Single Molecule Analysis of Bacterial Protein Transport

描述(由申请人提供)：蛋白质必须定位到正确的亚细胞室才能正常发挥作用。蛋白质转运机制通过调节依赖于信号的蛋白质分子跨膜运输来实现这一基本功能。这些转运过程对细胞生长和发育的重要性从它们在许多疾病状态中的直接作用可见一斑，包括细菌致病性和蛋白质错误折叠疾病，如囊性纤维化。蛋白质转运体结构的改变或被扰乱的运输是导致许多白血病和癌症的原因。在细菌中普遍存在但在人类中不存在的蛋白质运输机制是抗生素开发的极好靶点。这项拟议的研究的目的是促进我们对细菌中蛋白质运输机制的分子基础的理解。SEC和TAT机器将使用单周转停流荧光和单分子荧光显微镜技术进行检查，以剖析单个运输的动力学步骤。该项目的具体目标是：(1)开发一种基于荧光的实时动力学分析方法，用于大肠杆菌SEC设备；(2)通过停流荧光法测定SEC和TAT的转运时间；(3)开发一种适用于同时进行电学和单分子荧光测量的脂质双层系统；以及(4)通过单分子荧光显微镜来确定单个SEC和TAT底物的转运动力学。这一结果将广泛适用于我们对膜转运机制的理解，并将显著推进单分子生物物理领域的发展。

DESCRIPTION (provided by applicant): Proteins must be localized to the correct subcellular compartment for proper function. Protein translocation machineries fulfill this essential function by mediating signal-dependent transport of protein molecules across membranes. The importance of these transport processes for cell growth and development is evident from their direct role in a number of disease states including bacterial pathogenicity and protein misfolding diseases such as cystic fibrosis. Altered protein transporter structure or perturbed trafficking is responsible for a number of leukemias and cancers. Protein transport machineries ubiquitous in bacteria but absent from humans are excellent targets for antibiotic development. The goal of the proposed research is to advance our understanding of the molecular basis of protein transport mechanisms in bacteria. The Sec and Tat machineries will be examined using single-turnover stopped-flow fluorescence and single-molecule fluorescence microscopy techniques in order to dissect individual kinetic steps of transport. The specific aims of the project are: (1) to develop a real-time fluorescence-based kinetic assay for the Escherichia coli Sec machinery; (2) to determine the Sec and Tat transport times via stopped-flow fluorescence; (3) to develop a lipid bilayer system suitable for simultaneous electrical and single-molecule fluorescence measurements; and (4) to determine the transport kinetics of single Sec and Tat substrates via single-molecule fluorescence microscopy. The results will be widely applicable to our understanding of membrane transport mechanisms and will significantly advance the field of single-molecule biophysics.