Interaction of ECF and ABC transporter modules studied by time resolved fluorescence microscopy and spectroscopy

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The organisation and structure of ECF ("energy-coupling factor") transporters (BioMNY, EcfAA`T) and the histidine ABC transporter (HisJ/LAO-HisQMP2) during the catalytic cycle of the transporters will be investigated by time resolved fluorescence microscopy and spectroscopy. Various techniques including Fluorescence Lifetime Imaging, Förster-Resonance-Energy-Transfer, Fluorescence (Cross) Correlation spectroscopy and Fluorescence Recovery after Photobleaching will be employed to study the interaction of the various subunits and modules of transporters and their conformational dynamics. Transporter subunits will be labelled either by fluorescent proteins or by reactive fluorophores. By tagging of transporter subunits with fluorescent proteins, measurements to characterize subunit interactions can be performed on living cells (E. coli) and on detergent solubilized, liposome (Giant Unilamellar Vesicles) and NanoDisc reconstituted modules. NanoDiscs will be used to characterize the organisation of transporter at single molecule level by fluorescence bleaching. Biochemical assays provided by T. Eitinger and E. Schneider (see respective proposals) will be used to probe for function of fluorescently labelled transporters. Our studies complement those by Electron Spin Resonance (see project proposals of T. Eitinger, E. Schneider and H.J. Steinhoff)