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31P and 2H relaxation studies of helix VII and the cytoplasmic helix of the human cannabinoid receptors utilizing solid-state NMR techniques.

Tiburu EK, Karp ES, Birrane G, Struppe JO, Chu S, Lorigan GA, Avraham S, Avraham HK

Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA.

Cannabinoid receptors are G-protein-coupled receptors comprised of seven transmembrane helices. We hypothesized that the extended helix of the receptor interacts differently with POPC bilayers due to the differing distribution of charged amino acid residues. To test this, hCB1(T377-E416) and hCB2(K278-H316) peptides were studied with 31P and 2H solid-state NMR spectroscopy by incorporating them into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine bilayers. Lipid affinities of the 40- and 39-residue peptides were analyzed on the basis of 31P and 2H spectral line shapes, order parameters, and T1 relaxation measurements of the POPC bilayers. Lipid headgroup perturbations were noticed in the 31P NMR spectra in the lipid/peptide mixtures when compared with the pure lipids. 2H order parameters were calculated from the quadrupolar splitting of the de-Paked 2H NMR spectra. At the top of the acyl chain, pure lipids had an average S(CD) approximately = 0.20, whereas S(CD) approximately = 0.16 and S(CD) approximately = 0.18 were found in the presence of hCB1(T377-E416) and hCB2(K278-H316), respectively. S(CD) values decreased in the central part of the acyl chains when compared to the pure POPC lipids, indicating a change in the dynamic properties of the lipid membrane in the presence of the cannabinoid peptides. R(1Z) vs S2(CD) plots exhibited a linear dependency with and without the peptides, with an increase in slope upon addition of the peptides to the POPC, indicating that the dynamics of the lipid bilayer is dominated by fast axially symmetric motion. This study provides insights into the interaction of cannabinoid peptides with the membrane bilayer by investigating the headgroup and acyl chain dynamics.

Published 6 June 2006 in Biochemistry, 45(23): 7356-65.
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