HNCA-TOCSY-CANH experiments with alternate 13C-12C labeling: a set of 3D experiment w

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


Abstract Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak
3 \textJ\textCa\textCa 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 \textJ\textCa\textCa 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 \textCa of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions iâ??1, i + 1 and iâ??2. 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 iâ??2, iâ??1, 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.

• Content Type Journal Article
• DOI 10.1007/s10858-010-9456-2
• Authors
  • Koh Takeuchi, Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
  • Maayan Gal, Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
  • Hideo Takahashi, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, 135-0064 Japan
  • Ichio Shimada, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, 135-0064 Japan
  • Gerhard Wagner, Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR