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.

A Magic‐Angle‐Spinning NMR Spectroscopy Method for the Site‐Specific Measurement of Proton Chemical‐Shift Anisotropy in Biological and Organic Solids

“…However, in several instances multiple cross-correlation relaxation rates were measured leading to a highly defined set of principal components and directions. [9, 11, 59] …”

“…As far as we know, cross-correlation relaxation experiments led to about fifteen publications reporting quantitative proton CSA results. [5–6, 8–9, 11, 55, 59–67] Of particular interest are the measurements made since 1997 of multiple sites of amide protons in proteins [5–6, 9, 11, 67] and of hydrogen bonds in RNA kissing complexes. [59] None of the solid-state methods mentioned above has achieved multiple site analysis to this extent.…”

“…To obtain values of more than one CSA tensor component measurements of multiple LC alignments are needed. [11] Various experimental approaches, using temperature dependence or combinations of nematic solvents or smectic LCs are applied to determine the RCSA and the order parameters from which Δσ can readily be extracted. However, contributions arising from intermolecular interactions with the solvent molecules, as well as thermal motions, can have a substantial effect on the results.…”

“…[7, 11] In 2000, Cornilescu and Bax dissolved perdeuterated ubiquitin in a medium containing phospholipid bicelles, where 1 H- 15 N dipolar couplings of reprotonated amide groups provided a reference to determine the protein alignment tensor. Assuming that all atoms of a given type had the same CSA tensor, the average principal CSA components of α-helix and β-sheet amide protons, as well as the tensor orientation for each residue could be determined.…”

Layer selection effect on solid state 13C and 15N chemical shifts calculation using ONIOM approach

“…Combination of solid state nuclear magnetic resonance and quantum calculations has developed a strong tool applied in different field investigations, such as chemistry, biochemistry, and pharmacology [1][2][3][4][5][6][7][8][9][10][11][12][13]. Two important elements having accurate computation, especially in solid state, are molecular geometry and calculation method.…”