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Solution NMR spectroscopy of large protein systems is hampered by rapid signal decay, so most multidimensional studies focus on long-lived 1 H- 13 C magnetization in methyl groups and/or backbone amide 1 H- 15 N magnetization in an otherwise perdeuterated environment. Herein we demonstrate that it is possible to biosynthetically incorporate additional 1 H- 12 C groups that possess long-lived magnetization using cost-effective partially deuterated or unlabeled amino acid precursors added to Escherichia coli growth media. This approach is applied to the outer membrane enzyme PagP in membrane-mimetic dodecylphosphocholine micelles. We were able to obtain chemical shift assignments for a majority of side chain 1 H positions in PagP using nuclear Overhauser enhancements (NOEs) to connect them to previously assigned backbone 1 H- 15 N groups and newly assigned 1 H- 13 C methyl groups. Side chain methyl-to-aromatic NOEs were particularly important for confirming that the amphipathic α-helix of PagP packs against its eight-stranded β-barrel, as indicated by previous X-ray crystal structures. Interestingly, aromatic NOEs suggest that some aromatic residues in PagP that are buried in the membrane bilayer are highly mobile in the micellar environment, like Phe138 and Phe159. In contrast, Tyr87 in the middle of the bilayer is quite rigid, held in place by a hydrogen bonded network extending to the surface that resembles a classic catalytic triad: Tyr87-His67-Asp61. This hydrogen bonded arrangement of residues is not known to have any catalytic activity, but we postulate that its role is to immobilize Tyr87 to facilitate packing of the amphipathic α-helix against the β-barrel., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peter Hwang reports financial support was provided by NSERC., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Perdeuteration with selective 1 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 from 1 H- 12 C groups is sufficiently long-lived, and it can be transferred via through-space NOEs to slowly relaxing 1 H- 15 N or 1 H- 13 C methyl groups to achieve multidimensional solution NMR. We demonstrate stereoselective 1 H, 12 C-labeling by adding relatively inexpensive unlabeled carbon sources to Escherichia coli growth media in D 2 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 H β positions of serine and tryptophan. In contrast, using our FROMP media (fumarate, rhamnose, oxalate, malonate, pyruvate), stereoselective protonation of H β2 with deuteration at H α and H β3 was achieved in Asp, Asn, Lys, and Met residues. In solution NMR, stereospecific chemical shift assignments for H β are typically obtained in conjunction with χ 1 dihedral angle determinations using 3-bond J-coupling ( 3 J N-Hβ , 3 J CO-Hβ , 3 J Hα-Hβ ) experiments. However, due to motional averaging, the assumption of a pure rotameric state can yield incorrect χ 1 dihedral angles with incorrect stereospecific assignments. This was the case for three residues in the Pin1 WW domain (Lys28, Met30, and Asn44). Thus, stereoselective 1 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.