عنوان

Purification and Biophysical Characterization of Native Nicotinic Acetylcholine Receptor in Lipid-like Detergent Complexes

شماره

TLpq2445594511

زبان متن نوشتاري يا گفتاري و مانند آن

انگلیسی

عنوان اصلي

Purification and Biophysical Characterization of Native Nicotinic Acetylcholine Receptor in Lipid-like Detergent Complexes

نام عام مواد

[Thesis]

نام نخستين پديدآور

Maldonado-Hernandez, Rafael

نام ساير پديدآوران

Lasalde-Dominicci, José A.

نام ناشر، پخش کننده و غيره

University of Puerto Rico, Rio Piedras (Puerto Rico)

تاریخ نشرو بخش و غیره

2020

نام خاص و کميت اثر

322

جزئيات پايان نامه و نوع درجه آن

Ph.D.

کسي که مدرک را اعطا کرده

University of Puerto Rico, Rio Piedras (Puerto Rico)

امتياز متن

2020

متن يادداشت

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.

موضوع مستند نشده

Biochemistry

موضوع مستند نشده

Biophysics

موضوع مستند نشده

Molecular biology

مستند نام اشخاص تاييد نشده

Lasalde-Dominicci, José A.

مستند نام اشخاص تاييد نشده

Maldonado-Hernandez, Rafael

نام الکترونيکي

فرمت انتشار

p

نوع ماده

[Thesis]

کد کاربرگه

276903

سطح دسترسي

a

تكميل شده

Y