[NMR paper] Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional

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Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR.

Related Articles Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR.

J Biomol NMR. 1999 Sep;15(1):1-14

Authors: Hong M

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of 13C and 15N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to 13C and 15N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the 13C and 15N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields Ni-C alpha i-1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C beta chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a 13C spin diffusion experiment, which exhibits 13C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive 13C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive 13C labeling approach.

PMID: 10549131 [PubMed - indexed for MEDLINE]



Source: PubMed