Biophysics of kinesin motion by single-pair FRET

单对 FRET 驱动蛋白运动的生物物理学

• 批准号: 7334025
• 负责人: DOUGLAS S MARTIN
• 金额: $ 1.8万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7334025
• 关键词: ATP Hydrolysis; Address; Adenylyl Imidodiphosphate; Binding; Biophysics; Cells; Cephalometry; Chemicals; Condition; Enzymes; Equilibrium; Event; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Goals; Head; Hydrolysis; Kinesin; Label; Mechanics; Microtubules; Modeling; Motion; Motor; Nucleotides; Population; Proteins; Research; Testing; Time; Tubulin; Work; analog; dimer; research study

Kinesin is a motor protein which uses the energy provided by ATP hydrolysis to move processively along microtubules. While two kinesin head domains are required for processive motion, the precise sequence of kinesin head-microtubule binding during ATP turnover is not known. Currently, the equilibrium models used to observe kinesin configuration at presumed tumover intermediates detect kinesin-microtubule binding only indirectly. The goal of this research is to directly determine the kinesin head binding status in the various model states, and the head binding status during ATP turnover. Whether the kinesin head is bound will be ascertained using fluorescence resonance energy transfer between a fluorescent dye attached to one head of a kinesin dimer, and a second fluorescent dye periodically and uniformly labeling a microtubule. Equilibrium experiments using ensembles of kinesin dimers will provide the population of bound and unbound heads under constant nucleotide conditions. Head binding during turnover will be directly determined using single kinesin dimers. Direct observation of binding at equilibrium and during tumover will help establish the relationship between the equilibrium models and actual turnover events.

驱动蛋白是一种利用ATP水解提供的能量使前体沿着运动的运动蛋白 微管虽然进行性运动需要两个驱动蛋白头部结构域，但驱动蛋白头部结构域的精确序列是不确定的。 在ATP转换过程中驱动蛋白头-微管结合是未知的。目前，均衡模型 用于观察驱动蛋白在假定的微管移动器中间体处的构型 只是间接的约束。本研究的目的是直接确定驱动蛋白头部结合状态， 各种模型状态，以及ATP周转期间的头部结合状态。驱动蛋白头部是否 结合将使用荧光染料之间的荧光共振能量转移来确定 连接到驱动蛋白二聚体的一个头部的第二荧光染料，以及周期性地和均匀地标记驱动蛋白二聚体的第二荧光染料。 微管使用驱动蛋白二聚体系综的平衡实验将提供 在恒定核苷酸条件下结合和未结合的头部。在周转期间， 使用单个驱动蛋白二聚体直接测定。直接观察平衡时的结合和 tumover将有助于建立均衡模型与实际人员流动事件之间的关系。