Stereo-selective deuteration in aspartate, asparagine, lysine, and methionine amino acid residues using fumarate as a carbon source for E. coli in D2O

• Author(s) / Creator(s)

  • Danmaliki, Gaddafi I.
  • Liu, Philip B.
  • Hwang, Peter M.

• We propose an isotope labeling strategy for expressing proteins in E. coli using protonated natural abundance 12C carbon sources in D2O. The strategy eliminates dominant 1H-13C and 1H-1H dipolar relaxation mechanisms to produce long-lived magnetization for solution NMR spectroscopy. Isolated 1H magnetization can be transferred via through-space NOEs to slowly relaxing 1H-15N or 1H-13C methyl groups, providing the resolution needed for accurate structure determination of large protein systems by solution NMR. To fully characterize 1H/2H-isotope incorporation by NMR, we applied our labelling strategy to a model system, the WW domain from human Pin1 protein. Using glucose as sole carbon source in D2O, a very high level of protonation was observed in the aromatic side chains, as well as at the Hβ2 and Ηβ3 positions of serine and tryptophan, consistent with previous studies. With our specialized FROMP media (fumarate, rhamnose, oxalate, malonate, pyruvate), more aromatic ring positions were deuterated, and stereo-selective protonation of Hβ2 with deuteration at Ha and Hβ3 was achieved in Asp, Asn, Lys, and Met residues. In solution NMR, stereospecific chemical shift assignments for Ηβ are typically obtained in conjunction with χ1 dihedral angle determinations using 3-bond J-coupling (3JN-Hβ, 3JCO-Hβ, 3JHα-Hβ) experiments. However, due to motional averaging, the assumption of a pure rotameric state can yield incorrect χ1 dihedral angles with incorrect stereospecific assignments. In three residues, Lys28, Met30, and Asn44, the use of stereo-selective isotope labeling reversed the stereospecific chemical shift assignments and χ1 dihedral angles obtained in the traditional manner, demonstrating how stereo-selective isotope labeling can improve the accuracy of χ1 dihedral angles obtained by solution NMR.

• Date created

  2017-01-01

• Subjects / Keywords

  • Solution nuclear magnetic resonance (NMR) spectroscopy
  • Protein structural biology
  • Bacterial amino acid biosynthetic pathways
  • Protein side chain dynamics
  • Central metabolic pathways

• Type of Item

  Article (Draft / Submitted)

• DOI

  https://doi.org/10.7939/r3-dnxd-pe14

• License

  This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Biochemistry, copyright © 2017 American Chemical Society after peer review. To access the final edited and published work see https://doi.org/10.1021/acs.biochem.7b00991