Ein integrierter Ansatz zur Analyse von Struktur, Funktion und Dynamik von TRAP Transportern
Biophysik
Zusammenfassung der Projektergebnisse
All living cells are encapsulated by at least one lipid membrane. While this barrier is absolutely necessary for the cell’s survival, it also requires specialized proteins that are able to transport nutrients, ions and small molecules into- and out of the cell. The tripartite ATP-independent periplasmic transporters (TRAP) transporters are one of the many different classes of such transporters. They are utilized by some pathogenic bacteria to “harvest” a small sugar molecule, sialic acid, from the host organism. The bacteria incorporate the sialic acid into their cell wall, effectively hiding themselves from the immune system of the host. TRAP transporters are essential for the pathogen but not present in humans and are therefore an interesting target for the development of new antibiotics. The structure and function of TRAP transporters is poorly understood and we therefore aimed to investigate these macromolecules with an integrated approach of X-ray crystallography, PELDOR spectroscopy and single molecule fluorescence microscopy. We successfully studied the solution structure and dynamics of the substrate binding domain of TRAP transporters with pulsed EPR- and FRET spectroscopy. The results are important for the understanding of the transport mechanism of TRAP transporters. Surprisingly, we found that one of our crystal structures contained a short peptide bound to the substrate binding cleft of the P-domain. We found that the affinity of the peptide can be increased by changing its amino acid sequence. If we are able to further increase the affinity in future experiments, this finding might lead to a peptide-based inhibitor of TRAP transporters. Although the structure of a TRAP transporter is still not solved, we have made significant steps into this direction. The nanodiscs reconstituted samples were of excellent quality and we already performed very promising cryo-EM experiments to investigate the structure. Although cryo-EM has very high potential to solve this structure, the small size of the TRAP transporter is still a drawback. It is simply difficult to identify the orientation of the transporter in the EM images. At the end of the funding we have therefore initiated a collaboration with the nanobody core facility of the University of Bonn with the aim to create specific nanobodies against the TRAP transporter that can be used as markers in 3D reconstruction and as crystal contact enhancers for X-ray crystallography.
Projektbezogene Publikationen (Auswahl)
- (2017) “PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution”. Biophysical Journal, 112, 109–20
Glaenzer J, Peter MF, Thomas GH, Hagelueken G
(Siehe online unter https://doi.org/10.1016/j.bpj.2016.12.010) - (2018) “Studying structure and function of membrane proteins with PELDOR/DEER spectroscopy - A crystallographers' perspective”. Methods, 147, 163-175
Glaenzer J, Peter MF, Hagelueken G
(Siehe online unter https://doi.org/10.1016/j.ymeth.2018.03.002) - Untersuchung von Struktur, Funktion und Dynamik von Makromolekülen mittels Integrativer Strukturbiologie, 2018
Gregor Hagelueken
