Abstract

Secure, competitive, and sustainable energy production is a major challenge facing human societies. Biomimetic solutionssuch as the development of new biofuel cells are hampered by our thus far incomplete understanding of proton transferreactions. The same holds for health threats to humanity: Curing diseases like cancer, obesity, chronic gastritis, gastric andduodenal ulcers, requires to pharmacologically interfere - in their molecular details - with yet unresolved proton transferreactions.Here we aim at clarifying the molecular reaction mechanism in the confines of interfacial water layers and proteinaceouscavities with emphasis on arrangement and mobility of proton relay moieties. Achieving this requires an interdisciplinary,multi-level approach comprising cutting edge technologies like second harmonic imaging, single molecule and time resolvedfluorescence microscopy and spectroscopy, advanced calculations of proton transfer, bioengineering of membrane channeland transporter containing systems, synthetic design of biomimetic proton channels, solving protein structures and rational drug design.PROTON will train 15 PhD students, who will acquire a solid state-of-the-art multidisciplinary scientific training in all kinds ofproton migration/reaction systems, covering from basic science to industrial applications, thus preparing them to generatenew scientific knowledge of the highest impact. In addition, practical training on transferable skills will increase theiremployability and qualify them for responsible positions in private and public sectors. Cross-disciplinary strategies and closecollaboration with industry will enable them to resolve the molecular details of proton driven processes in all kinds of settings- enabling the improvement of biomimetic applications – up to fuel cells - and to identify lead substances which may serve topharmacologically interfere with proton transport through membrane channels and transporters.

ESR7

Mechanism of the proton transport activation in UCPs (WP1, WP2)

Objectives: The molecular mechanisms of proton recruitment from the bulk/surface and protein-mediated proton transport through the mitochondrial membrane are still not understood. The presence of long-chain fatty acids (FA) is a conditio sine qua non for the activation of H+ transport. The objectives of this project are: (1) Identify the H+ transport pathways in uncoupling proteins 1 (UCP1-UCP3) based on the analysis of amino acid homology, crystallographic structure of ANT, modelling efforts as well as MD simulations (cooperation with WG Hub); (2) Site-directed mutagenesis of acidic surface residues that serve as proton antennae, i.e. collect protons from the surface into the channel. Compare H+ transport rates based on measurements of proton/total conductance of membranes reconstituted with recombinant UCPs.

Expected Results: (i) The impact of mutated amino acids on membrane proton conductance is evaluated; (ii) Proton pathways are characterized for UCP1-UCP3; (iii) MD model of UCP is developed;

Planned secondment(s): USAAR (Jochen Hub), M18 (6 months): MD model development, integration of experimental data; EVCYT (Johannes Grillari), M36 (3 months): Production of mitochondrial recombinant proteins (incl. UCPs) in human cell lines.

Publications as an introduction to the project

1. Kreiter, J., Beitz, E., Pohl, E.E.@ (2020). A Fluorescence‐Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes. Biomolecules: 10(5):E685, doi: 10.3390/biom10050685
2. Kreiter, J., Rupprecht, A., Zimmermann, L., Moschinger, M., Rokitskaya, T. I., Antonenko, Y. N., Gille, L., Fedorova, M., Pohl, E. E.@ (2019). Molecular mechanisms responsible for the dual pharmacological effect of genipin on mitochondrial proteins. Biophys. J. 117, 10, 1845‐1857. doi: 10.1016/j.bpj.2019.10.021.
3. Pohl, EE., Rupprecht, A., Macher, G., Hilse, KE (2019) Important Trends in UCP3 Investigation. Front Physiol. 2019; 10:470
4. Macher, G., Koehler, M., Rupprecht, A., Kreiter, J., Hinterdorfer, P., Pohl, E.E. (2018) Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate. Biochim Biophys Acta 1860, 664–672
5. Jovanovic, O., Pashkovskaya, A.A., Annibal, A., Vazdar, M., Burchardt, N., Sansone, A., Gille, L., Fedorova, M., Ferreri, C., Pohl, E.E. (2015) The molecular mechanism behind reactive aldehyde action on transmembrane translocations of proton and potassium ions. Free Radical Biology and Medicine
6. Rupprecht et al. (2010) Role of the transmembrane potential in the membrane proton leak. Biophys. J. 98, 1503-1511

Publications as Results from the project

Zuna 2021

vanBergen_Zuna_2024

Link JKU

Link VetDoc