In vitro analysis of Tat protein transport using single molecule fluorescence methods

一些蛋白质作用于细菌细胞的外部，例如细菌病原体产生的毒素。因为所有的蛋白质都是在细菌内部制造的，所以细胞外的蛋白质必须穿过通常不透水的细胞膜移出细胞。这项任务是由位于细胞膜上的名为蛋白质转运体的机器执行的。一种类型的转运体移动未折叠的蛋白质，将它们像线一样穿过细胞膜，穿过针眼。相比之下，第二种类型的转运蛋白，我们称之为TAT系统，通过膜运输折叠的蛋白质。这比穿线更具挑战性，因此人们认为TAT系统是通过一种不寻常的机制运行的。TAT系统在许多细菌过程中都是必需的，包括能量产生、细胞分裂、致病以及土壤细菌与植物的固氮共生。TAT蛋白运输系统不仅存在于细菌中，也存在于植物的叶绿体中，在叶绿体中形成和维持进行光合作用所需的蛋白质是必不可少的。TAT系统是一个可能的药物靶点，因为它是细菌发病所必需的，但在人类或动物中没有发现。它还具有生物技术价值，因为它可以被用来分泌有用的蛋白质产品。该项目旨在使用尖端技术来阐明TAT机械如何工作的主要特征。我们方法的关键是修改TAT机器的蛋白质成分，使它们在被适当的光源照射时发出光。然后，我们将使用先进的显微镜来显示单个TAT蛋白进行运输时的情况。我们希望能够观察不同的蛋白质在运输过程中如何聚在一起，然后又如何分离，并跟踪运输的蛋白质穿过膜的过程。分析这些事件是如何发生的，以及完成每个步骤需要多少时间，将极大地促进我们对TAT运输过程的理解。这些知识将有助于将TAT途径用于有用目的的开发。

Some proteins operate on the outside of the bacterial cell, for example the toxins produced by bacterial pathogens. Since all proteins are made inside the bacterium the extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by machines termed protein transporters that are located in the cell membrane. One type of transporter moves unfolded proteins, threading them across the membrane like string through the eye of a needle. By contrast, a second type of transporter, which we term the Tat system, moves folded proteins across the membrane. This is much more challenging than threading and so it is thought that the Tat system operates by an unusual mechanism. The Tat system is required for many bacterial processes including energy generation, cell division, pathogenesis, and the nitrogen-fixing symbiosis of soil bacteria with plants. The Tat protein transport system is not only found in bacteria but is also present in the chloroplasts of plants where it is essential to form and maintain the proteins required to carry out photosynthesis. The Tat system is a possible drug target because it is required for bacterial pathogenesis but is not found in humans or animals. It is also of biotechnological interest because it could be utilised to secrete useful protein products. This project aims to use cutting edge technology to elucidate major features of how the Tat machinery works. The key to our method is to modify the protein components of the Tat machinery so that they emit light when illuminated by an appropriate light source. We will then used advanced microscopy to visualise individual Tat proteins as they carry out transport. We hope to be able to watch how the different proteins come together and then apart again during the transport process and to follow the transported protein as it travels across a membrane. Analysis of how these events occur, and how much time it takes to complete each step, will significantly advance our understanding of the Tat transport process. This knowledge will help underpin the exploitation of the Tat pathway for useful purposes.