Molecular basis of protein translocation through outer membrane porins

革兰氏阴性细菌被两层膜包围，最外层(通常被称为外膜)是一种高效的屏障，可以抵御有毒分子；例如，哺乳动物肠道内存在的胆盐。外膜的有效性对生物体来说是一把双刃剑，因为它也非常有效地阻止了必要的营养物质，如糖，这些营养物质是有机体生长和分配所需的。因此，所有有外膜的生物体在外膜内也有特殊的蛋白质，其工作是允许营养物质和代谢物与环境交换。这些蛋白质被称为孔蛋白，这是一种桶状的膜蛋白，有一个孔或穿过膜的孔。革兰氏阴性菌的外膜中有许多桶状蛋白(据估计，2-3%的大肠杆菌基因组编码这种蛋白)，它们具有多种功能。那些允许营养交换的蛋白通常被称为普通或经典的孔蛋白，其中最被理解的是蛋白OmpF和OMPC。这些孔蛋白也是常用抗生素(如氨苄西林)扩散到细胞内的主要途径。事实上，在耐多药细菌中，这些孔蛋白的通道经常被发现发生突变。这项研究建议的重点是我们最近发现的一般孔蛋白OmpF的一种新功能，这表明孔蛋白可以做的不仅仅是让小分子通过毛孔扩散。我们已经发现，OmpF也可以用来允许蛋白质通过(转位)进入细胞，只要蛋白质是展开的(即随机卷曲)，并且可以蜿蜒穿过孔蛋白的狭窄毛孔。更值得注意的是，这种转位蛋白蜿蜒穿过OmpF的三个毛孔中的两个，它由三个桶状亚单位组成；也就是说，它进入细胞，然后又出来。鉴于孔蛋白对细菌和真核生物的生理学的重要性(哺乳动物和植物的一些细胞器的外膜中有孔蛋白)，我们的发现对我们理解有外膜的生物体的生物学具有重要的影响，因为它表明蛋白质可以利用它们的孔蛋白。蛋白质的功能通常是通过它们的三维结构或折叠来赋予的。尽管通过OmpF的蛋白质是未折叠的多肽，因此没有任何功能，但这些多肽可以通过与蛋白质等其他分子结合而变得有功能，从而以这种方式改变细胞行为。这项提议的双重目的是了解肠道细菌大肠杆菌中通过孔蛋白OmpF转运蛋白质的分子基础(例如确定有多少多肽可以通过毛孔)，并通过观察其他生物的孔蛋白和孔蛋白来发现这种现象有多普遍。另一个需要研究的重要问题是，是什么推动了蛋白质通过孔蛋白的转运？这是一个重要的问题，因为外膜没有能量来源来将蛋白质拖入细胞(不同于最内层的膜)，因此回答这个问题不仅会为我们提供对细菌孔洞本身以及它们所在的外膜的基本新见解。

Gram-negative bacteria are surrounded by two membranes, the outer most of which (usually referred to as the 'outer membrane') is a highly effective barrier against toxic molecules; for example, bile salts which exist within the gut of a mammal. The effectiveness of the outer membrane is a double-edged sword for the organism since it is also very effective at keeping out essential nutrients such as sugars, which the organism needs to grow and divide. Hence all organisms that have an outer membrane also have specialised proteins within this membrane whose job it is to allow the exchange of nutrients and metabolites with the environment. These proteins are known as porins, which are barrel-like membrane proteins that have a hole or pore running through them which traverses the membrane. There are many barrel proteins in the outer membrane of Gram-negative bacteria (it is estimated that 2-3% of the E. coli genome encode such proteins) which serve a variety of functions. Those that allow the exchange of nutrients are usually referred to as general or classical porins, the best understood of which are the proteins OmpF and OmpC. These porins are also the major routes by which commonly used antibiotics (e.g. ampicillin) diffuse into the cell. Indeed, the channels of these porins are frequently found to be mutated in multidrug resistant bacteria. The focus of this research proposal is a novel function we have recently discovered for the general porin OmpF which shows that porins can do more than just let small molecules diffuse through their pores. We have discovered that OmpF can also be used to allow proteins to pass (translocate) into the cell, as long as the protein is unfolded (i.e. a random coil) and can snake its way through the narrow pores of the porin. Even more remarkably, this translocating protein snakes through two of the three pores of OmpF, which is composed of three barrel subunits; i.e. it goes into the cell then comes back out again. Given the importance of porins to the physiology of bacteria and eukaryotes (mammals and plants have porins in the outer membranes of some of their organelles) our discovery has important ramifications for our understanding of the biology of organisms that have outer membranes since it shows that proteins can exploit their porins. Proteins have functions which are usually imparted by their having a three dimensional structure or fold. Although the proteins that pass through OmpF are unfolded polypeptides and so by implication have no function, these can become functional by virtue of their being able to bind to other molecules such as proteins, and hence in this way alter cellular behaviour. The twin aims of this proposal are to understand the molecular basis for protein translocation through the porin OmpF in the gut bacterium Escherichia coli (determining for example how much polypeptide can pass through the pore) and to discover how common this phenomenon is by looking at other porins and porins from other organisms. Another important question to be investigated is what drives protein translocation through porins? This is an important question since the outer membrane has no energy source to call upon in order to drag a protein into a cell (unlike the inner most membrane which does) and so answering this question will not only provide us with fundamental new insights into bacterial porins themselves but also the outer membrane in which they reside.