Single Molecule Analysis of Bacterial Protein Transport

描述（由申请方提供）：蛋白质必须定位于正确的亚细胞区室以实现正常功能。蛋白质易位机制通过介导蛋白质分子跨膜的信号依赖性转运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中显而易见，包括细菌致病性和蛋白质错误折叠疾病，如囊性纤维化。改变的蛋白转运体结构或扰乱的运输是许多白血病和癌症的原因。细菌中普遍存在但在人类中不存在的蛋白质转运机制是抗生素开发的极好目标。拟议研究的目标是促进我们对细菌中蛋白质转运机制的分子基础的理解。将使用单次翻转停流荧光和单分子荧光显微镜技术检查Sec和达特机械，以剖析运输的各个动力学步骤。本项目的具体目标是：（1）开发一种基于荧光的大肠杆菌Sec机制的实时动力学测定方法;（2）通过停流荧光测定Sec和达特转运时间;（3）开发一种适合于同时进行电学和单分子荧光测量的脂质双层系统;和（4）通过单分子荧光显微术确定单个Sec和达特底物的转运动力学。这些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学领域。

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.