Abstract - Krüger

Tripartite ATP-independent periplasmic (TRAP) transporters play a crucial role in bacterial and archaeal uptake of different substrates. They comprise three different domains, a soluble substrate-binding domain (P-domain) and two membraneinserted domains (Q- and M-domain). In contrast to ATPdependent transporters, they rely on an electrochemical gradient as energy source for the substrate import. The Pdomain binds and delivers the substrate to the membraneinserted domains to initiate import. HiSiaPQM and its homologs act as TRAP transporters for sialic acid, which makes them crucial for host colonization by pathogenic bacteria. Extensive structural insights were already gained by crystallography and cryo-electron-microscopy, but the real-time mechanism of the import remains elusive. As the P-domain undergoes conformational changes upon substrate binding a nanobody could be created, which specifically binds to the substrate bound P-domain, but not to the empty P-domain. By conjugating donor and acceptor fluorophores to the nanobody and the P-domain, respectively, a substratedetecting sensor was created that exhibits high Förster resonance energy transfer (FRET) efficiency in presence of the substrate. This sensor was trapped in surface-immobilized large unilamellar vesicles (LUVs), which contained the reconstituted QM-domain in their membrane. With this assay, we aim at quantifying the import of sialic acid by HiSiaPQM into the vesicle lumen over time by single-molecule alternating laser excitation (ALEX) FRET using a TIRF microscope.

Annika M. Krüger (Clausius Institute, University of Bonn), Philipp Hendricks (Institute of Structural Biology, University of Bonn), Jiajun Li (Institute of Structural Biology, University of Bonn), Niels Schneberger (Institute of Structural Biology, University of Bonn), Jan Gerhartz (Institute of Structural Biology, University of Bonn), Martin F. Peter (Institute of Structural Biology, University of Bonn), Jan Peter Siebrasse (Clausius Institute, University of Bonn), Gregor Hagelueken (Institute of Structural Biology, University of Bonn), Ulrich Kubitscheck (Clausius Institute, University of Bonn)