Journal of Biomolecular NMR

, Volume 65, Issue 1, pp 15–27 | Cite as

Facilitating unambiguous NMR assignments and enabling higher probe density through selective labeling of all methyl containing amino acids

  • Andrew Proudfoot
  • Andreas O. Frank
  • Fiorella Ruggiu
  • Mulugeta Mamo
  • Andreas Lingel


The deuteration of proteins and selective labeling of side chain methyl groups has greatly enhanced the molecular weight range of proteins and protein complexes which can be studied using solution NMR spectroscopy. Protocols for the selective labeling of all six methyl group containing amino acids individually are available, however to date, only a maximum of five amino acids have been labeled simultaneously. Here, we describe a new methodology for the simultaneous, selective labeling of all six methyl containing amino acids using the 115 kDa homohexameric enzyme CoaD from E. coli as a model system. The utility of the labeling protocol is demonstrated by efficiently and unambiguously assigning all methyl groups in the enzymatic active site using a single 4D 13C-resolved HMQC–NOESY–HMQC experiment, in conjunction with a crystal structure. Furthermore, the six fold labeled protein was employed to characterize the interaction between the substrate analogue (R)-pantetheine and CoaD by chemical shift perturbations, demonstrating the benefit of the increased probe density.


Selective labeling 4D NOESY CoaD Methyl groups Assignments NMR 



The authors would like to thank Dr. Clemens Anklin and Dr. Eric Johnson at the Bruker BioSpin Corporation for their assistance with the implementation of the 4D methyl–methyl 13C-resolved HMQC–NOESY–HMQC experiment, Dr. Lili Xie and Kyoko Uehara for their help in designing the purification protocol for CoaD, and Dr. Dirksen Bussiere for his assistance with refinement of the CoaD structure. All images produced of the CoaD structure were done so using Pymol (Schrödinger, LLC 2015).

Compliance with ethical standards

Conflict of interest

AP, AOF, FR, MM and AL are employees of Novartis.

Supplementary material

10858_2016_32_MOESM1_ESM.pdf (3.6 mb)
Supplementary material 1 (PDF 3658 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Andrew Proudfoot
    • 1
  • Andreas O. Frank
    • 1
  • Fiorella Ruggiu
    • 1
  • Mulugeta Mamo
    • 1
  • Andreas Lingel
    • 1
  1. 1.Novartis Institutes for BioMedical ResearchEmeryvilleUSA

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