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

细胞健康是由其生物大分子的结构、运动和相互作用决定的。能够询问生物大分子在自然细胞环境中的行为将使我们能够更好地理解这些分子是如何决定细胞的行为和功能的。蛋白质是一类重要的生物大分子，它执行广泛的细胞过程，如酶催化、细胞信号和支架以及DNA复制。它们由一条线性氨基酸链组成，定义为多肽，它们的序列由编码它们的遗传序列决定。蛋白质的一个重要子集是整膜蛋白，它存在于细胞膜中，约占细胞蛋白的30%。细胞膜是一种动态结构，主要由蛋白质和脂类组成，它们起到分隔细胞的作用，为细胞及其细胞器的外部环境提供屏障。完整的膜蛋白是由其疏水性多肽伸展段的含量定义的，该伸展段使其部分结构能够嵌入细胞膜中或与细胞膜相关联。它们负责各种动态的细胞过程，如感觉、细胞调节和细胞对细胞的黏附。膜蛋白的功能及其表达水平将在很大程度上决定离子组成，从而决定特定细胞类型的代谢水平，使它们成为所有生命所必需的，以及关键的药物靶点。我的主要目标是直接确定膜蛋白在其天然细胞膜环境中的结构动态信息，包括在活细胞中。了解蛋白质的结构动力学是很重要的，因为它们的波动经常代表着对蛋白质功能至关重要的运动和状态。为了做到这一点，我将开发通用的策略，使膜蛋白质的结构和动力学能够在复杂的环境中通过先进的结构质谱学方法进行破译。结构质谱学使用关于多肽及其多肽构件的高分辨率质量信息来推断蛋白质分子的结构属性--它们的形状、相互作用和运动。使用氢/氢交换质谱学(测量蛋白质主链酰胺氢与氢交换的程度和速率)等技术，可以提供有关蛋白质相互作用、配体结合和结构动力学的全球和局部信息。在这里，我提出了发展化学生物学和先进的质谱学方法来研究不同天然膜环境中的膜蛋白结构。完整的膜蛋白的一个关键领域是在抗菌素耐药性的发展中。抗击抗菌素耐药性是一项关键的社会挑战，如果不加以解决，有可能成为一场全球健康危机。在细菌细胞系中，对结构无关化学物质的多重耐药性的形成与多药外排膜蛋白转运体的功能有关，该转运体可以排出广泛的有毒物质，并导致抗生素的抑制作用减弱。我的研究将集中于在多药外排膜蛋白系统的背景下开发上述方法，这些系统已知在细菌抗生素耐药性中发挥主要作用。这将使人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，以及临床上相关的突变。更广泛地说，能够对细胞内的生物大分子进行结构洞察，将是我们在理解它们如何塑造健康和患病细胞的功能方面向前迈出的一大步。

Cellular health is determined by the structure, movement, and interplay of its biomacromolecules. Being able to interrogate the behaviour of biomacromolecules within a native cellular context would enable us to gain an enhanced understanding of how these molecules dictate a cells behaviour and function. Proteins are an essential class of biomacromolecule which perform a wide range of cellular processes such as enzyme catalysis, cell signalling and scaffolding, and DNA replication. They consist of a linear chain of amino acids, defined as a polypeptide, their sequence being determined by the genetic sequence which encodes them. An important subset of proteins is integral membrane proteins which reside within cellular membranes and account for about 30% of cellular proteins. Cellular membranes are dynamic structures consisting mostly of protein and lipid which act to compartmentalise the cell, providing barriers to the external environments of the cell and its organelles. Integral membrane proteins are defined by their content of hydrophobic polypeptide stretches which enable parts of their structure to be embedded within, or associated with, the cellular membrane. They are responsible for a variety of dynamic cellular processes, such as sensation, cellular regulation, and cell-to-cell adhesion. A membrane protein's functional capability and their level of expression will largely decide the ionic composition, and therefore the metabolic levels of a given cell type, making them essential for all life, as well as key drug targets. My main aim is to determine structural dynamic information of membrane proteins directly within their native cellular membrane environment, including within live cells. It is important to understand the structural dynamics of proteins, as their fluctuations frequently represent motions and states that are critical for protein function. To do this I will develop general strategies which enable membrane protein structure and dynamics to be deciphered within complex environments by advanced structural mass spectrometry methods. Structural mass spectrometry uses high-resolution mass information on polypeptides and their peptide building blocks to infer on the structural properties of a protein molecule - their shape, interactions, and movements. Using techniques such as hydrogen/deuterium exchange mass spectrometry (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. Here, I propose the development of chemical biology and advanced mass spectrometry strategies for membrane protein structural investigation within different native membrane environments.One key area in which integral membrane proteins are important is in the development of antimicrobial resistance. Combating antimicrobial resistance is a key societal challenge which, if not addressed, has the potential to become a global health crisis. In bacterial cell lines, the development of multiple drug resistance to structurally unrelated chemicals have been correlated to the function of multidrug efflux membrane protein transporters, which expel a broad range of toxic substances and result in reduced inhibitory effects of antibiotics. My research will focus on developing the aforementioned methods in the context of multidrug efflux membrane protein systems which are known to play major roles in bacterial antibiotic resistance. 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. More generally, the ability to achieve structural insight into biomacromolecules within cells would be a huge step forward in our understanding of how they shape the function of healthy and diseased cells.