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

Abstract

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.

Keywords: 17O NMR; auxotrophic strains; isotope labeling; recombinant protein; ubiquitin.