Identification of the walking mechanism of conventional kinesin by single molecule fluorescence resonance energy transfer (smFRET) measurement on heterodimeric kinesins constructs

通过对异二聚驱动蛋白构建体进行单分子荧光共振能量转移 (smFRET) 测量来鉴定传统驱动蛋白的行走机制

• 批准号: 5427581
• 负责人: Professor Dr. Arne Gennerich
• 金额: --
• 依托单位: Department of Cellular and Molecular Pharmacology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2006-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5427581?language=en
• 关键词: Identification; walking; mechanism; conventional; kinesin

The objective of the project is to identify the walking mechanism of conventional kinesin and to find substantial evidence for the neck-linker structural change that has been proposed to enable the processive movement of the motor enzymes. The issues of the project will be addressed by the generation of both a kinesin heterodimer in which one of the heads is a mutant motor with impaired neck-linker mobility and two dimeric kinesin constructs with specific dye-labeled residues for single molecule fluorescence resonance energy transfer (smFRET) measurements. The kinesin constructs will be used in single and multiple molecule processivity assays. Quantitative information on motor movement and structural changes within the dye-labeled dimeric kinesins will be obtained using single particle tracking (SPT) and smFRET techniques together with total internal reflection fluorescence (TIRF) microscopy. By simultaneously measuring, under various biochemical conditions, the spatial movement and specific distances between carefully chosen FRET donor- and acceptor-labeled positions in the catalytic cores and the neck-linkers of processive kinesin enzymes, these studies will offer the detailed conformational information needed to unequivocally identify the correct walking mechanism of conventional kinesin.

该项目的目的是确定传统驱动蛋白的行走机制，并找到大量的证据，颈连接器的结构变化，已提出使运动酶的进行性运动。该项目的问题将得到解决的两个驱动蛋白异源二聚体的产生，其中的头部之一是一个突变的电机与受损的颈连接器的流动性和两个二聚体驱动蛋白结构与特定的染料标记的残基单分子荧光共振能量转移（smFRET）测量。驱动蛋白构建体将用于单分子和多分子持续合成能力测定。将获得定量信息的电机运动和结构变化的染料标记的二聚体驱动蛋白内使用单粒子跟踪（SPT）和smFRET技术与全内反射荧光（TIRF）显微镜。通过同时测量，在各种生化条件下，空间运动和特定的FRET供体和受体标记的位置之间的催化核心和颈接头的进行性驱动蛋白酶的距离，这些研究将提供详细的构象信息需要明确地确定正确的步行机制的常规驱动蛋白。