HNCA-TOCSY-CANH experiments with alternate (13)C- (12)C 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 ³J(CαCα) coupling. These pulse sequences, which resemble recently described (13)C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in (1)H(2)O, and use (1)H excitation and detection. These experiments require alternate (13)C-(12)C labeling together with perdeuteration, which allows utilizing the small ³J(CαCα) scalar coupling that is otherwise masked by the stronger (1)J(CC) couplings in uniformly (13)C 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-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the (15)N-(1)H 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.
Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Koh
K
Takeuchi
Maayan
M
Gal
Hideo
H
Takahashi
Ichio
I
Shimada
Gerhard
G
Wagner
D000595:Amino Acid Sequence; D002247:Carbon Isotopes / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009419:Nerve Tissue Proteins / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D002247:Carbon Isotopes; C029152:G-substrate; D009419:Nerve Tissue Proteins; D011522:Protons; D011993:Recombinant Fusion Proteins