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

2000-09-30T00:00:00Z (GMT) by Gabriel Cornilescu Ad Bax
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 HN 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 rCN = 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 HN, 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 1HN, the scatter is completely dominated by intrinsic variations in the CSA tensor at the different sites.