Determination of Peptide Backbone Torsion Angles Using Double-Quantum Dipolar Recoupling Solid-State NMR Spectroscopy

Several approaches for utilizing dipolar recoupling solid-state NMR (ssNMR) techniques to
determine local structure at high resolution in peptides and proteins have been developed. However, many
of these techniques measure only one torsion angle or are accurate for only certain classes of secondary
structure. Additionally, the efficiency with which these dipolar recoupling experiments suppress the
deleterious effects of chemical shift anisotropy (CSA) at high magnetic field strengths varies. Dipolar
recoupling with a windowless sequence (DRAWS) has proven to be an effective pulse sequence for exciting
double-quantum (DQ) coherences between adjacent carbonyl carbons along the peptide backbone. By
allowing this DQ coherence to evolve, it is possible to measure the relative orientations of the CSA tensors
and subsequently use this information to determine the Ramachandran torsion angles φ and ψ. Here, we
explore the accuracies of the assumptions made in interpreting DQ-DRAWS data and demonstrate their
fidelity in measuring torsion angles corresponding to a variety of secondary structures irrespective of
hydrogen-bonding patterns. It is shown how a simple choice of isotopic labels and experimental conditions
allows accurate measurement of backbone secondary structures without any prior knowledge. This approach
is considerably more sensitive for determining structure in helices and has comparable accuracy for β-sheet
and extended conformations relative to other methods. We also illustrate the ability of DQ-DRAWS to
distinguish between structures in heterogeneous samples.