Understanding the TatA channel of the twin-arginine protein translocase

一些细菌蛋白质在细胞外起作用，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产生的，所以细胞外的蛋白质必须通过通常不渗透的细胞膜被移出细胞。这项任务是由位于细胞膜上的蛋白质转运体来完成的。一种转运蛋白移动未折叠的蛋白质，将它们穿过细胞膜，就像线穿过针眼一样。相比之下，第二种转运体，我们称之为Tat系统，将折叠的蛋白质穿过细胞膜。Tat蛋白运输系统也存在于植物叶绿体中。TatA系统的一个重要组成部分是蛋白质TatA。多个复制的塔塔组合成一个大的综合体。我们用电子显微镜观察了塔塔复合体。塔塔形成了一个甜甜圈形状的结构。这表明TatA在细胞膜上形成了一个通道，蛋白质通过中心孔运输。塔塔的一个令人惊讶的特点是，单个塔塔复合物的大小不同，显然是因为它们含有不同数量的塔塔蛋白。这种大小的变化可能使TatA能够紧密地包裹在正在运输的蛋白质周围，从而阻止其他分子的共同运输。然而，塔塔集团可能会因为其他一些貌似合理的原因而变得灵活。在这个项目中，我们想增加我们对TatA蛋白如何工作的理解。我们特别想了解Tat复合物大小变化的意义。电子显微镜图像太模糊，无法让我们确定TatA复合体内单个蛋白质的位置或形状。在复合体中，亚基的组织将被探测，使用的方法是通过将两种蛋白质化学结合在一起来确定TatA分子的哪些部分与其他TatA分子接触。这些链接还将用于确定TatA蛋白的哪些部分必须相对于蛋白质的其他部分移动，以使TatA能够发挥其功能。我们将通过询问在运输不同大小的蛋白质时环的大小是否会变化，来研究TatA复合物大小的可变性是否与根据底物定制孔的大小有关。我们还将测试单个TatA蛋白在运输过程中是否进入和离开TatA复合体，因为这是对大小变化的底物拟合解释的预测。这些和其他的实验有望让人们更清楚地了解塔塔集团的运作。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。

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. The Tat protein transport system is also found in plant chloroplasts. One essential component of the Tat system is the protein TatA. Multiple copies of TatA assemble to form a large complex. We have used electron microscopy to visualize the TatA complex. TatA forms a doughnut-shaped structure. This suggests that TatA forms a channel across the cell membrane with proteins being transported through the central hole. A surprising feature of TatA is that individual TatA complexes have different sizes, apparently because they contain different numbers of TatA proteins. It is possible that this size variation allows TatA to pack tightly around the protein that is being transported and thus prevent the co-transport of other molecules. However, the TatA complex may be flexible for a number of other plausible reasons. In this project we want to increase our understanding of how the TatA protein works. In particular we want to learn the significance of the variation in the size of the Tat complexes. The electron microscopy images are too fuzzy to allow us to determine the position or shape of individual proteins within the TatA complex. The organization of the subunits in the complex will instead be probed using methods that determine which parts of the TatA molecule are in contact with other TatA molecules by chemically tying the two proteins together. These links will also be used to determine which parts of the TatA protein have to move relative to other parts of the protein to allow TatA to carry out its function. We will investigate whether the variability in the size of the TatA complexes is linked to tailoring the size of the hole to the substrate by asking whether the size of the ring varies when transporting proteins of differing sizes. We will also test whether individual TatA proteins enter and leave the TatA complex during transport since this is a prediction of the substrate-fitting explanation of the size variations. These and other experiments are expected to give a clearer picture of the operation of TatA. 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 used to secrete useful protein products.