Investigation of the transport mechanism of ECF transporters by static and dynamic structure determination
Final Report Abstract
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.
