Measurement of 15N chemical shift anisotropy in a protein dissolved in a dilute liquid crystalline medium with the application of magic angle sample spinning.
The chemical shifts of nuclei that have chemical shielding anisotropy, such as the 15N amide in a protein, show significant changes in their chemical shifts when the sample is altered from an isotropic state to an aligned state. Such orientation-dependent chemical shift changes provide information on the magnitudes and orientation of the chemical shielding tensors relative to the molecule's alignment frame. Because of the extremely high sensitivity of the chemical shifts to the sample conditions, the changes in chemical shifts induced by adding aligned bicelles do not arise only from the protein alignment but should also include the accumulated effects of environmental changes including protein-bicelle interactions. With the aim of determining accurate 15N chemical shielding tensor values for solution proteins, here we have used magic angle sample spinning (MAS) to observe discriminately the orientation-dependent changes in the 15N chemical shift. The application of MAS to an aligned bicelle solution removes the torque that aligns the bicelles against the magnetic field. Thus, the application of MAS to a protein in a bicelle solution eliminates only the molecular alignment effect, while keeping all other sample conditions the same. The observed chemical shift differences between experiments with and without MAS therefore provide accurate values of the orientation-dependent 15N chemical shifts. From the values for ubiquitin in a 7.5% (w/v) bicelle medium, we determined the 15N chemical shielding anisotropy (CSA) tensor. For this evaluation, we considered uncertainties in measuring the 1H-15N dipolar couplings and the 15N chemical shifts and also structural noise present in the reference X-ray structure, assuming a random distribution of each NH bond vector in a cone with 5 degrees deviation from the original orientation. Taking into account these types of noise, we determined the average 15N CSA tensor for the residues in ubiquitin as Delta sigma=-162.0+/-4.3 ppm, eta=0.18+/-0.02, and beta=18.6+/-0.5 degrees, assuming a 1H-15N bond length of 1.02 A. These tensor values are consistent with those obtained from solid-state NMR experiments.
Kurita
Jun-ichi
J
Center for New Materials, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, 923-1292 Ishikawa, Japan.
Shimahara
Hideto
H
Utsunomiya-Tate
Naoko
N
Tate
Shin-ichi
S
D000577:Amides / Q000737:chemistry; D016880:Anisotropy; D003460:Crystallization / Q000379:methods; D003461:Crystallography / Q000379:methods*; D006801:Humans; D008823:Micelles; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D011522:Protons; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013113:Spin Labels; D025801:Ubiquitin / Q000737:chemistry*
D000577:Amides; D008823:Micelles; D009587:Nitrogen Isotopes; D010743:Phospholipids; D011506:Proteins; D011522:Protons; D012996:Solutions; D012997:Solvents; D013113:Spin Labels; D025801:Ubiquitin