Year: 2026
Type: preprint
Abstract: 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 the use of NMR pulse schemes. Here, we describe a novel cell-free protein expression protocol that allows specific isotopic labelling of glycine residues, whilst all other amino acids are independently labelled. This technique enables us to prevent amino acid scrambling and cross-protonation from the solvent resulting in high labelling selectivity of glycine residues as attested by quantification of 15N amide scrambling and residual Ca protonation. We then highlight the potential applications of these samples in the field of biomolecular NMR. We produced a 1 H, 13 C glycine specifically labelled sample of a subunit of the heavy metal ATPase HMA8, in a completely perdeuterated 2 H background and demonstrate how the CH 2 -TROSY experiment recorded on such a sample improves the spectral quality of glycine residues.
Source: ChemRxiv
Institutions: Centre National de la Recherche Scientifique, Université Paris Sciences et Lettres, École Normale Supérieure - PSL, Sorbonne Université, Chimie Physique et Chimie du Vivant, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, European Molecular Biology Laboratory, Institut de Biologie Structurale, Integrated Structural Biology Grenoble, Université Grenoble Alpes
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7220463920