title: Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles.
abstract: Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.
authors.affiliation: Laboratory of Molecular Biology and Biochemistry, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Sourabh
authors.initials: S
authors.lastname: Banerjee
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D032701:Biomimetics / Q000379:methods; D002417:Cattle; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000032:analysis; D012234:Rho Factor / Q000502:physiology; D012243:Rhodopsin / Q000493:pharmacokinetics* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish / Q000235:genetics
chemical_list: D016632:Apolipoprotein A-I; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D043562:Receptors, G-Protein-Coupled; D012234:Rho Factor; D012243:Rhodopsin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine