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

Research output: Contribution to journalArticlepeer-review

Abstract

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak 3JCα Cα 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 3JCα Cαscalar 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 13Calpha of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i-1, i + 1 and i-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 i-2, i-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.

Original languageEnglish
Pages (from-to)17-26
Number of pages10
JournalJournal of Biomolecular NMR
Volume49
Issue number1
DOIs
StatePublished - Jan 2011
Externally publishedYes

Keywords

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

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