Journal of Biomolecular NMR

, Volume 49, Issue 1, pp 17–26

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

Authors

  • Koh Takeuchi
    • Department of Biochemistry and Molecular PharmacologyHarvard Medical School
    • Biomedicinal Information Research CenterNational Institute of Advanced Industrial Science and Technology
  • Maayan Gal
    • Department of Biochemistry and Molecular PharmacologyHarvard Medical School
  • Hideo Takahashi
    • Biomedicinal Information Research CenterNational Institute of Advanced Industrial Science and Technology
    • Graduate School of NanobioscienceYokohama City University
  • Ichio Shimada
    • Biomedicinal Information Research CenterNational Institute of Advanced Industrial Science and Technology
    • Department of Physical Chemistry, Graduate School of Pharmaceutical SciencesThe University of Tokyo
    • Department of Biochemistry and Molecular PharmacologyHarvard Medical School
Article

DOI: 10.1007/s10858-010-9456-2

Cite this article as:
Takeuchi, K., Gal, M., Takahashi, H. et al. J Biomol NMR (2011) 49: 17. doi:10.1007/s10858-010-9456-2

Abstract

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.

Keywords

Alternate 13C labelingTOCSYNuclear magnetic resonance (NMR)Sequential assignmentTriple resonanceThree dimensionalSupra sequential assignments

Copyright information

© Springer Science+Business Media B.V. 2010