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Energetics and Structural Aspects of Cation-Coupled Drug Transport by NorM Multidrug Transporters 10.17863/CAM.32283 Raturi, Sagar Van Veen, Hendrik NorM multidrug transport proteins belong to the multiple antibiotics and toxins extrusion (MATE) family of secondary active transporters. Members of this family are present across all species including bacteria, plants and humans. In bacteria, their over-expression can lead to antibiotic resistance, whereas in the human body, the transporters can alter the plasma levels of drugs. NorM proteins are therefore relevant for the pharmacokinetic properties of drugs. Previously, NorM from Vibrio cholerae (NorM-VC) was shown to export drug (ethidium) in an antiport reaction that is coupled to the simultaneous uptake of protons and sodium ions down their electrochemical gradients across the plasma membrane. But NorM from Pseudomonas stutzeri (NorM-PS) was shown to transport DAPI by utilising proton cycling exclusively. NorM-VC and NorM-PS share 42% identical amino-acid residues and yet their functions differ in terms of their ion coupling properties. These differences in functionality of two highly homologous proteins provide an excellent opportunity to carry out a comparative study. The work presented in this thesis investigates the energetics of drug transport processes by NorM-VC and NorM-PS and the structural basis for ion-coupled drug transport by NorM-VC. Ethidium efflux assays in intact Lactococcus lactis cells were used to study the effect of the magnitude and composition of the proton- and sodium-motive force on transport activity. Furthermore, ethidium binding assays were used to study partial reactions in drug efflux processes. These biochemical data were supplemented by computational studies and analyses of current protein structures. Based on the observations detailed here, a novel transport model for NorM-VC is proposed, which explains published findings for NorM-VC and other MATE transporters. The model represents a potentially universal mechanism for MATE transporters that can be used to predict further structure-function relationships in this important family of member transporters. University of Cambridge 2018-11-21 Thesis Doctoral Doctor of Philosophy (PhD) en Cambridge Commonwealth Trust, Nehru Trust for Cambridge University https://www.repository.cam.ac.uk/handle/1810/284914 https://www.repository.cam.ac.uk/bitstreams/478b1dbd-312a-458d-bbf5-1d5dacac18b9/download 2f90c7eb353841debfcefa9805ede30e https://www.repository.cam.ac.uk/bitstreams/8efbbdfd-6d89-4525-b9ef-9e92ec965f9c/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Multi-drug Transporters NorM MATE transporters