Expanding the Toolset of Biomolecular NMR with Efficient and Cost‐Effective ¹⁷O‐Labeling via Bacterial Expression

Oxygen plays a central role in biomolecular structures and functions, with ¹⁷O NMR emerging as a powerful tool for elucidating biomolecular properties. However, the low natural abundance of the NMR-active isotope, ¹⁷O (0.0373 %), presents a significant hurdle to its widespread application. Here, we introduce a rapid and cost-effective approach for amino acid-specific ¹⁷O-labeling of recombinant proteins. Using a common bacterial expression system and with a 30-minute rapid synthesis protocol of ¹⁷O-labeled amino acids via mechanochemical saponification, we have generated Leu- and Phe-specific ¹⁷O-labeled recombinant proteins derived from diverse organisms, including CrgA and FtsQ from Mycobacterium tuberculosis and E protein from SARS-CoV-2 virus, demonstrating the applicability of our technique for amino acids known to be isotopically labeled without scrambling. We have acquired magic-angle-spinning ¹⁷O NMR of these proteins to confirm the successful ¹⁷O labeling and illustrate the sensitivity of ¹⁷O NMR to the protein's local structural environments. Our work significantly broadens the accessibility of ¹⁷O-NMR, empowering researchers to delve deeper into protein biophysics and biochemistry. This approach opens new avenues for understanding cellular processes at the molecular level by providing an effective tool for investigating oxygen-related interactions and chemistry within biomolecules.