A transmembrane helix-bundle from G-protein coupled receptor CB2: biosynthesis, purification, and NMR characterization

Biopolymers
HaiAn ZhengXiang-Qun Xie

Abstract

The cannabinoid receptor subtype 2 (CB2) is a member of the G-protein coupled receptor (GPCR) superfamily. As the relationship between structure and function for this receptor remains poorly understood, the present study was undertaken to characterize the structure of a segment including the first and second transmembrane helix (TM1 and TM2) domains of CB2. To accomplish this, a transmembrane double-helix bundle from this region was expressed, purified, and characterized by NMR. Milligrams of this hydrophobic fragment of the receptor were biosynthesized using a fusion protein overexpression strategy and purified by affinity chromatography combined with reverse phase HPLC. Chemical and enzymatic cleavage methods were implemented to remove the fusion tag. The resultant recombinant protein samples were analyzed and confirmed by HPLC, mass spectrometry, and circular dichroism (CD). The CD analyses of HPLC-purified protein in solution and in DPC micelle preparations suggested predominant alpha-helical structures under both conditions. The 13C/15N double-labeled protein CB2(27-101) was further verified and analyzed by NMR spectroscopy. Sequential assignment was accomplished for more than 80% of residues. The 15N HSQC NMR results show...Continue Reading

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Oct 1, 2011·Analytical Chemistry·Chunming LiuPaul S Cremer
Jun 15, 2007·Journal of the American Chemical Society·Susan DanielPaul S Cremer
Apr 1, 2008·Protein Expression and Purification·Yuxun Zhang, Xiang-Qun Xie
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Oct 19, 2010·Biochimica Et Biophysica Acta·Satita TapaneeyakornAnthony Watts
Apr 23, 2009·Biophysical Journal·Alexey NeumoinFred Naider
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