Site-Resolved Determination of Peptide Torsion Angle φ 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 φ in peptides. The technique is based on the measurement of the relative orientation of the N−H N and C α −H α bonds, which is manifested in the rotational sideband spectrum of the sum and difference of the two corresponding dipolar couplings. The method exploits 15 N− 13 C 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 15 N-labeled N -acetyl- d, l -valine, the experiment yielded φ = −135°, which agrees well with the X-ray crystal structure. Simulations indicate that the accuracy of the measured angle φ is within ±10° when the N−H N and C α −H α 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 15 N and to larger polypeptides that are uniformly and randomly labeled in both 15 N and 13 C. It allows φ angles in various residues to be measured simultaneously and resolved by the C α chemical shifts.