We report a general method for amino acid-type specific ¹⁷ O-labeling of recombinant proteins in Escherichia coli. In particular, we have prepared several [1-¹³ C,¹⁷ O]-labeled yeast ubiquitin (Ub) samples including Ub-[1-¹³ C,¹⁷ O]Gly, Ub-[1-¹³ C,¹⁷ O]Tyr, and Ub-[1-¹³ C,¹⁷ O]Phe using the auxotrophic E. coli strain DL39 GlyA λDE3 (aspC^- tyrB^- ilvE^- glyA^- λDE3). We have also produced Ub-[η-¹⁷ O]Tyr, in which the phenolic group of Tyr59 is ¹⁷ O-labeled. We show for the first time that ¹⁷ O NMR signals from protein terminal residues and side chains can be readily detected in aqueous solution. We also reported solid-state ¹⁷ O NMR spectra for Ub-[1-¹³ C,¹⁷ O]Tyr and Ub-[1-¹³ C,¹⁷ O]Phe obtained at an ultrahigh magnetic field, 35.2 T (1.5 GHz for ¹ H). This work represents a significant advance in the field of ¹⁷ O NMR studies of proteins.

• PMID: 33058374
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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Lin B, Hung I, Gan Z, Chien PH, Spencer HL, Smith SP, Wu G

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