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 <italic>Mycobacterium tuberculosis</italic> 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. [ABSTRACT FROM AUTHOR]