Stereoselective Deuteration in Aspartate, Asparagine, Lysine, and Methionine Amino Acid Residues Using Fumarate as a Carbon Source for Escherichia coli in D 2 O.

Perdeuteration with selective1 H,13 C-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 from1 H-12 C groups is sufficiently long-lived, and it can be transferred via through-space NOEs to slowly relaxing1 H-15 N or1 H-13 C methyl groups to achieve multidimensional solution NMR. We demonstrate stereoselective1 H,12 C-labeling by adding relatively inexpensive unlabeled carbon sources to Escherichia coli growth media in D2 O. 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, and Met residues. In solution NMR, stereospecific chemical shift assignments for Hβ are typically obtained in conjunction with χ1 dihedral angle determinations using 3-bond J-coupling (3 JN-Hβ ,3 JCO-Hβ ,3 JHα-Hβ ) experiments. However, due to motional averaging, the assumption of a pure rotameric state can yield incorrect χ1 dihedral angles with incorrect stereospecific assignments. This was the case for three residues in the Pin1 WW domain (Lys28, Met30, and Asn44). Thus, stereoselective1 H,12 C-labeling will be useful not only for NMR studies of large protein systems, but also for determining side chain rotamers and dynamics in any protein system.