Abstract
In the present study, we characterized the effect of detergent solubilization and affinity column purification on Torpedo californica nicotinic acetylcholine receptor (nAChR) by using a series of lipid-like detergent with similar acyl composition to the most abundant fatty acid found in the native tissue of Torpedo (18:1, 18:0, 16:1, 16:0) as well as cholesterol-analog detergents. Fatty acid analogs included members of the Fos-choline (FC) family of detergents (FC-12, −14, −16 and lyso-FC-16), while cholesterol analogs were represented by cholate, taurocholate and CHAPS. Each detergent was used to solubilize and purify the nAChR using established affinity column protocols, followed by analytical size exclusion chromatography (A-SEC) to probe the stability and aggregation state of the nAChR in solution, as well as planar lipid bilayers to probe ion channel function. The overall results showed that the stability and function of the solubilized and purified receptor is preserved in lipid-analog detergents that have acyl chains similar to the most abundant lipid (18:1, 18:0, 16:1, and 16:0) in the endogenous Torpedo lipid environment, providing a suitable set of detergents for future structural studies. We also performed lipidic cubic phase (LCP) fluorescence recovery after photo bleaching (FRAP) experiments using fluorescently-tagged-Torpedo nAChR solubilized and affinity purified in FC-12, −14 and lyso-FC-16 detergents to estimate the mobile fraction and diffusion coefficient with the goal of using these data to perform LCP crystallization trials within a narrower crystallization space. These detergents display a linear increase in mobile fraction and diffusion coefficient (LFC-16>FC-12>FC-14) which is inversely proportional to the percentage of dimmer in these detergents (FC-14>FC-12>LFC-16). These experiments will serve to establish correlations to define suitable conditions for nAChR crystallization in LCP.
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