How do ATP-independent chaperones assist OMP folding and assembly? Insights from mass spectrometry and other approaches

每一个活细胞都被一个被称为膜的包膜所包围，它是外界环境的屏障。这些膜由脂质和蛋白质组成，它们调节分子进出细胞：特别是调节营养物质的进入，以确保生物体能够生长和存活。有些细菌，称为革兰氏阴性细菌，被两层膜包围，一层在另一层里面，称为内膜和外膜，中间有一个细胞区室称为周质。此类细菌的外膜（OM）含有不同选择的脂质，并且充满了对细菌生存至关重要的OM蛋白（OMP）。新的外膜蛋白一直由细菌制造，但用于制造这些蛋白质的机器位于细胞的主隔室（细胞质）中，位于内膜内。一旦制造出来，外膜蛋白就必须长途跋涉，到达细胞中需要它们的地方，即外膜。细胞机制已经进化到协助这一过程;特别是，周质中称为Skp和SurA的伴侣蛋白与新产生的外膜蛋白结合，并将它们护送到外膜。然后将OMP插入OM中，并通过称为BAM（β-桶组装机）的分子机器折叠成正确的结构。干扰这一途径导致细菌活力的丧失，因此代表了一种潜在的新途径，用于控制革兰氏阴性病原体负责infection.The分子伴侣蛋白Skp和SurA的结构，和BAM的所有组成蛋白，是已知的。目前尚不确定的是，这些蛋白质如何共同作用，形成新的、折叠的和功能性的外膜蛋白。我们建议利用质谱（MS）（沿着其他生物物理技术）的最新令人兴奋的发展来解决这个基本问题。我们的工作将集中在如何两个伴侣Skp和SurA绑定和识别其底物外膜蛋白。已知Skp包含对于捕获OMP重要的大空腔;然而，该空腔如何能够容纳不同尺寸的OMP仍然未知。SurA是一种关键的分子伴侣，它如何结合和递送OMP以折叠成OM，在分子细节上也是未知的。在这里，我们将使用MS方法来了解这些分子伴侣的功能。我们还将首次使用类似的方法来确定BAM的结构组织。最后，在具有挑战性但令人兴奋的实验中，我们将研究分子伴侣结合的OMPs如何被传递到BAM，以及BAM的亚基如何重新排列，以便OMPs可以插入OM。最后，我们的目标是了解BAM功能如何被抑制。从这项工作中获得的对这种基本细胞机制的见解可能会导致确定急需的抗生素新靶标，以对抗对人类，植物和动物造成疾病的革兰氏阴性细菌。

Every living cell is surrounded by an envelope, called a membrane, which acts as a barrier to the external environment. These membranes are comprised of lipids and proteins that regulate the entry and exit of molecules in to and out of the cell: in particular regulating the entry of nutrients that ensure that the organism is able to grow and survive. Some bacteria, called gram-negative bacteria, are surrounded by two membranes, one inside the other, called the inner and outer membranes, with a cellular compartment in-between called the periplasm. The outer membrane (OM) of such bacteria contains a distinct selection of lipids, and is crowded with OM proteins (OMPs) that are vital for bacterial survival. New OMPs are made by the bacteria all the time, but the machinery that is used to make these proteins is located in the main compartment of the cell (the cytoplasm), inside the inner membrane. Once made, the OMPs have to undertake a long journey to where they are needed in the cell, the OM. Cellular machinery has evolved to assist this process; in particular, chaperone proteins in the periplasm called Skp and SurA bind to the newly made OMPs and escort them to the OM. The OMPs are then inserted into the OM and folded to the correct structure by a molecular machine called BAM (beta-barrel assembly machinery). Perturbing this pathway leads to a loss of bacterial viability, and therefore represents a potential new avenue for controlling gram-negative pathogens responsible for infections.The structures of the chaperone proteins Skp and SurA, and all the component proteins of BAM, are known. What remains undetermined is how these proteins all work together to make new, folded and functional OMPs. We propose to address this fundamental question by exploiting recent exciting developments in mass spectrometry (MS) (along with other biophysical techniques). Our work will focus on how the two chaperones Skp and SurA bind and recognise their substrate OMPs. It is known that Skp contains a large cavity important for trapping OMPs; however, how this cavity is able to accommodate OMPs of different sizes remains unknown. How SurA, a key chaperone, binds and delivers OMPs for folding into the OM is also unknown in molecular detail. Here we will use MS methods to understand how these chaperones function. We will also use similar approaches to determine the structural organisation of BAM for the first time. Finally, in challenging but exciting experiments, we will investigate how chaperone-bound OMPs are delivered to BAM, and how the subunits of BAM rearrange so that OMPs can be inserted into the OM. Finally, we aim to understand how BAM function can be inhibited. The insights into this essential cellular machinery gained from this work may lead to the identification of much-needed new targets for antibiotics against gram-negative bacteria that cause disease to humans, plants and animals.