Determination of solid-state NMR structures of proteins by means of three-dimensional 15N-13C-13C dipolar correlation spectroscopy and chemical shift analysis.

In this paper, a three-dimensional (3D) NMR-based approach for the determination of the fold of moderately sized proteins by solid-state magic-angle spinning (MAS) NMR is presented and applied to the alpha-spectrin SH3 domain. This methodology includes the measurement of multiple (13)C-(13)C distance restraints on biosynthetically site-directed (13)C-enriched samples, obtained by growing bacteria on [2-(13)C]glycerol and [1,3-(13)C]glycerol. 3D (15)N-(13)C-(13)C dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C(alpha)-C(alpha), CO-CO, C(alpha)-CO, and CO-C(alpha) type appear. Additional restraints for confining the structure were obtained from phi and psi backbone torsion angles of 29 residues derived from C(alpha), C(beta), CO, NH, and H(alpha) chemical shifts. Using both distance and angular restraints, a refined structure was calculated with a backbone root-mean-square deviation of 0.7 A with respect to the average structure.