Combining Selective Dnp-Nmr And Site-Specific Labeling To Study Active Sites Of Large Biomolecular...

The PhD student will be involved in a highly interdisciplinary research project at the frontiers of Chemistry, Biology and Physics. The aim is to continue the development of selective hyperpolarization NMR approaches for their application to large biomolecular complexes.

Dynamic Nuclear Polarization (DNP) has emerged as a promising hyperpolarization technique for solid-state NMR. Nevertheless, for biomolecules, the spectral resolution is often strongly impacted by the use of cryogenic temperature. The MEM DNP-NMR team recently introduced a new methodology, called Selective DNP (SelDNP), which allows recovering high- resolution DNP spectra for specific parts of the protein 1. This targeted DNP-NMR approach 2 combines paramagnetic tagging (radical-functionalized ligand, spin label, paramagnetic metal ion, etc.) and differential spectroscopy. It was demonstrated to reveal the carbohydrate-binding site of the protein LecA (12 kDa). Thanks to the expertise of NMR-Bio in advanced specific isotopic spin labeling (e.g. 2H, 15N, 13C, 13CH3, etc.) 3 and of our collaborators from the University of Island in radical chemistry (Prof. Snorri Siggurdsson's group), we propose to extend this emerging and promising SelDNP methodology to large biomolecules. As a proof of concept, we will first focus on a chondroitin sulfate 4-endosulfatase (ENDO-4sulfatase, 58 kDa) expressed by Prevotella oris, a bacteria of the human gut microbiota. Sulfatases are important and yet poorly understood enzymes, which play a key role in regulating the sulfation states of many important substrates, including complex cell-surface polysaccharides. Beyond the mentioned consortium, the project will also involve collaboration with R. Vivès (IBS) specialized in sulfatases. Ultimately, the development of the SelDNP methodology should provide new solutions for investigating protein/glycan interactions and glycoenzymes.

1 I. Marin-Montesinos, D. Goyard, E. Gillon, O. Renaudet, A. Imberty, S. Hediger and G. De Paëpe, Chem. Sci., 2019, 10, 3366. 2 D. Gauto, O. Dakhlaoui, I. Marin-Montesinos, S. Hediger and G.De Paëpe, Chem. Sci., 2021,12, 6223. 3 R. Kerfah, MJ. PLevin, R. Sounier, P. Gans and J.Boisbouvier, Current Opinion in structural Biology, 2015, 32:113-122.

The PhD student will be involved in methyl specifically labelled proteins production and purification (under the supervision of NMR-Bio) and their investigation with solid-state NMR and DNP within the MEM laboratory. Notably he/she will be trained to become autonomous on the DNP-NMR spectrometer, including specific DNP sample preparation, recording and processing of DNP-NMR experiments, as well as their assignment and interpretation. He/she will be actively contributing to the design of the strategy to combine specific labelling and selective DNP, such to obtain new structural insights on the sulfatase.

Funding category: Contrat doctoral

PHD Country: France

Safety: work in the presence of magnetic fields and cryogenic fluids; standard chemical and biological risks.