17 O NMR Studies of Yeast Ubiquitin in Aqueous Solution and in the Solid State.

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.
(© 2020 Wiley-VCH GmbH.)