Delivery and clearance of outer membrane proteins to the bacterial outer membrane

革兰氏阴性菌是自然界中绝对普遍存在的一种微生物，我们的生活与它们的生物学密切相关。这些细菌对我们的日常生活有着巨大的影响，因为它们生活在我们的肠道中，感染我们的身体，存在于我们的环境中。因此，它们影响人类的健康和福祉，对生物技术、可持续农业经济等等都很重要。革兰氏阴性细菌与其他细菌的不同之处在于它们具有富含蛋白质和复杂脂多糖（LPS）的外膜（或OM）。这种OM在保护细菌免受环境影响方面起着至关重要的作用，例如，它是毒素的强大屏障，包括那些我们故意用来毒害它们的抗生素。OM对它们的正常生长和它们在植物、动物和人类疾病中的发病机制至关重要。然而，OM的创造具有独特的挑战性，因为制造它的每一种成分，无论是蛋白质还是脂质分子，都是在细菌细胞内制造的，并通过内膜出口到内膜和外膜之间的空间，称为周质。然后，这些组件必须在合并到OM之前进行。外周质缺乏ATP，这种分子通常用于推动自然界的这些过程，因此OM生物发生中不同步骤的编排必须以与细胞内典型蛋白质机器不同的方式提供动力和控制。因此，细菌已经进化出复杂的机制来建立、维持和适应它们的OMs，这取决于生长条件。需要一种单一的、必需的蛋白质复合物，即β -桶组装机制（或BAM复合物），来折叠并将外膜蛋白（omp）插入到外膜中。尽管最近的重大进展已经揭示了BAM的结构，并初步了解了OMP可能插入到OM中的机制，但BAM功能的两个关键领域仍然没有得到很好的描述，即OMP如何传递到BAM以及如果OMP折叠停止会发生什么？OMP的合成、折叠和降解速率是精细平衡的，它们的失调可能具有杀菌作用。因此，了解这些过程如何运作对于理解微生物的生物发生至关重要，并可能揭示阻止细菌生长的致命弱点。这个应用程序的重点是为这些重要问题提供新的见解。我们将使用综合结构分子生物学方法来确定BAM与向BAM提供omp的质周分子伴侣SurA之间复合物的3D结构，以及包含底物omp的复合物。然后，我们将使用质谱法来观察这些复合物中的动力学和相互作用，并进行功能分析以了解OMP的递送方式。我们还将研究当OMP折叠出错时会发生什么，以及BAM如何使用相同的技术工具包与降解错误折叠的OMP的蛋白质合作。

Gram-negative bacteria are a type of micro-organism that are absolutely pervasive in Nature, and we lead lives that are intimately entangled with their biology. such bacteria and have enormous implications for our everyday lives, because they live in our guts, infect our bodies, and are in our environment. They therefore impact human health and wellbeing, are important for biotechnology, sustainable agricultural economies and much more. Gram-negative bacteria differ from other bacteria in that they possess an outer membrane (or OM) that is rich in proteins and complex lipopolysaccharides (LPS). This OM plays a vital role in protecting the bacteria from their environment, for example it is a formidable barrier to toxins, including those such antibiotics that we intentionally use to try to poison them. The OM is critical for their normal growth, and for their pathogenesis in plant, animal and human disease. However, the creation of the OM is uniquely challenging because each component from which it is made, whether that be a protein or lipid molecule, is made inside the bacterial cell, and exported across the inner membrane to a space between the inner and outer membranes called the periplasm. Those components then have to before be incorporated into the OM. The periplasm is devoid of ATP, the molecule that is typically used to power such processes across Nature and so the choreography of the different steps in OM biogenesis must be powered and controlled in a different way to typical protein machineries inside the cell. Bacteria have thus evolved elaborate machineries to build, maintain and adapt their OMs, dependent on growth conditions. A single, essential protein complex, the beta-barrel assembly machinery (or BAM complex), is required to fold and insert outer membrane proteins (OMPs) into the OM. Although substantial recent progress has revealed the structure of BAM and given initial insights into the mechanisms by which OMPs may be inserted into the OM, two critical areas of BAM function remain poorly characterised, namely how are OMPs delivered to BAM and what happens if OMP folding stalls? The rates of OMP synthesis, folding and degradation are finely balanced, and their dysregulation can be bactericidal1. Understanding how these processes operate is thus crucial to understanding OM biogenesis and may reveal an Achilles heel by which bacterial growth can be halted. The focus of this application is to provide new insight into these important questions. We will use an integrated structural molecular biology approach to determine the 3D structure of complexes between BAM and the periplasmic molecular chaperone SurA, which delivers OMPs to BAM, and complexes that include substrate OMPs. We will then use mass spectrometry methods to look at the dynamics and interactions made in such complexes, and functional assays to understand how OMP delivery works. We will also look at what happens when OMP folding goes wrong, and how BAM cooperates with proteins that degrade misfolded OMPs, using the same toolkit of techniques.