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

Autor: Rosati M; CERM ─ Magnetic Resonance Center, Università degli Studi di Firenze, Sesto Fiorentino, Italy., Barbieri L; CERM ─ Magnetic Resonance Center, Università degli Studi di Firenze, Sesto Fiorentino, Italy.; Consorzio Interuniversitario Risonanze Magnetiche di Metallo Proteine ─ CIRMMP, Sesto Fiorentino, Italy., Hlavac M; MAG-LAB GmbH, Vienna, Austria., Kratzwald S; MAG-LAB GmbH, Vienna, Austria., Lichtenecker RJ; MAG-LAB GmbH, Vienna, Austria.; Institute of Organic Chemistry, University of Vienna, Vienna, Austria., Konrat R; MAG-LAB GmbH, Vienna, Austria.; Department of Structural and Computational Biology, Max Perutz Laboratories, University of Vienna, Vienna, Austria., Luchinat E; CERM ─ Magnetic Resonance Center, Università degli Studi di Firenze, Sesto Fiorentino, Italy. eluchinat@cerm.unifi.it.; Consorzio Interuniversitario Risonanze Magnetiche di Metallo Proteine ─ CIRMMP, Sesto Fiorentino, Italy. eluchinat@cerm.unifi.it.; Dipartimento di Chimica, Università degli Studi di Firenze, Sesto Fiorentino, Italy. eluchinat@cerm.unifi.it., Banci L; CERM ─ Magnetic Resonance Center, Università degli Studi di Firenze, Sesto Fiorentino, Italy. banci@cerm.unifi.it.; Consorzio Interuniversitario Risonanze Magnetiche di Metallo Proteine ─ CIRMMP, Sesto Fiorentino, Italy. banci@cerm.unifi.it.; Dipartimento di Chimica, Università degli Studi di Firenze, Sesto Fiorentino, Italy. banci@cerm.unifi.it.
Jazyk: angličtina
Zdroj: Journal of biomolecular NMR [J Biomol NMR] 2024 Aug 22. Date of Electronic Publication: 2024 Aug 22.
DOI: 10.1007/s10858-024-00447-6
Abstrakt: 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.
(© 2024. The Author(s).)
Databáze: MEDLINE
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