American Chemical Society
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Site-Specific Backbone Amide 15N Chemical Shift Anisotropy Tensors in a Small Protein from Liquid Crystal and Cross-Correlated Relaxation Measurements

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journal contribution
posted on 2010-03-31, 00:00 authored by Lishan Yao, Alexander Grishaev, Gabriel Cornilescu, Ad Bax
Site-specific 15N chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide 15N nuclei in the B3 domain of protein G (GB3) from residual chemical shift anisotropy (RCSA) measured in six different mutants that retain the native structure but align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension. This information is complemented by measurement of cross-correlated relaxation rates between the 15N CSA tensor and either the 15N−1H or 15N−13C′ dipolar interaction. In agreement with recent solid state NMR measurements, the 15N CSA tensors exhibit only a moderate degree of variation from averaged values, but have larger magnitudes in α-helical (−173 ± 7 ppm) than in β-sheet (−162 ± 6 ppm) residues, a finding also confirmed by quantum computations. The orientations of the least shielded tensor component cluster tightly around an in-peptide-plane vector that makes an angle of 19.6 ± 2.5° with the N−H bond, with the asymmetry of the 15N CSA tensor being slightly smaller in α-helix (η = 0.23 ± 0.17) than in β-sheet (η = 0.31 ± 0.11). The residue-specific 15N CSA values are validated by improved agreement between computed and experimental 15N R relaxation rates measured for 15N-{2H} sites in GB3, which are dominated by the CSA mechanism. Use of residue-specific 15N CSA values also results in more uniform generalized order parameters, S2, and predicts considerable residue-by-residue variations in the magnetic field strengths where TROSY line narrowing is most effective.