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 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.
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Acknowledgments
This work was supported by the NIH (grants AI37581, GM47467 and EB 002026). M.G would like to thank the Human Frontier science Program (HFSP) for a postdoctoral fellowship.
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Takeuchi, K., Gal, M., Takahashi, H. et al. HNCA-TOCSY-CANH experiments with alternate 13C-12C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment. J Biomol NMR 49, 17–26 (2011). https://doi.org/10.1007/s10858-010-9456-2
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DOI: https://doi.org/10.1007/s10858-010-9456-2