MdfA - a model multidrug efflux pump for single-molecule investigations of transport

MdfA is an Escherichia coli multidrug efflux pump that has a broad - substrate profile that encompasses cationic, neutral and zwitterionic antimicrobials. MdfA is proposed to operate by an alternating access mechanism, a major substrate translocation mechanism thought to occur throughout a diverse range of transport proteins. However, direct visual evidence for this transport mechanism has not been obtained for any protein. The aim of this research is to explore MdfA as a model for conducting single - molecule Förster resonance energy transfer (sm - FRET) experiments to allow for direct visualisation of the alternating access mechanism, by direct observation of fluorescent substrate transport in parallel with protein domain movement. A prerequisite for this work is to identify a highly fluorescent substrate. This thesis aimed to determine whether MdfA and a Cys - variant of MdfA designed for FRET studies can recognise and transport the highly fluorescent monovalent cation rhodamine 6G (R6G) using fluorescence binding and transport assays. The results demonstrate that R6G is a substrate of MdfA, and that the Cys - variant of MdfA retains sufficient activity as a model for future sm - FRET studies. Moreover, this thesis reports on the first direct visualisation of fluorescence transport mediated by a multidrug efflu x pump at the single - cell level in real - time.