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

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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.