O-17 Nmr Studies Of Yeast Ubiquitin In Aqueous Solution And In The Solid State

We report a general method for amino acid-type specific O-17-labeling of recombinant proteins in Escherichia coli. In particular, we have prepared several [1-C-13,O-17]-labeled yeast ubiquitin (Ub) samples including Ub-[1-C-13,O-17]Gly, Ub-[1-C-13,O-17]Tyr, and Ub-[1-C-13,O-17]Phe using the auxotrophic E. coli strain DL39 GlyA lambda DE3 (aspC(-) tyrB(-) ilvE(-) glyA(-) lambda DE3). We have also produced Ub-[eta-O-17]Tyr, in which the phenolic group of Tyr59 is O-17-labeled. We show for the first time that O-17 NMR signals from protein terminal residues and side chains can be readily detected in aqueous solution. We also reported solid-state O-17 NMR spectra for Ub-[1-C-13,O-17]Tyr and Ub-[1-C-13,O-17]Phe obtained at an ultrahigh magnetic field, 35.2 T (1.5 GHz for H-1). This work represents a significant advance in the field of O-17 NMR studies of proteins.