1. Isotopic labelling and quantification of bacterial membrane labelling

Dror Warschawski, Delphine Ravault, Laila Zaatouf, Hugo Legras-Hemonnot

In order to study bacterial membranes using solid-state NMR, we must first isotopically label these membranes, mainly with deuterium, and quantify this labelling according to the culture conditions. Over the years, we have developed and refined several labelling and lipidomics techniques: quantification of lipids, free fatty acids, esterified fatty acids, and labelled fatty acids, using 31P NMR in solution and gas chromatography coupled with mass spectrometry (GCMS). To this end, we obtained funding in 2022 to purchase a GCMS spectrometer. Collaboration: Isabelle Marcotte (UQAM, Montréal, Canada).

2. Metabolomics and lipidomics of aquatic organisms by 1H solution NMR

Dror Warschawski, Delphine Ravault

Solution NMR is gaining ground alongside mass spectrometry for mapping the metabolome of plants or animals, and its changes in response to environmental conditions, particularly in “untargeted” cases, i.e. when the metabolites being sought are not known in advance. This is the case for a large number of aquatic organisms, which are much less studied than terrestrial organisms, yet very important in the food chain, but also as sentinels of environmental disruption. With Isabelle Marcotte’s laboratory in Quebec, we are mainly studying the metabolome of mussels and the effects of pollution, but also noise. With Antoine Danon’s laboratory at Sorbonne University, we want to monitor changes in the metabolome of different microalgae mutants under the effect of different stresses. These experiments are complemented by monitoring variations in the lipidome using more traditional techniques: thin-layer chromatography (TLC) and gas and liquid chromatography coupled with mass spectrometry (GCMS and HPLC). Collaboration: Antoine Danon (Sorbonne Université, Paris), Isabelle Marcotte (UQAM, Montréal, Canada).

3. NMR experiments on specifically labelled proteins

Emeric Miclet, Jean-Jacques Lacapère, Delphine Ravault

Specific isotopic labelling of proteins has greatly advanced biomolecular NMR. Protein domains, individual residues or certain chemical groups with specific patterns of isotopes can be incorporated to optimize NMR pulse schemes. In collaboration with J. Boisbouvier and Lionel Imbert, we have developed novel cell-free protein expression protocols that allows specific isotopic labelling of glycine residues, whilst all other amino acids are independently labelled. Our approach enables us to prevent amino acid scrambling and cross-protonation from the solvent resulting in high labelling selectivity of glycine residues. In a second approach, we aim to introduce fluorine atoms to tryptophan residues using heterologous expression in E. coli. Real-time monitoring of the bacterial cultures allows us to fine-tune the expression protocols for better fluorine incorporation. We are developing applications of these samples in the field of biomolecular NMR, including the use of CH2-TROSY experiments.