Single-molecule fluorescence spectroscopy to study structure and conformational dynamics of model proteins and the transporter BetP from Corynebacterium glutamicum

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Although static structures are known for many proteins, their functions are often governed by their dynamic character. In this proposal we want establish new fluorescence spectroscopic methods based on single molecules, multiparameter detection and fluorescence correlation spectroscopy (FCS). We want to develop new robust concepts of filtered FCS (fFCS) to study in a wide time range (50 ns -10 ms) the conformation dynamics of proteins using fluorescence anisotropy and/or fluorescence resonance energy transfer (FRET). We will use and verify these new tools to monitor conformational exchange in selected model proteins (e.g. T4 Lysozyme and Klenow Fragment of DNA Polymerase I). Thereby our results will be compared with those of other techniques. This preliminary work will enable us to analyze the complex structure and conformational dynamics of the homotrimer transporter BetP from Corynebacterium glutamicum. BetP is an ideal model system for the functional analysis of a transporter, because there is a broad background of knowledge on its function and structure available. Nevertheless the mechanisms for sensing and transport by BetP are still not clear. Studying single BetP molecules reconstituted in liposomes, the following questions will be addressed: (I) Analysis of the conformational changes of its C-terminal domain during sensing and catalysis; (II) Identification and structural characterization of functional intermediates in the catalytic cycle; and (III) Establishment of a full view of the catalytic cycle by monitoring FRET trajectories of immobilized proteoliposomes.