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Towards cost-effective side-chain isotope labelling of proteins expressed in human cells.

Martina RosatiLetizia BarbieriMatus HlavacSarah KratzwaldRoman J LichteneckerRobert KonratEnrico LuchinatLucia Banci
Published in: Journal of biomolecular NMR (2024)
Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1 H, 13 C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.
Keyphrases
  • amino acid
  • gas chromatography
  • escherichia coli
  • stem cells
  • cell therapy
  • molecular dynamics
  • mesenchymal stem cells
  • bone marrow
  • room temperature
  • heat shock
  • tandem mass spectrometry
  • fluorescent probe
  • drug induced