Functional analysis and NMR studies of multi-drug efflux pumps

Efflux pumps play an important role in bacterial multi-drug resistance by actively expelling antibiotics and toxic compounds from the cell. We describe an integrated workflow for the characterisation of inner membrane efflux pumps, consisting of an in vivo whole-cell functional efflux assay and an in vitro liposomal system for structural characterisation of protein/substrate interactions. The fluorescence-based assay uses the uncoupler CCCP to depolarise cell membranes, allowing fluorescent substrate to accumulate within the cell and bind to cellular DNA. In cells re-energised with glucose, restoration of PMF reactivates efflux, and the fluorescence of DNA-bound substrate provides a real-time measure of transport. The assay can inform on the impact of mutations and knockouts on efflux efficiency, facilitating mechanistic studies of substrate recognition and the functional roles of key residues. Insights into the structural and dynamic aspects of pump-substrate interactions are obtained using solid state NMR from membrane-reconstituted liposomal systems, where stable isotope-enriched recombinant protein is produced, purified and embedded in proteoliposomes. These are characterised by dynamics-sensitive and dipolar recoupling NMR methods to distinguish pump-associated substrate, and changes in pump NMR spectra inform on direct molecular contacts. The combined in vivo/in vitro approach can be extended to studies of other membrane transporter proteins, for which suitable fluorescent substrates can be identified.