Unravelling BamA Function Using Fluorescence & Single Molecule Force Experiments

细菌的外膜是保护细菌免受周围环境影响的重要外层。与此同时，细菌必须吸收特定的营养物质才能生长和分裂。为了实现这一点，一类细菌的OM--所谓的革兰氏阴性型--是由特殊的脂类组成的，其中驻留着稳定OM并使营养物质能够运输到细菌中的蛋白质。因此，这些外膜蛋白(所谓的外膜蛋白)对细菌的生长和生存至关重要。OM并不是具有稀有漂浮蛋白质岛的脂质海洋，而是现在已知的是挤满了OMP，形成了一个非常拥挤的环境。细菌在生长和分裂过程中不断产生新的OM，一种迷人的纳米机器已经进化出来，这对于这些蛋白质成功地插入拥挤的OM并折叠到正确的结构是必不可少的，这样它们就可以执行它们的重要功能。这台机器被称为BAM(Beta-Barrel组装机械)，是这项提案的重点。我们建议使用最新的生化和生物物理技术来首次发现这种机器是如何工作的。许多革兰氏阴性细菌是致病的，会导致人类、动物和植物的疾病。自从80年前发现青霉素以来，许多这样的生物已经或正在对抗生素产生抗药性，这些抗生素成功地保护了我们免受革兰氏阴性细菌的入侵。我们现在迫切需要开发能够预防细菌感染的新抗生素。BAM就是这样一个令人兴奋的新目标。然而，BAM是如何发挥作用的尚不清楚。已知的是所有蛋白质部分的结构(在细菌的情况下是5个)，这为新的实验打开了大门，以找出OMPS是如何折叠的，以及BAM如何使这一过程在细菌OM中有效地发生。在这项拟议的工作中，我们的目标是使用最新的技术，包括荧光、FRET和单分子“拉动”实验来解开OMPS如何折叠以及BAM如何发挥作用。具体来说，我们将专注于BAM的一个组件，即BAMA，它是BAM辅助OMP折叠和组装的动力源。我们的目标是通过开发分析方法来了解这种膜蛋白的功能，利用这些方法我们可以监测折叠或功能事件期间发生的结构变化。除了提供有关生物如何进化这种聪明的纳米机器的新的基本信息外，从长远来看，我们的目标是利用所获得的信息为开发抗击由革兰氏阴性细菌引起的疾病的新途径铺平道路。

The outer membrane (OM) of bacteria is an important outer coat which protects the bacterium from its surrounding environment. At the same time, the bacterium has to take up specific nutrients in order to grow and divide. To achieve this, the OM of one class of bacteria- the so-called called Gram negative type - is uniquely built of special lipids in which proteins that stabilise the OM and enable transport of nutrients into the bacterium reside. These Outer Membrane Proteins (so-called OMPs) are thus vital for bacterial growth and survival. Rather than being a sea of lipids with rare floating protein islands, the OM is now known to be cram-packed with OMPs, forming a very crowded environment. Bacteria continually make new OMPs as they grow and divide, and a fascinating nano-machine has evolved which is essential for these proteins to be inserted successfully into the crowded OM and to fold to the correct structure so that they can carry out their vital functions. This machine - called BAM (beta-barrel assembly machinery) - is the focus of this proposal. We propose to use the very latest biochemical and biophysical techniques to discover, for the first time, how this machinery works. Many Gram negative bacteria are pathogenic, causing diseases in humans, animals and plants. Many such organisms have become, or are becoming, resistant to antibiotics that have so successfully protected us from the invasion of Gram negative bacteria since the discovery of penicillin >80 years ago. We now urgently need to develop new antibiotics able to prevent bacterial infection. BAM is one such exciting new target. How BAM functions, however, is not known. What is known is the structure of all of the protein parts (five in the case of the bacterium E.coli) opening the door to new experiments to work out how OMPs fold and how BAM allows this to happen efficiently in the bacterial OM. In the proposed work, we aim to use the very latest techniques, including fluorescence, FRET and single molecule 'pulling' experiments to unravel how OMPs fold and how BAM functions. Specifically we will focus on one component of BAM, known as BamA, which is the powerhouse for BAM-assisted OMP folding and assembly. Our aim is to understand the way in which this membrane protein functions by developing analytical methods with which we can monitor structural changes taking place during a folding or functional event. As well as providing new and fundamental information about how biology has evolved this clever nano-machine, in the long term we aim to use the information gained to pave the way towards developing new routes to combatting diseases caused by Gram negative organisms.