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Location, structure, and dynamics of the synthetic cannabinoid ligand CP-55,940 in lipid bilayers.

ABSTRACT: The widely used hydrophobic cannabinoid ligand CP-55,940 partitions with high efficiency into biomembranes. We studied the location, orientation, and dynamics of CP-55,940 in POPC bilayers by solid-state NMR. Chemical-shift perturbation of POPC protons from the aromatic ring-current effect, as well as 1H NMR cross-relaxation rates, locate the hydroxyphenyl ring of the ligand near the lipid glycerol, carbonyls, and upper acyl-chain methylenes. Order parameters of the hydroxyphenyl ring determined by the 1H-13C DIPSHIFT experiment indicate that the bond between the hydroxyphenyl and hydroxycyclohexyl rings is oriented perpendicular to the bilayer normal. 2H NMR order parameters of the nonyl tail are very low, indicating that the hydrophobic chain maintains a high level of conformational flexibility in the membrane. Lateral diffusion rates of CP-55,940 and POPC were measured by 1H magic-angle spinning NMR with pulsed magnetic field gradients. The rate of CP-55,940 diffusion is comparable to the rate of lipid diffusion. The magnitude of cross-relaxation and diffusion rates suggests that associations between CP-55,940 and lipids are with lifetimes of a fraction of a microsecond. With its flexible hydrophobic tail, CP-55,940 may efficiently approach the binding site of the cannabinoid receptor from the lipid-water interface by lateral diffusion.

SUBMITTER: Kimura T

PROVIDER: S-EPMC2712040 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Location, structure, and dynamics of the synthetic cannabinoid ligand CP-55,940 in lipid bilayers.

Kimura Tomohiro T Cheng Kejun K Rice Kenner C KC Gawrisch Klaus K

Biophysical journal 20090601 12

The widely used hydrophobic cannabinoid ligand CP-55,940 partitions with high efficiency into biomembranes. We studied the location, orientation, and dynamics of CP-55,940 in POPC bilayers by solid-state NMR. Chemical-shift perturbation of POPC protons from the aromatic ring-current effect, as well as 1H NMR cross-relaxation rates, locate the hydroxyphenyl ring of the ligand near the lipid glycerol, carbonyls, and upper acyl-chain methylenes. Order parameters of the hydroxyphenyl ring determined ...[more]

PMID: 19527650

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Project description:Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB(2), and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB(2), here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB(2) were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of (2)H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB(2) in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB(2) reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.

Dataset Information

Location, structure, and dynamics of the synthetic cannabinoid ligand CP-55,940 in lipid bilayers.

Publications

Location, structure, and dynamics of the synthetic cannabinoid ligand CP-55,940 in lipid bilayers.

Similar Datasets

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