X-ray crystallographic studies of the TatBC complex - linchpin of the twin-arginine protein transport system

一些细菌蛋白质作用于细胞外，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系统的核心是由两种名为TatB和TatC的蛋白质组成的复合体。这种复合体位于细胞膜上，识别并结合将要运输的蛋白质。然后，它与另一种名为TATA的蛋白质结合，形成活性转运蛋白。我们的目标是确定包含TatB和TatC的复合体的详细结构。我们还将尝试单独确定TatC的结构，因为这可能更容易，而且TatC似乎形成了TAT系统的稳定功能核心。为了获得这些结构，我们首先需要生长纯化蛋白质的晶体。这很可能是相当具有挑战性的，因为在膜中发现的蛋白质不容易形成晶体。为此，我们提出了一些或不同的结晶方法，以最大化我们的成功机会。我们还将充分利用最新的高通量技术来加快工作。如果我们要了解TAT系统运输的不寻常机制，确定TatBC和/或TatC的结构是必不可少的。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。

Some bacterial proteins operate on the outside of the 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 protein transport system is not only found in bacteria it is also present in the chloroplasts of plants where it is essential to form and maintain the proteins required to carry out photosynthesis. The heart of the Tat system is a complex formed by two proteins called TatB and TatC. This complex sits in the cell membrane and recognises and binds the proteins that are to be transported. It then binds another protein called TatA to form the active transporter. We aim to determine a detailed structure of the complex containing TatB and TatC. We will also try to determine the structure of TatC alone since this may be easier and TatC appears to form the stable functional core of the Tat system. To obtain these structures we first need to grow crystals of the purified proteins. This is likely to be quite challenging because it is not easy to form crystals of proteins found in membranes. For this reason we have proposed a number or different crystallization approaches to maximize our chances of success. We will also make full use of the latest high-throughput technologies to speed the work. Determination of a structure for TatBC and/or TatC is essential if we are to understand the unusual mechanism of transport by the Tat system. The Tat system is a possible drug target because it is required for bacterial pathogenesis but is not found in humans. It is also of biotechnological interest because it could be utilised to secrete useful protein products.