Single Molecule Analysis of Bacterial Protein Transport

蛋白质靶向和跨脂质双层转运是一个基本的能量需求 在所有生物体中。生物体蛋白质组中大约一半的蛋白质 通过蛋白质易位系统插入或转运穿过膜，或 translocons。存在许多不同类型的易位系统， 分子穿过膜而不损害膜作为渗透性的作用 离子、代谢中间体和其他大分子的屏障。为进一步 扩大我们的知识的分子机制，存在易位大 分子穿过膜，拟议的研究将检查细菌双胞胎- 精氨酸转运（达特）输出系统。达特系统运输完全折叠的 组装蛋白质。由达特系统转运的蛋白质的数量是高度物种化的 依赖性，从没有到很多（> 100）。功能性达特系统的缺失 通常导致宿主细菌的生长缺陷。此外，达特机器负责 为众多对人类健康有重要意义的细菌毒力因子的输出。中 一个特别戏剧性的例子，一个功能性的达特系统是生长所必需的。 结核分枝杆菌，结核病的病原体。由达特运输 系统最低限度需要三种蛋白质，TalA、TatS和TatC。主要的假设是 TatBC复合物作为受体，识别运输的前序， 底物，并且TalA寡聚体提供了门控孔，货物蛋白通过该门控孔被转运。 穿过膜双层。为了进一步理解基本的机械原理， 通过达特系统控制运输，我们将：（1）探测前体与脂质的相互作用 和转座子，通过将荧光染料连接到前序列;（2）研究 货物尺寸和形状对运输速度和运输效率的作用;（3）调查 通过荧光共振能量转移的translocon-货物相互作用。这些 调查预计将大大增加我们对货物如何 由达特系统识别，以及什么类型的货物可以转运。此外，本发明还提供了一种方法， 它们将进一步阐明转座子组分和构象的作用。 运输所需的变化。这种对达特基本性质的描述 易位系统将为未来的工作提供必要的基础，如 开发靶向达特系统的药物或利用达特系统的可能性 系统在生物技术应用中的应用，例如蛋白质治疗剂的表达。

Protein targeting and transport across lipid bilayers is a fundamental energy-requiring process in all organisms. Up to approximately half of the proteins in an organism's proteome are inserted into or transported across membranes by protein translocation systems, or translocons. Many distinct types of translocation systems exist that allow large protein molecules to cross membranes without compromising the membranes' role as a permeability barrier to ions, metabolic intermediates, and other macromolecules. In order to further expand our knowledge of the molecular mechanisms that exist to translocate large molecules across membranes, the proposed research will examine the bacterial twin- arginine translocation (Tat) export system. The Tat system transports fully-folded and assembled proteins. The number of proteins transported by the Tat system is highly species dependent, ranging from none to many (> 100). The absence of a functional Tat system often leads to growth defects in the host bacterium. Further, the Tat machinery is responsible for the export of numerous bacterial virulence factors of human health significance. In a particularly dramatic example, a functional Tat system is required for the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis. Transport by the Tat system minimally requires three proteins, TalA, TatS and TatC. The dominant hypothesis is that a TatBC complex acts as a receptor, which recognizes the presequence of transport substrates, and a TalA oligomer provides a gated pore through which the cargo protein crosses the membrane bilayer. To further understand the basic mechanistic principles governing transport via the Tat system, we will: (1) probe precursor interactions with the lipid and the translocon, by attaching a fluorescence dye to the presequence; (2) investigate the role of cargo size and shape on transport rate and transport efficiency; and (3) investigate translocon-cargo interactions, by fluorescence resonance energy transfer.These investigations are expected to substantially increase our understanding of how cargos are recognized by the Tat system, and what types of cargos can be translocated. In addition, they will further elucidate the role of the translocon components and the conformational changes required for transport. This characterization of the basic properties of the Tat translocation system will provide an essential foundation for future work, such as the possibility of developing drugs that target the Tat system, or for the utilization of the Tat system in biotechnological applications, such as the expression of protein therapeutics.