PUBLICATION
Rapid Incorporation of Functional Rhodopsin into Nanoscale Apolipoprotein Bound Bilayer (NABB) Particles
- Authors
- Banerjee, S., Huber, T., and Sakmar, T.P.
- ID
- ZDB-PUB-080306-38
- Date
- 2008
- Source
- Journal of molecular biology 377(4): 1067-1081 (Journal)
- Registered Authors
- Huber, Tara
- Keywords
- G-protein-coupled receptor, rhodopsin, high-density lipoprotein, single-particle imaging, electron microscopy
- MeSH Terms
-
- Animals
- Molecular Sequence Data
- Rhodopsin/pharmacokinetics*
- Rhodopsin/physiology
- Nanoparticles*/chemistry
- Cattle
- Receptors, G-Protein-Coupled/analysis
- Humans
- Lipid Bilayers/metabolism*
- Fluorescent Dyes/pharmacology
- Sequence Homology, Amino Acid
- Biomimetics/methods
- Amino Acid Sequence
- Rho Factor/physiology
- Zebrafish/genetics
- Apolipoprotein A-I/chemistry
- Apolipoprotein A-I/genetics
- Apolipoprotein A-I/isolation & purification
- Apolipoprotein A-I/metabolism*
- Phosphatidylcholines/chemistry
- Phosphatidylcholines/metabolism
- PubMed
- 18313692 Full text @ J. Mol. Biol.
Citation
Banerjee, S., Huber, T., and Sakmar, T.P. (2008) Rapid Incorporation of Functional Rhodopsin into Nanoscale Apolipoprotein Bound Bilayer (NABB) Particles. Journal of molecular biology. 377(4):1067-1081.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping