Dataset Information

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.

ABSTRACT: Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³J(C?C?) 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(C?C?) 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(?) 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.

SUBMITTER: Takeuchi K

PROVIDER: S-EPMC3072286 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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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.

Takeuchi Koh K Gal Maayan M Takahashi Hideo H Shimada Ichio I Wagner Gerhard G

Journal of biomolecular NMR 20101126 1

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 b ...[more]

PMID: 21110064

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Project description:One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called NASCA: (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), NASCA: extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that NASCA: assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by NASCA: have backbone RMSD 0.8-1.5 Å from the reference structures determined by traditional NMR approaches.

Dataset Information

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.

Publications

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.

Similar Datasets

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