Membranes 2017, 7(1), 10; https://doi.org/10.3390/membranes7010010
Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques
1
Department of Biomedical Engineering, University of Ghana, P.O. Box LG 25, Legon, Accra, Ghana
2
Department of Chemistry, College of Staten Island, New York, NY 10314, USA
3
Department of Biochemistry, Cell and Molecular Biology; University of Ghana, P.O. Box LG 25, Legon, Accra, Ghana
4
West Africa Center for Cell Biology of Infectious Pathogens (WACCBIP), Accra, Ghana
*
Author to whom correspondence should be addressed.
Academic Editor: Chuyang Y. Tang
Received: 16 December 2016 / Revised: 26 January 2017 / Accepted: 6 February 2017 / Published: 17 February 2017
(This article belongs to the Section Membrane Applications in Water Treatment)
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 uniformly 15N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8). As the concentration of dimyristoylphosphocholine (DMPC) was increased such that the q ratio was between 0.16 and 0.42, there was less crowding in the cross peaks with increasing q ratio. In bicelles of q = 0.42, the maximum number of cross peaks were obtained and the cross peaks were uniformly dispersed. The receptor domain in bicelles beyond q = 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. View Full-TextKeywords:
selective and uniformly labeling; insect cells; bicelles; NMR
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Tiburu, E.K.; Zhuang, J.; Fleischer, H.N.A.; Arthur, P.K.; Awandare, G.A. Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques. Membranes 2017, 7, 10.
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