Determination of polypeptide backbone dihedral angles in solid state NMR by double quantum 13C chemical shift anisotropy measurements

A solid state NMR technique for the determination of peptide backbone conformations at specific sites in unoriented samples under magic angle spinning (MAS) is described and demonstrated on a doubly labeled polycrystalline sample of the tripeptide AlaClyGly and a sextuply labeled lyophilized sample of the 17-residue peptide MB(i + 4)EK. The technique is applicable to peptides and proteins that are labeled with C-13 at two (or more) consecutive backbone carbonyl sites. Double quantum (DQ) coherences are excited with a radiofrequency-driven recoupling sequence and evolve during a constant-time t(1) period at the sum of the two anisotropic chemical shifts. The relative orientation of the two chemical shift anisotropy (CSA) tensors, which depends on the phi and psi backbone dihedral angles, determines the t(1)-dependence of spinning sideband intensities in the DQ-filtered C-13 MAS spectrum. Experiments and simulations show that both dihedral angles can be extracted from a single data set. This technique, called DQCSA spectroscopy, may be especially useful when analyzing the backbone conformation of a polypeptide at a particular doubly labeled site in the presence of additional labeled carbons along the sequence.