In this dissertation, I employed a combination of in vitro and in silico techniques to characterize two bacterial membrane proteins: the MtrE efflux conduit from Neisseria gonorrhoeae and the AmtB ammonia transporter from Escherichia coli. MtrE is an efflux conduit located in the outer membrane of N. gonorrhoeae. It can form a protein complex with MtrC (a periplasmic adapter) and MtrD (an inner membrane active transporter). This tripartite machinery actively extrudes a diverse set of compounds from the periplasm to the exterior of the bacteria. The MtrCDE complex and homologous tripartite efflux pumps are amongst the major contributors to the emergence of super-resistant Gram-negative bacteria. The efforts to abolish the activity of these protein complexes have so far concentrated on the inhibition of the active pump, but had little clinical success to date. I carried out a combination of Planar Lipid Bilayer electrophysiology experiments, Molecular Dynamics simulations, and protein homology modeling on the MtrE and the MtrC proteins. I characterized the MtrE conduit as being slightly cation selective, as opposed to other homologous proteins which exhibit a much stronger selectivity. Additionally, I demonstrated that the opening of MtrE is modulated by the binding of the adapter protein MtrC. These results have a critical importance because they imply that the tripartite pump activity can be diminished not only by the MtrD inhibition, but by targeting the interface between MtrC and MtrE. The AmtB ammonia transporter is a protein embedded in the inner membrane of E. coli in situations of growth-limiting low levels of ammonia. The transport has been shown to be electrogenic in similar orthologous proteins, but the exact mechanism has so far remained elusive. The only inner pathway that has been identified in AmtB is lined by hydrophobic amino acids and for this reason it has been proposed to carry neutral ammonia. My Molecular Dynamics simulations reveal the opening of a water wire separated from the well known hydrophobic pore. This finding suggests a transport mechanism in which the neutral ammonia and the proton travel along different conduits. My simulations on several AmtB mutants gave additional important insights into the protein function. Notably my results explain the structural determinants of the switching of substrate from NH4+ to K+ observed for the H168D/H318E double mutant. Lastly, I identified several 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) binding sites on AmtB, which could possibly contribute to modulating the transport. Importantly, these in silico experiments were guided and confirmed by Solid Supported Membrane-electrophysiology measurements carried out in collaboration with the Javelle group. The data presented in this Thesis highlights the strength of a coordinated approach, in which experimental and computational findings direct, and integrate with each other.

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