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" Purification and Biophysical Characterization of Native Nicotinic Acetylcholine Receptor in Lipid-like Detergent Complexes "
Maldonado-Hernandez, Rafael
Lasalde-Dominicci, José A.
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Latin Dissertation
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English
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| Record Number
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1054253
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TL53370
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| Main Entry
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Maldonado-Hernandez, Rafael
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| Title & Author
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Purification and Biophysical Characterization of Native Nicotinic Acetylcholine Receptor in Lipid-like Detergent Complexes\ Maldonado-Hernandez, RafaelLasalde-Dominicci, José A.
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| College
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University of Puerto Rico, Rio Piedras (Puerto Rico)
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| Date
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2020
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| Degree
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Ph.D.
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2020
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322 p.
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| Abstract
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Over the past ten 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. These receptors are essential as a biological target for the therapeutic development of a large number of diseases. We have significantly improved and optimized the purity and yield of the native nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising structure, stability and functionality. Additionally, in collaboration with CDI, we have prepared and characterized a new mAbs against the impurities of nAChR-DC. We implemented novel 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 Lysophoscholine 16 (LFC-16), followed by three sequential steps of chromatography purification. A total of 105 target peaks were detected for the nAChR peptide mass fingerprinting, resulting in confident protein identification by Proteome Discoverer software version 2.1 in the mass spectrometry analysis. Likewise, we performed the N-glycans release experiment and confirmed the presence of glycans, specifically the oligommanose population Man8-9GlcNac2 with peaks at m/z 1742 and 1904 ([M + Na]+ ion. Furthermore, we used dynamic light scattering observing the polydispersity index (PDI) and obtaining a sample without aggregation. Additionally, 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 the lipid cubic phase. The behavior was reversed by Methyl-β-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus laevis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. However, the Methyl-β-Cyclodextrin treatment was incapable of reverse functionality but passing the nAChR-LFC-16 through a delipidation column achieved a functional protein similar to nAChR-LFC-16 without CHS treatment. Finally, we provided a pure, stable and complete functional native nAChR-DC sample for protein crystallizability purposes with the possibility of obtaining the X-ray atomic structure of the nAChR-DC in future studies.
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| Descriptor
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Biochemistry
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Biophysics
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Molecular biology
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Lasalde-Dominicci, José A.
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University of Puerto Rico, Rio Piedras (Puerto Rico)
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