BIOPHYSICS AND ENZYMOLOGY OF KINESIN MOVEMENT

驱动蛋白是一种机械酶，其驱动基于微管的细胞内 细胞器运输过程。 驱动蛋白结合了一种释放自由能的 化学反应（ATP的水解）到机械循环 使酶分子和附着的细胞器沿着运动的过程 微管 我们想描述机械过程的循环 并确定这些过程是如何耦合的 ATP水解的反应。 我们开发了新颖的 实验系统可以让我们直接监测纳米尺度的 机械过程、畴运动和化学步骤， 分离的驱动蛋白分子。 驱动蛋白及其同源物参与的细胞内细胞器转运 在真核细胞的生理学中起着重要作用。 其职能 包括物质运输、染色体和细胞核运动， 有丝分裂/减数分裂和膜细胞器的形态发生。 探讨 这些功能在分子水平上，我们将： 描述激肽α-螺旋的构象变化 卷曲螺旋颈域，其是进行性运动沿着 微管 测试驱动蛋白运动机制的双手交替假说 检测和表征隔离驱动蛋白头的动力冲程 域 这些实验将结合联合收割机单分子生物物理学技术， 常规的酶学方法包括蛋白质化学修饰， 定点诱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.