The Impact of Hydrogen Bonding on Amide 1H Chemical Shift Anisotropy Studied by Cross-Correlated Relaxation and Liquid Crystal NMR Spectroscopy

Abstract

Site-specific 1H chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide moieties in the B3 domain of protein G (GB3). Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the 1HN CSA tensor and either the 1H−15N, the 1H−13C′, or the 1H−13Cα dipolar interactions. Analyses with the assumption that the 1HN CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. 1HN CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N−H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of N-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the 1HN CSA. Using experimentally derived 1HN CSA tensors, the optimal relaxation interference effect needed for narrowest 1HN TROSY line widths is found at ∼1200 MHz.