Measurement of multiple ψ torsion angles in uniformly 13C,15N-labeled α-spectrin SH3 domain using 3D 15N-13C-13C-15N MAS dipolar-chemical shift correlation spectroscopy

We demonstrate the simultaneous measurement of several backbone torsion angles psi in the uniformly C-13, N-15-labeled alpha-Spectrin SH3 domain using two different 3D N-15-C-13-C-13-N-15 dipolar-chemical shift magic-angle spinning (MAS) NMR experiments. The first NCCN experiment utilizes double quantum (DO) spectroscopy combined with the INADEQUATE type C-13-C-13 chemical shift correlation. The decay of the DO coherences formed between 13C(i)(') and C-13(alphai) spin pairs is determined by the correlated dipolar field due to N-15(i)-C-13(alphai) and 13C(i)(')-N-15(i+1) dipolar couplings and is particularly sensitive to variations of the torsion angle in the regime \psi\ > 140degrees. However, the ability of this experiment to constrain multiple psi-torsion angles is limited by the resolution of the C-13(alpha)-(CO)-C-13 correlation spectrum. This problem is partially addressed in the second approach described here, which is an NCOCA NCCN experiment. In this case the resolution is enhanced by the superior spectral dispersion of the N-15 resonances present in the N-15(i+1)-C-13(alphai) part of the NCOCA chemical shift correlation spectrum. For the case of the 62-residue alpha-spectrin SH3 domain, we determined 13 psi angle constraints with the INADEQUATE NCCN experiment and 22 psi constraints were measured in the NCOCA NCCN experiment.