Stereoselective Deuteration in Aspartate, Asparagine, Lysine, and Methionine Amino Acid Residues Using Fumarate as a Carbon Source for Escherichia coli in D2O

Perdeuteration with selective 1H,13C-enrichment of methyl groups has enabled solution NMR studies of large (>30 kDa) protein systems. However, we propose that for all non-methyl positions, only magnetization originating from 1H–12C groups is sufficiently long-lived, and it can be transferred via through-space NOEs to slowly relaxing 1H–15N or 1H–13C methyl groups to achieve multidimensional solution NMR. We demonstrate stereoselective 1H,12C-labeling by adding relatively inexpensive unlabeled carbon sources to Escherichia coli growth media in D2O. Using our model system, a mutant WW domain from human Pin1, we compare deuteration patterns in 19 amino acids (all except cysteine). Protein grown using glucose as the sole carbon source had high levels of protonation in aromatic rings and the Hβ positions of serine and tryptophan. In contrast, using our FROMP media (fumarate, rhamnose, oxalate, malonate, pyruvate), stereoselective protonation of Hβ2 with deuteration at Hα and Hβ3 was achieved in Asp, Asn, Lys, ...