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.