化一种特定生物标记的动蛋白融合蛋白。 此前的研究表明，这一物种应该完全 机械力化学功能。我们将描述泛函 这种重组机械酶的性质，以确保这是 这个案子。 *合成一种新型的酶标记试剂，由100 nm组成 直径的聚苯乙烯珠子，每个珠子包含一个单一的位置 生物酶的附着。 *制备和表征1：1机械酶：珠结合物。这个 然后将使用共轭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。我们会 建立一种定量的运动测定方法，它将测量 通过测量运动的比例来测量运动的特定活动 酶-珠偶联物能够移动。 *在化验中寻找单次成交变动，方法是检查 机械酶的运动：纳米尺度的微珠复合体 精确度。这些测量将在以下条件下进行 (例如，限制ATP)，其中对应于 应该检测到单次周转运动。 *描述被观测对象的空间和时间特性 跳跃并确定他们是否有 单周转动作的特点。