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

摘要

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³JCαCα coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY () 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 ³JCαCα 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 ¹³C^α 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 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.