Backbone and side-chain 13C and 15N signal assignments of the alpha-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 Tesla.
The backbone and side-chain 13C and 15N signals of a solid 62-residue (u-13C,15N)-labelled protein containing the alpha-spectrin SH3 domain were assigned by two-dimensional (2D) magic angle spinning (MAS) 15N-13C and 13C-13C dipolar correlation spectroscopy at 17.6 T. The side-chain signal sets of the individual amino acids were identified by 2D 13C-13C proton-driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton-nitrogen and a nitrogen-carbon cross-polarisation step followed by a carbon-carbon homonuclear transfer unit. Interresidue correlations leading to sequence-specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7-61, while the signals of the N- and C-terminal residues 1-6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor-bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three-dimensional versions of the applied or analogous techniques.
Pauli
J
J
Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Baldus
M
M
van Rossum
B
B
de Groot
H
H
Oschkinat
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes; D011232:Chemical Precipitation; D002645:Chickens; D008280:Magnetics; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D013049:Spectrin / Q000737:chemistry*; D018909:src Homology Domains
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D013049:Spectrin