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

使用单分子荧光方法体外分析 Tat 蛋白转运

• 批准号: BB/H018050/1
• 负责人: Benjamin Berks
• 金额: $ 87.45万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH018050%2F1
• 关键词: vitro; analysis; Tat; protein; transport

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.

一些蛋白质在细菌细胞的外部起作用，例如细菌病原体产生的毒素。由于所有的蛋白质都是在细菌内部制造的，因此胞外蛋白质必须穿过通常不可渗透的细胞膜移出细胞。这一任务是由位于细胞膜上的蛋白质转运蛋白完成的。一种类型的转运蛋白移动未折叠的蛋白质，将它们穿过细胞膜，就像穿过针眼的线一样。相比之下，第二种类型的转运蛋白，我们称之为达特系统，将折叠的蛋白质跨膜移动。这比线程化更具挑战性，因此人们认为达特系统通过一种不寻常的机制运行。达特系统是许多细菌过程所必需的，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。达特蛋白转运系统不仅存在于细菌中，而且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需的。达特系统是一个可能的药物靶点，因为它是细菌致病所必需的，但在人类或动物中没有发现。它也是生物技术的兴趣，因为它可以用来分泌有用的蛋白质产品。本项目旨在利用尖端技术阐明达特机械的主要特点。我们的方法的关键是修改达特机器的蛋白质成分，使它们在适当的光源照射下发光。然后，我们将使用先进的显微镜来可视化个别达特蛋白质，因为他们进行运输。我们希望能够观察不同的蛋白质如何在运输过程中聚集在一起，然后再次分开，并在运输蛋白质穿过膜时跟踪它。分析这些事件是如何发生的，以及完成每一步需要多少时间，将大大促进我们对达特运输过程的理解。这些知识将有助于为有用的目的开发达特途径。