Site-resolved determination of peptide torsion angle phi from the relative orientations of backbone N–H and C–H bonds by solid-state NMR

We describe a method for determining the torsion angle in peptides. The technique is based on the measurement of the relative orientation of the N-HN and CR-HR bonds, which is manifested in the rotational sideband spectrum of the sum and difference of the two corresponding dipolar couplings. The method exploits 15N-13C double-quantum and zero-quantum coherences, which evolve simultaneously under the N-H and C-H dipolar interactions. The magnitudes of these dipolar couplings scaled by the proton homonuclear decoupling sequence are directly extracted from control experiments that correlate the dipolar interactions with the isotropic chemical shifts. Applied to 15N-labeled N-acetyl-D,L-valine, the experiment yielded )-135°, which agrees well with the X-ray crystal structure. Simulations indicate that the accuracy of the measured angle is within (10 ° when the N-HN and CR-HR bonds are approximately antiparallel and (20 ° when they are roughly parallel. The technique is sufficiently sensitive to be applied to small peptides that are only labeled in 15N and to larger polypeptides that are uniformly and randomly labeled in both 15N and 13C. It allows angles in various residues to be measured simultaneously and resolved by the CR chemical shifts. 1