Determining structural dynamics of membrane proteins in their native environment: focus on bacterial antibiotic resistance

细胞由膜分隔，膜为细胞及其细胞器的外部环境提供屏障。它们是主要由蛋白质和脂质组成的动态结构。它们包含一个重要的蛋白质亚类，即膜蛋白。膜蛋白位于脂质双层内，负责各种基本的细胞过程，如感觉、细胞调节和代谢，使其成为所有生命所必需的，也是关键的药物靶点。本项目旨在揭示与细菌抗菌素耐药性（AMR）有关的膜蛋白的结构动力学。AMR被世界卫生组织和联合国确认为全球卫生紧急情况。开发和销售针对耐药细菌的新型化学抗生素的传统模式由于成本高昂（每种药物15亿美元）而瓦解。这些项目的重点是多药外排膜蛋白系统，已知这些系统在细菌抗生素耐药性中起主要作用，特别是耐药-增殖-分裂（RND）外排泵。它们将多种有毒物质排出细菌的能力显着有助于对结构和功能多样的抗菌药物的多药耐药性。了解它们的结构动力学是很重要的，因为这些基本波动经常代表对蛋白质功能和药物外排至关重要的运动和状态。为了做到这一点，化学生物学和先进的质谱分析策略正在开发中，使膜蛋白动力学在复杂的环境中，包括在活细胞中被破译。使用诸如氢/氘交换质谱（HDX-MS）的技术，其测量蛋白质骨架酰胺氢交换氘的程度和速率，可以提供关于蛋白质相互作用、配体结合和结构动力学的全局和局部信息。这将使我们能够前所未有地深入了解这些系统的结构、动力学和功能，特别是药物和脂质相互作用的影响以及临床相关突变。随着细胞结构动态洞察力的实现，我们在理解它们如何塑造健康和患病细胞的功能方面迈出了巨大的一步。到目前为止，我们已经探索了原型耐药-增殖-分裂（RND）多药外排系统内的一个质子背景。浮游细菌在悬浮液中“自由生活”或“自由流动”，通常在实验室的烧瓶培养中生长。它们并不固定在一个细菌群落中，而是被设计成殖民新的生态位，但存活的机会较低。然而，自然发现的细菌种群通常形成称为生物膜的结构化细胞群落，这为细菌繁殖和生存提供了更安全的方式。生物膜通常对植入物造成很大的问题，因为它们提供了理想的固体支持以促进生长，因此治疗这种感染非常困难，通常导致植入物的移除。在这次更新中，我们计划以两种方式扩大我们的研究：1）探索相关的外排蛋白和系统，远离原型，以拓宽我们对结构动力学在多药耐药表型中所起的基本作用的理解，以及2）使我们的方法适应与生物膜系统的界面，以便获得这些外排系统在细菌感染和耐药性中所起作用的“真实”背景。总之，有大量的证据支持RND泵在细菌致病性中的影响。通过了解这些致病模式，可以设计治疗方法来克服它们并治疗感染。通过利用不同的方法，专注于用外排泵抑制剂（EPI）靶向RND功能，可以改善对新的、更有效的抗生素的依赖。

Cells are compartmentalized by membranes, which provide a barrier to the external environments of the cell and its organelles. They are dynamic structures consisting mostly of protein and lipid. They contain an important subset of proteins, integral membrane proteins, which reside within a lipid bilayer and are responsible for a variety of essential cellular processes, such as sensation, cellular regulation, and metabolism - making them essential for all life, as well as key drug targets.This project aims to uncover the structural dynamics of membrane proteins involved in antimicrobial resistance (AMR) of bacteria. AMR is recognised by the WHO and United Nations as a global health emergency. The traditional model for the development and marketing of new chemical antibiotics against drug-resistant bacteria has disintegrated due to the high cost ($1.5 billion per drug). The projects' focus is on multidrug efflux membrane protein systems which are known to play major roles in bacterial antibiotic resistance, specifically the resistance-nodulation-division (RND) efflux pumps. Their ability to expel a broad range of toxic substances out of bacteria significantly contributes to multidrug resistance against structurally and functionally diverse antimicrobial drugs. Understanding their structural dynamics is important, as these fundamental fluctuations frequently represent motions and states that are critical for protein function and drug efflux. To do this, chemical biology and advanced mass spectrometry strategies are being developed which enable membrane protein dynamics to be deciphered within complex environments, including in live cells. Using techniques such as Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS), which measures the extent and rate of exchange of protein backbone amide hydrogens for deuterium, both global and local information on protein interactions, ligand binding, and structural dynamics can be delivered. This will enable an unprecedented insight into the structure, dynamics, and function of these systems, particularly on the impact of drug and lipid interactions, and clinically relevant mutations. With the achievement of cellular structural dynamic insight offering a huge step forward in our understanding of how they shape the function of healthy and diseased cells. So far, we have explored prototypical resistance-nodulation-division (RND) multidrug efflux systems within a planktonic context. Planktonic bacteria are 'free-living' or 'free flowing' in suspension, commonly grown in flask cultures in the laboratory. They are not fixed to a community of bacteria and are designed to colonize new niches, but with a lower chance of survival. However, bacterial populations found naturally often form structured communities of cells called biofilms, which provide a more secure way for bacteria to reproduce and survive. Biofilms typically pose a great issue for implants as they provide an ideal solid support to promote growth, thus treatment of such infections is extremely difficult, normally resulting in the removal of the implant. Within this renewal we plan to expand our research in two ways: 1) the exploration of related efflux proteins and systems, away from the prototypical, to broaden our understanding of the fundamental role structural dynamics plays in the multidrug resistance phenotype, and 2) adapt our methods to interface with biofilm systems, so as to gain a 'true' context of the role these efflux systems play in bacterial infection and resistance. In conclusion there is an abundance of evidence to support the influence of RND pumps in the pathogenicity of bacteria. By understanding these modes of pathogenicity therapeutic methods can be designed to overcome them and treat infection. By utilizing different methods, focused on targeting RND function with efflux pump inhibitors (EPIs), reliance on new, more potent antibiotics can be ameliorated.