Biophysics of kinesin motion by single-pair FRET

描述（由申请人提供）： 驱动蛋白是一种利用ATP水解提供的能量沿沿着运动的运动蛋白。虽然两个驱动蛋白头部结构域所需的进行性运动，驱动蛋白头部微管结合的ATP周转过程中的精确序列是未知的。目前，用于观察驱动蛋白构型的平衡模型只能间接检测驱动蛋白-微管结合。本研究的目的是直接确定驱动蛋白头部在各种模型状态下的结合状态，以及ATP转换过程中的头部结合状态。驱动蛋白头部是否被结合将使用连接到驱动蛋白二聚体的一个头部的荧光染料与周期性且均匀地标记微管的第二荧光染料之间的荧光共振能量转移来确定。使用驱动蛋白二聚体系综的平衡实验将在恒定核苷酸条件下提供结合和未结合头部的群体。周转期间的头部结合将使用单个驱动蛋白二聚体直接确定。直接观察平衡时和周转期间的约束将有助于建立平衡模型与实际周转事件之间的关系。

DESCRIPTION (provided by applicant): 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 turnover 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 turnover will help establish the relationship between the equilibrium models and actual turnover events.