jp3049229_si_001.pdf (316.36 kB)

Determination of 15N Chemical Shift Anisotropy from a Membrane-Bound Protein by NMR Spectroscopy

Download (316.36 kB)
journal contribution
posted on 20.02.2016, 18:56 by Manoj Kumar Pandey, Subramanian Vivekanandan, Shivani Ahuja, Kumar Pichumani, Sang-Choul Im, Lucy Waskell, Ayyalusamy Ramamoorthy
Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the 15N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction, and the amino acid sequence, there is a need for accurately determined CSA tensors from proteins. In this study, we report the backbone amide-15N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit Cytochrome b5 (cytb5), determined using the 15N CSA/15N–1H dipolar transverse cross-correlation rates. The mean values of 15N CSA determined for residues in helical, sheet, and turn regions are −187.9, −166.0, and −161.1 ppm, respectively, with an overall average value of −171.7 ppm. While the average CSA value determined from this study is in good agreement with previous solution NMR experiments on small globular proteins, the CSA value determined for residues in helical conformation is slightly larger, which may be attributed to the paramagnetic effect from Fe­(III) of the heme unit in cytb5. However, like in previous solution NMR studies, the CSA values reported in this study are larger than the values measured from solid-state NMR experiments. We believe that the CSA parameters reported in this study will be useful in determining the structure, dynamics, and orientation of proteins, including membrane proteins, using NMR spectroscopy.