Journal of Biomolecular NMR

, Volume 49, Issue 1, pp 17–26 | Cite as

HNCA-TOCSY-CANH experiments with alternate 13C-12C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

  • Koh Takeuchi
  • Maayan Gal
  • Hideo Takahashi
  • Ichio Shimada
  • Gerhard Wagner


Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak \( ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} \) coupling. These pulse sequences, which resemble recently described 13C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in 1H2O, and use 1H excitation and detection. These experiments require alternate 13C-12C labeling together with perdeuteration, which allows utilizing the small \( ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} \) scalar coupling that is otherwise masked by the stronger 1JCC couplings in uniformly 13C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the \( ^{ 1 3} {\text{C}}^{\alpha } \) of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i1, i + 1 and i2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the 15N-1H spin pair of residue i to adjacent amide protons and nitrogens at positions i2, i1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.


Alternate 13C labeling TOCSY Nuclear magnetic resonance (NMR) Sequential assignment Triple resonance Three dimensional Supra sequential assignments 



This work was supported by the NIH (grants AI37581, GM47467 and EB 002026). M.G would like to thank the Human Frontier science Program (HFSP) for a postdoctoral fellowship.


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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Koh Takeuchi
    • 1
    • 2
  • Maayan Gal
    • 1
  • Hideo Takahashi
    • 2
    • 3
  • Ichio Shimada
    • 2
    • 4
  • Gerhard Wagner
    • 1
  1. 1.Department of Biochemistry and Molecular PharmacologyHarvard Medical SchoolBostonUSA
  2. 2.Biomedicinal Information Research CenterNational Institute of Advanced Industrial Science and TechnologyTokyoJapan
  3. 3.Graduate School of NanobioscienceYokohama City UniversityKanagawaJapan
  4. 4.Department of Physical Chemistry, Graduate School of Pharmaceutical SciencesThe University of TokyoTokyoJapan

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