title: Probing the transmembrane structure and topology of microsomal cytochrome-p450 by solid-state NMR on temperature-resistant bicelles.
abstract: Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochrome-P4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.
authors.affiliation: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Melissa
authors.initials: M
authors.lastname: Gildenberg
authors.forename: Shivani
authors.initials: S
authors.lastname: Ahuja
authors.forename: Sang-Choul
authors.initials: SC
authors.lastname: Im
authors.forename: Paige
authors.initials: P
authors.lastname: Pearcy
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
authors.forename: Ayyalusamy
authors.initials: A
authors.lastname: Ramamoorthy
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008862:Microsomes, Liver / Q000737:chemistry*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D008051:Lipid Bilayers; D003577:Cytochrome P-450 Enzyme System