Biophysics of kinesin motion by single-pair FRET

描述(由申请人提供)： 动蛋白是一种利用ATP水解提供的能量沿微管进行运动的马达蛋白。虽然前进运动需要两个Kinesin头部结构域，但在ATP转换过程中，Kinesin头部与微管结合的准确序列尚不清楚。目前，用于观察假定周转中间体上的运动蛋白构型的平衡模型只能间接地检测运动蛋白与微管的结合。本研究的目的是直接确定不同模型状态下的Kinesin头部结合状态，以及ATP周转过程中的头部结合状态。通过连接到运动蛋白二聚体的一个头部的荧光染料和周期性且均匀地标记微管的第二个荧光染料之间的荧光共振能量转移，可以确定运动蛋白头部是否被结合。使用动蛋白二聚体系综的平衡实验将在恒定的核苷酸条件下提供结合和未结合的头部的种群。翻转过程中的头部结合将直接使用单个动蛋白二聚体来确定。直接观察平衡时和周转期间的绑定将有助于建立均衡模型和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.