XB-ART-57249
Anal Biochem
2020 Dec 01;610:113887. doi: 10.1016/j.ab.2020.113887.
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Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies.
Maldonado-Hernández R
,
Quesada O
,
Colón-Sáez JO
,
Lasalde-Dominicci JA
.
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Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of β-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-β-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-β-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.
???displayArticle.pubmedLink??? 32763308
???displayArticle.pmcLink??? PMC7669685
???displayArticle.link??? Anal Biochem
???displayArticle.grants??? [+]
P20 GM103642 NIGMS NIH HHS , R01 GM098343 NIGMS NIH HHS , R25 GM061151 NIGMS NIH HHS , U54 MD007600 NIMHD NIH HHS
Species referenced: Xenopus laevis
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