CAREER: Single Molecule Studies to Dissect the Mechanochemistry of Cytoplasmic Dynein

科学价值：细胞质动力蛋白是一种二聚体运动蛋白，负责真核细胞中几乎所有沿微管的负端定向运输。动力蛋白形成一个大的二聚体复合体，它在起源、结构、机械化学和调控方面与动力蛋白有很大的不同。与对动力蛋白的结构和作用机制越来越了解的动力蛋白相比，我们对动力蛋白的作用机制了解相对较少。研究动力蛋白运动性的主要障碍是缺乏重组表达系统和缺乏具有足够空间和时间分辨率的工具来观察单个马达的运动和构象状态。本项目的目的是利用最先进的体外单分子方法阐明细胞质动力蛋白运动的基本机制。关于动力蛋白运动的三个突出问题将被解决：1)两个运动域的运动是如何协调以保持高进程性2)ATP在环中的水解如何导致单向运动以及哪些AAA位点负责动力蛋白运动3)动力蛋白负端方向性的结构基础是什么？解剖动力蛋白运动的机制将显著促进对动力蛋白在不同细胞过程中的功能的理解。更广泛的影响：该项目还将包括以下教育和推广部分：1)为来自加州奥克兰代表性不足地区的高中生建立一个教育推广计划。2)在当地公立学校组织夏季研究机会和科学博览会，以吸引年轻一代对科学感兴趣。3)在生物物理成像方法方面开设新的多学科研究生课程。4)为物理专业的学生开设以生物物理学为重点的新课程。

Scientific Merit:Cytoplasmic dynein is a dimeric motor protein responsible for nearly all minus-end directed transport along microtubules in eukaryotic cells. Dynein forms a large dimeric complex and it differs significantly from kinesin motor protein in origin, structure, mechanochemistry and regulation. In comparison with kinesin, whose structure and mechanism are increasingly well understood, we know relatively little about dynein mechanism. The major barriers to studying dynein's motility were the lack of a recombinant expression system and a paucity of tools for observing the movement and conformational states of single motors with sufficient spatial and temporal resolution. The goal of this project is to elucidate the fundamental mechanism of cytoplasmic dynein motility by using state-of-the-art single molecule methods in vitro. Three outstanding questions will be addressed about dynein motility: 1) How is movement of the two motor domains coordinated to maintain high processivity 2) How does ATP hydrolysis in the ring lead to unidirectional motion and which AAA sites are responsible for dynein motility 3) What is the structural basis for dynein's minus-end directionality? Dissecting the mechanism of dynein motility will significantly advance understanding of the way dynein functions in diverse cellular processes.Broader Impacts: This project will also include the following educational and outreach components: 1) Establish an educational outreach program for high school students from underrepresented areas of Oakland, CA. 2) Organize summer research opportunities and science fairs in local public schools to attract young generations to science. 3) Develop a new multidisciplinary graduate level course in biophysical imaging methods. 4) Develop a new curriculum with designated emphasis in Biophysics for Physics majors.