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Reconstitution of Human Peripheral Cannabinoid Receptor CB2 into Phosphatidylcholine Proteoliposomes
Author(s) -
Kimura Tomohiro,
Yeliseev Alexei A,
Rhodes Steven D,
Gawrisch Klaus
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a983
Subject(s) - cannabinoid receptor , cannabinoid receptor type 2 , peripheral , cannabinoid , phosphatidylcholine , endocannabinoid system , chemistry , pharmacology , receptor , medicine , biochemistry , phospholipid , agonist , membrane
For structural and functional studies on G‐protein‐coupled receptors (GPCR), it is highly desirable to reconstitute GPCR into a membrane of controlled composition. We attempted functional reconstitution of the human peripheral cannabinoid receptor CB2, expressed in E. coli , into 1‐palmitoyl‐2‐oleoyl‐ sn ‐glycero‐3‐phosphocholine(POPC) proteoliposomes from various detergent solutions. The method includes expression and purification of the protein in milligram quantities, exchange of detergents, and rapid removal of detergents from micellar solutions of CB2, lipid, and detergents by column chromatography. The packing material separates detergents from proteoliposomes both by size exclusion as well as specific detergent adsorption. The reconstitution process was monitored by fluorescence from trace amounts of labeled lipid and CB2 that were added to the mixtures. Efficiency of detergent removal was established by high‐resolution 1 H NMR on small aliquots of proteoliposomes dissolved in organic solvent. The protein‐to‐lipid molar ratio of proteoliposomes was systematically varied and the density and homogeneity of resulting proteoliposomes monitored by sucrose gradient centrifugation. Liposome size and homogeneity were further monitored by measurement of liposome diffusion rates using 1 H NMR with application of pulsed magnetic field gradients. Preliminary results of structural studies on the proteoliposomes by solid state NMR and circular dichroism will be shown.