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

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak (3)J(C alpha C alpha) coupling. These pulse sequences, which resemble recently described C-13 detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in (H2O)-H-1, and use H-1 excitation and detection. These experiments require alternate C-13-C-12 labeling together with perdeuteration, which allows utilizing the small (3)J(C alpha C alpha) scalar coupling that is otherwise masked by the stronger (1)J(CC) couplings in uniformly C-13 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 C-13(alpha) 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-TOC-SY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the N-15-H-1 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.