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。我们已经开发出了小说 允许我们直接监测纳米级的实验系统 机械过程、磁区移动和化学步骤 使用光学显微镜仪器分离动蛋白分子。 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： 表征激动素α-螺旋的构象变化 前向运动所需的卷曲颈区 微管。 检验Kinesin运动机制的手-手假说 检测和表征孤立运动蛋白头部的力量冲程 域。 这些实验将结合单分子生物物理技术和 传统的酶学方法包括蛋白质化学修饰， 定点突变和化学动力学技术。

Kinesin is 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 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 novel experimental systems that allow us to directly monitor nanometer-scale mechanical processes, domain movements, and chemical steps in single isolated kinesin molecules using light microscope-based instruments. 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: Characterize the conformational changes in the kinsin alpha-helical coiled-coil neck domain that are required for processive motility along microtubules. Test the hand-over-hand hypothesis for the kinesin movement mechanism Detect and characterize the power stroke of an isolated kinesin head domain. The experiments will combine single-molecule biophysics techniques with conventional enzymology methods including protein chemical modification, site-directed mutagenesis, and chemical kinetics techniques.