Investigation of the transport mechanism of ECF transporters by static and dynamic structure determination

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In this project, the structural basis for the function of energy-coupling factor (ECF) transporters, which were recently classified as members of the ATP-binding cassette (ABC) transporter family, will be investigated using the example of the folate ECF transporter (ECF-FolT) from different Lactobacillus species. Based on recent crystal structures it was hypothesized that ECF transporters use an unprecedented transport mechanism in which a complete subunit rotates in the lipid bilayer (Topples over). To test this intriguing hypothesis I will study structures of ECF-FolT in novel conformational states by x-ray crystallography and investigate the dynamic conformational changes of the transporter during the transport cycle using single molecule fluorescence spectroscopy. For site-directed fluorescence labelling of ECF-FolT the in vivo incorporation of non-natural amino acids in combination with azide-alkyne click chemistry and classical maleimide crosslinking at defined cysteine residues will be applied. In addition, reconstitution of ECF-FolT into proteoliposomes allows not only the dynamic structure determination in the membrane environment, but also the biochemical characterization of the transporter and the establishment of an in vitro system to investigate ligand-protein interactions. The understanding of the transport mechanism at molecular level may enable the development of novel antibiotics against human pathogens, which are dependent on ECF transporters for their vitamin requirements due to lack of biosynthetic pathways.