title: Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques.
abstract: This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly15N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Differentqratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8). As the concentration of dimyristoylphosphocholine (DMPC) was increased such that theqratio was between 0.16 and 0.42, there was less crowding in the cross peaks with increasingqratio. In bicelles ofq= 0.42, the maximum number of cross peaks were obtained and the cross peaks were uniformly dispersed. The receptor domain in bicelles beyondq= 0.42 resulted in peak crowding. These studies demonstrate that GPCRs folding especially in bicelles is protein-specific and requires the right mix of the longer chain and shorter chain lipids to provide the right environment for proper folding. These findings will allow further development of novel membrane mimetics to provide greater diversity of lipid mixtures than those currently being employed for GPCR stability and folding, which are critical for both X-ray and NMR studies of GPCRs.
authors.affiliation: Department of Biomedical Engineering, University of Ghana, P.O. Box LG 25, Legon, Accra, Ghana. etiburu@ug.edu.gh.
authors.forename: Elvis K
authors.initials: EK
authors.lastname: Tiburu
authors.affiliation: Department of Chemistry, College of Staten Island, New York, NY 10314, USA. jianqinzhuang@gmail.com.
authors.forename: Jianqin
authors.initials: J
authors.lastname: Zhuang
authors.affiliation: Department of Biomedical Engineering, University of Ghana, P.O. Box LG 25, Legon, Accra, Ghana. 369hmnaf@gmail.com.
authors.forename: Heidimarie N A
authors.initials: HN
authors.lastname: Fleischer
authors.affiliation: Department of Biochemistry, Cell and Molecular Biology; University of Ghana, P.O. Box LG 25, Legon, Accra, Ghana. parthur14@gmail.com.|West Africa Center for Cell Biology of Infectious Pathogens (WACCBIP), Accra, Ghana. parthur14@gmail.com.
authors.forename: Patrick K
authors.initials: PK
authors.lastname: Arthur
authors.affiliation: Department of Biochemistry, Cell and Molecular Biology; University of Ghana, P.O. Box LG 25, Legon, Accra, Ghana. gawandare@ug.edu.gh.|West Africa Center for Cell Biology of Infectious Pathogens (WACCBIP), Accra, Ghana. gawandare@ug.edu.gh.
authors.forename: Gordon A
authors.initials: GA
authors.lastname: Awandare
keywords: NMR; bicelles; insect cells; selective and uniformly labeling