Cost-effective selective deuteration of aromatic amino acid residues produces long-lived solution H NMR magnetization in proteins.

Solution NMR studies of large proteins are hampered by rapid signal decay due to short-range dipolar H-H and H-C interactions. These are attenuated by rapid rotation in methyl groups and by deuteration (H), so selective H,C-isotope labelling of methyl groups in otherwise perdeuterated proteins, combined with methyl transverse relaxation optimized spectroscopy (methyl-TROSY), is now standard for solution NMR of large protein systems > 25 kDa. For non-methyl positions, long-lived magnetization can be introduced as isolated H-C groups. We have developed a cost-effective chemical synthesis for producing selectively deuterated phenylpyruvate and hydroxyphenylpyruvate. Feeding these amino acid precursors to E. coli in DO, along with selectively deuterated anthranilate and unlabeled histidine, results in isolated and long-lived H magnetization in the aromatic rings of Phe (HD, HZ), Tyr (HD), Trp (HH2, HE3) and His (HD2 and HE1). We are additionally able to obtain stereoselective deuteration of Asp, Asn, and Lys amino acid residues using unlabeled glucose and fumarate as carbon sources and oxalate and malonate as metabolic inhibitors. Combining these approaches produces isolated H-C groups in Phe, Tyr, Trp, His, Asp, Asn, and Lys in a perdeuterated background, which is compatible with standard H-C labeling of methyl groups in Ala, Ile, Leu, Val, Thr, Met. We show that isotope labeling of Ala is improved using the transaminase inhibitor L-cycloserine, and labeling of Thr is improved through addition of Cys and Met, which are known inhibitors of homoserine dehydrogenase. We demonstrate the creation of long-lived H NMR signals in most amino acid residues using our model system, the WW domain of human Pin1, as well as the bacterial outer membrane protein PagP.