The Saupe matrix describing protein alignment in a liquid crystalline medium contains five independent elements, enabling the generation of up to five linearly independent alignment conditions. Measurement of internuclear residual dipolar couplings (RDCs) by NMR spectroscopy under these conditions, orthogonal in five-dimensional alignment space, provides access to the amplitude, asymmetry, and direction of motions of the internuclear vector. It is demonstrated for the small protein domain GB3 (56 residues) that suitably orthogonal alignment conditions can be generated in a single liquid crystalline medium of Pf1 phage, by generating a series of conservative mutants that have negligible impact on the time-averaged backbone structure of the domain. Mutations involve changes in the charge of several solvent-exposed sidechains, as well as extension of the protein by either an N- or C-terminal His-tag peptide, commonly used for protein purification. These protein mutants map out the five-dimensional alignment space, providing unique insights into the structure and dynamics, and providing access to anisotropic parameters such as the 13C, 15N and 1H chemical shielding tensors. 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 alpha-helical (-173 7 ppm) than in beta-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 19.62.5 with the N-H bond, with the asymmetry of the 15N CSA tensor being slightly smaller in alpha-helix (eta=0.230.17) than in beta-sheet (eta=0.310.11). The residue-specific 15N CSA values are validated by improved agreement between computed and experimental 15N R1rho 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. The N-H bond length in backbone peptide groups of the protein GB3 has also been studied by liquid crystal NMR, using five of the above mentioned structurally conserved mutants of this protein. In the absence of additional information, the impact of dynamic fluctuations of the N-H vector orientation on the 15N-1H dipolar interaction cannot be separated from a change in N-H bond length. However, a change in N-H bond length directly impacts the orientation of C'-H vectors in the peptide group, and simultaneous analysis of 13C'-HN and 15N-HN residual dipolar couplings, measured under five different alignment orientations, permitted modelfree determination of the average equilibrium N-H bond length in GB3, yielding rNHeq = 1.008 0.006 . Anharmonicity of the bond stretching resulted in a slightly longer time-averaged bond length
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