Integrated Approach to Study Structure, Function and Dynamics of TRAP Transporters

• Applicant: Privatdozent Dr. Gregor Hagelueken
• Subject Area: Structural Biology ; Biophysics
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 277679584

The tripartite ATP-independent periplasmic transporters (TRAP) are an important and widespread class of transporters in bacteria and archaea. Typically TRAPs consist of two transmembrane domains and a soluble substrate binding protein (SBP). The individual members translocate a diverse array of substrates, including C4-dicarboxylates, amino acids or sialic acid. The latter molecule plays an important role in infection processes of pathogenic bacteria, such as Vibrio cholerae or Haemophilus influenzae. Together with the fact that TRAP transporters are absent in eukaryotes, this makes them an interesting target for drug discovery. However, in contrast to other transporter classes (e.g. ABC transporters or secondary active transporters), TRAPs are not as well studied, especially from a structural point of view. Therefore, this proposal aims to study the structure, function and dynamics of TRAP transporters by an integrated approach, consisting of PELDOR spectroscopy, X-ray crystallography and single molecule fluorescence microscopy.The transport mechanism of TRAPs will be investigated by PELDOR spectroscopy. The current working hypothesis for the transport cycle includes multiple conformational changes which are triggered by substrate translocation or interactions between transporter and the SBP. These processes will be analysed by PELDOR measurements on the spin labelled proteins. At the same time, the purified transporter will be crystallised and X-ray crystallography will be employed to gather high-resolution information. Finally, the spin labelling mutants will be reused for single particle fluorescence microscopy experiments. For this purpose, fluorescence labels will be attached to both transporter and SBP and the transporter will be reconstituted into giant unilamellar vesicles (GUVs). The interaction between transporter and SBP will then be analysed under different conditions by single particle tracking and co-localization experiments. The combined results from all three methods will deliver a very detailed picture of TRAP transporters.

• DFG Programme: Research Grants