Backbone and side-chain 13C and 15N signal assignments of the α-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 tesla

The backbone and side-chain C-13 and N-15 signals of a solid 62-residue (u-C-13,N-15)-labelled protein containing the alpha -spectrin SH3 domain were assigned by two-dimensional (2D) magic angle spinning (MAS) N-15- C-13 and C-13- C-13 dipolar correlation spectroscopy at 17.6 T. The side-chain signal sets of the individual amino acids were identified by 2D C-13-C-13 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.