Biophysical characterization dataset of native nicotinic acetylcholine receptor in lipid-like detergent complexes

Abstract

For a long time, traditional purification and extraction methods for the native Torpedo californica nicotinic acetylcholine receptor in lipid-like detergent complex (nAChR-DC) have compromised its purity, functionality and X-ray structural studies possibility. The dataset presented in this article provide a characterization of the Torpedo californica nAChR-DC purified using a sequential purification processes developed in our laboratory [1] . This purification takes in consideration all of the physicochemical and functional requirements stablished by several researchers for the past three decades for the nAChR. These requirements were addressed in order to preserve the stability and functionality of nAChR-DC while ensuring the highest degree of protein purity. We focused on the effect of cholesteryl hemisuccinate (CHS) supplementation on nAChR conformational changes during the purification process. Data from the size exclusion chromatography of the nAChR-DC supplemented with CHS in concentrations ranging from 0.01 mM, 0.1 mM, 0.2 mM and 0.5 mM consistently demonstrated that 0.5 mM CHS affects receptor stability via disassemble of the pentameric oligomer. However, 0.2 mM CHS produced negligible nAChR-DC subunit disruption. The purified nAChR-DC has been characterized by circular dichroism (CD) and fluorescence recovery after photobleaching (FRAP) , in order to assess its stability. The CD data was recorded in the wavelength range of 190 - 250 nm, showed that CHS induce a ⍺-helix to β-sheet transition of the nAChR-DC. The nAChR-LFC-16 delipidation with Methyl-β-Cyclodextrin decreased the percentage of α-helix and increased the β-sheet antiparallel secondary structure and levels the percentage of turns to that of the nAChR-DC without CHS treatment. Additionally, the stability of the nAChR-DC supplemented with CHS and incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of FRAP. The LCP-FRAP data allowed to establish possible optimal crystallization conditions for the development of crystals from purified nAChR-conjugated to α-Bungarotoxin, Alexa Fluor ™ 488 (α-BTX) in order to obtain a high-resolution atomic structure by X-ray diffraction.

Pursuing High-Resolution Structures of Nicotinic Acetylcholine Receptors: Lessons Learned from Five Decades

“…Recently, our laboratory developed a novel, straightforward, and sequential automated purification process to obtain highly pure and functional nAChR-DCs [ 77 , 78 ]. First, the protein is solubilized using LysoFos Choline 16 (LFC-16), a phospholipid analog detergent, supplemented with CHS, and sodium chloride at high concentrations to assist the solubilization process, thus removing impurities that have weak or non-specific interactions with the nAChR.…”

“…Moreover, the column was placed within an insulated jacket using a recirculation system to control the temperature inside the column, in addition to performing experiments in a cold room. The combination of all of these steps and strategies has substantially improved the quality and functional parameters of purified nAChR-DCs as a possible final product for the production of high-quality crystals [ 77 , 78 ]. It has been postulated that the main impediment to achieving a high-resolution structure of the nAChR is the purity, homogeneity, function, and stability of the nAChR-DCs [ 52 , 54 , 55 , 73 , 79 , 80 , 81 , 82 , 83 ].…”

“…However, few have focused on the importance of generating functional nAChR detergent complexes. The above-described purification protocols proved capable of producing a stable and functional nAChR-DC, suitable as a starting material for crystallization studies [ 77 , 78 ]. Among the most common methods used in these cases are sucrose density gradient, alkali treatment, ion exchange, gel filtration, and different affinity chromatography methodologies using nAChR ligands [ 62 , 84 , 85 , 86 ], some of which have the disadvantage of short lifespans one (1) month or less [ 87 ].…”

“…Another organism used to obtain nAChR was Electrophorus electricus (Ee), from which only 3–4 mg of purified nAChR was recovered from 500 g of tissue [ 93 ]. In our method, a significant reduction in the amount of Tc tissue used for solubilization and purification (20–40 g) is possible, from which we were able to produce 2–4 mg of pure, stable, and functional nAChRs [ 77 , 78 ]. As mentioned above, procedures that have been applied to the purification of nAChR from other tissue sources have invariably yielded nAChRs with several co-purifying contaminants.…”

“…We have developed and characterized a highly reproducible purification process for native nAChRs with final purity levels of ~94% [ 77 , 78 ]. We implemented, for the first time in native nAChR samples, a robust purity analysis process using microfluidics-based capillary gel electrophoresis using the Agilent 2100 Bioanalyzer system.…”