Unravelling BamA Function Using Fluorescence & Single Molecule Force Experiments

利用荧光揭示 BamA 功能

• 批准号: BB/N007603/1
• 负责人: David Brockwell
• 金额: $ 45.69万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN007603%2F1
• 关键词: Unravelling; BamA; Function; Using; Fluorescence

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.

细菌的外膜（OM）是一件重要的外层，可保护细菌免受其周围环境的影响。同时，细菌必须吸收特定的营养才能生长和分裂。为了实现这一目标，一类细菌的OM（称为革兰氏负类型）是由特殊脂质构建的，其中稳定OM并使营养物质能够驻留到细菌中。因此，这些外膜蛋白（所谓的OMP）对于细菌生长和存活至关重要。现在众所周知，该OM不再是具有稀有浮动蛋白质岛的脂质的海洋，而是塞满了Omps，形成了一个非常拥挤的环境。细菌在生长和分裂时不断地产生新的OMP，而引人入胜的纳米光明已经进化了，这对于这些蛋白质成功插入了拥挤的OM并折叠到正确的结构，以便它们可以执行其重要功能。该机器称为BAM（β-桶装配机械） - 是该提案的重点。我们建议使用最新的生化和生物物理技术，以发现这种机械的工作原理。许多革兰氏阴性细菌是致病性的，会导致人类，动物和植物的疾病。自从发现青霉素> 80年前以来，许多这样的生物已经成为对抗生素的抗药性，这些生物已经成功地保护了我们免受革兰氏阴性细菌的入侵。现在，我们迫切需要开发能够预防细菌感染的新抗生素。 BAM是一个令人兴奋的新目标。但是，BAM的功能尚不清楚。众所周知，所有蛋白质部分的结构（在细菌e.coli的情况下）打开了新实验的大门，以弄清楚如何折叠以及BAM允许BAM如何在细菌OM中有效地发生这种情况。在拟议的工作中，我们旨在使用最新技术，包括荧光，FRET和单分子“拉动”实验，以揭示如何折叠以及BAM的功能。具体而言，我们将专注于BAM的一个组成部分，即BAMA，这是BAM辅助OMP折叠和组装的强大组成部分。我们的目的是通过开发分析方法来了解该膜蛋白功能的方式，我们可以在折叠或功能事件中监视发生的结构变化。除了提供有关生物学如何进化这种聪明的纳米机器的新的基本信息，从长远来看，我们旨在利用所获得的信息为开发新的途径铺平道路，以打击由革兰氏阴性生物引起的疾病。

项目成果

Dynamic interplay between the periplasmic chaperone SurA and the BAM complex in outer membrane protein folding.

• DOI: 10.1038/s42003-022-03502-w
• 发表时间: 2022-06-08
• 期刊: Communications biology
• 影响因子: 5.9

Outer membrane protein folding from an energy landscape perspective.

• DOI: 10.1186/s12915-017-0464-5
• 发表时间: 2017-12-21
• 期刊: BMC biology
• 影响因子: 5.4
• 作者: Schiffrin B;Brockwell DJ;Radford SE
• 通讯作者: Radford SE

Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding.

• DOI: 10.1016/j.jmb.2017.09.008
• 发表时间: 2017-11-24
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Schiffrin B;Calabrese AN;Higgins AJ;Humes JR;Ashcroft AE;Kalli AC;Brockwell DJ;Radford SE
• 通讯作者: Radford SE

Distortion of the bilayer and dynamics of the BAM complex in lipid nanodiscs.

• DOI: 10.1038/s42003-020-01419-w
• 发表时间: 2020-12-14
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Iadanza MG;Schiffrin B;White P;Watson MA;Horne JE;Higgins AJ;Calabrese AN;Brockwell DJ;Tuma R;Kalli AC;Radford SE;Ranson NA
• 通讯作者: Ranson NA

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

使用标签转移光交联剂快速绘制蛋白质相互作用图

• DOI: 10.1002/ange.201809149
• 发表时间: 2018
• 期刊: Angewandte Chemie
• 作者: Horne J