Measurement of Proton, Nitrogen, and Carbonyl Chemical Shielding Anisotropies in a Protein Dissolved in a Dilute Liquid Crystalline Phase

The changes in a solute's chemical shifts between an isotropic and a liquid crystalline phase provide information on the magnitude and orientation of the chemical shielding tensors relative to the molecule's alignment frame. Such chemical shift changes have been measured for the polypeptide backbone C‘, N, and H^N resonances in the protein ubiquitin. Perdeuterated ubiquitin was dissolved in a medium containing a small volume fraction of phospholipid bicelles, which switches from an isotropic to a liquid crystalline phase at ca. 25 °C. The one-bond ¹H−¹⁵N dipolar couplings provide a reference to determine the protein alignment tensor, using a vibrationally corrected ¹H−¹⁵N bond length of 1.04 Å, corresponding to r_CN = 1.33 Å. Assuming all atoms of a given type have the same chemical shielding anisotropy (CSA) tensor, the average C‘ tensor values are σ₁₁ = −75 ppm, σ₂₂ = −12 ppm, and σ₃₃ = 87 ppm for ¹³C‘ with an angle between σ₁₁ and the C‘−N bond of 38°, and σ₃₃ orthogonal to the peptide plane. Similarly, for ¹⁵N, σ₁₁ = −108 ppm, σ₂₂ = 46 ppm, and σ₃₃ = 63 ppm, with an angle of 19° between the H−N vector and the σ₁₁ axis, and σ₂₂ orthogonal to the peptide plane. For H^N, the commonly used approximation of an axially symmetric shielding tensor is found to be invalid, and best fit tensor values are σ₁₁ = −6 ppm, σ₂₂ = 0 ppm, and σ₃₃ = 6 ppm, with the σ₁₁ axis orthogonal to the peptide plane and σ₃₃ roughly parallel to the H−N bond vector. Considerable differences in CSA are found when separately considering residues in helical and extended regions of the polypeptide chain. For ¹³C‘ and ¹⁵N, the scatter in the correlation between experimental chemical shift changes and those predicted on the basis of the structure and a uniform CSA tensor is dominated by uncertainty in the protein structure and by the fact that the uniform CSA assumption is not valid. Upper limits for the degree of this intrinsic variation in the CSA tensor are obtained from these correlations. For ¹H^N, the scatter is completely dominated by intrinsic variations in the CSA tensor at the different sites.