Untersuchung des Transportmechanismus von ECF Transportern mittels statischer und dynamischer Strukturbestimmung
Zusammenfassung der Projektergebnisse
The aim of this project was to understand the transport mechanism of group II energy-coupling factor (ECF) transporters. Shortly before, the Slotboom group postulated a mechanism based on the toppling of the substrate-binding S-component. To proof this hypothesis, on the one hand a Cystein-less mutant was generated and the incorporation of the non-natural amino acid n-propargyl-lysine into ECF-FolT and ECF-Pdx of different Lactobacillus species with the amber stop codon mechanism was successfully demonstrated. The mutants were investigated by functional transport assay using radio-labled substrates. On the other hand preliminary X-ray crystallographic studies in lipid environment were performed. The lipids mimic the physiologically in vivo situation and might stabilize the transporter in novel conformations such as substrate- and/or nucleotide-bound state. Initial crystals were obtained of ECF-FolT, ECF-Pdx and ECF-ThiT by lipidic cubic phase crystallization in monolein as well as by the bicelle technique. However, the diffraction was not sufficient to obtain a novel structure of a group II ECF transporter.
