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 phi in peptides . The technique is based on the measurement of the relative orientatio n of the N-H-N and C-alpha-H-alpha bonds, which is manifested in the r otational sideband spectrum of the sum and difference of the two corre sponding dipolar couplings. The method exploits N-15-C-13 double-quant um and zero-quantum coherences, which evolve simultaneously under the N-H and C-H dipolar interactions. The magnitudes of these dipolar coup lings scaled by the proton homonuclear decoupling sequence are directl y extracted from control experiments that correlate the dipolar intera ctions with the isotropic chemical shifts. Applied to N-15-labeled N-a cetyl-D,L-valine, the experiment yielded phi = -135 degrees, which agr ees well with the X-ray crystal structure. Simulations indicate that t he accuracy of the measured angle phi is within +/-10 degrees when the N-H-N and C-alpha-H-alpha bonds are approximately antiparallel and +/ -20 degrees when they are roughly parallel. The technique is sufficien tly sensitive to be applied to small peptides that are only labeled in N-15 and to larger polypeptides that are uniformly and randomly label ed in both N-15 and C-13. It allows phi angles in various residues to be measured simultaneously and resolved by the C-alpha chemical shifts .