title: Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.
abstract: 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 (15)N 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-(15)N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit Cytochrome b(5) (cytb(5)), determined using the (15)N CSA/(15)N-(1)H dipolar transverse cross-correlation rates. The mean values of (15)N 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 cytb(5). 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.
authors.affiliation: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Pandey
authors.forename: Subramanian
authors.initials: S
authors.lastname: Vivekanandan
authors.forename: Shivani
authors.initials: S
authors.lastname: Ahuja
authors.forename: Kumar
authors.initials: K
authors.lastname: Pichumani
authors.forename: Sang-Choul
authors.initials: SC
authors.lastname: Im
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
authors.forename: Ayyalusamy
authors.initials: A
authors.lastname: Ramamoorthy
mesh_terms: D000818:Animals; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011817:Rabbits
chemical_list: D005290:Ferric Compounds; D009587:Nitrogen Isotopes; D006418:Heme; D015786:Cytochromes b5