Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

30-POS Board 30 ASSESSMENT OF THE FUNCTIONALITY AND STABILITY OF SOLUBILIZED NICOTINIC ACETYLCHOLINE RECEPTOR DETERGENT COMPLEXES IN LIPIDIC CUBIC PHASE (LCP) AS A GUIDE FOR CRYSTAL FORMATION Jose A. Lasalde-Dominicci 1,2,4 ; Orestes Quesada-Gonzalez 3,4 ; 1 University of Puerto Rico, Department of Biology, San Juan, Puerto Rico 2 University of Puerto Rico, Chemistry, San Juan, Puerto Rico LCP is a crystallization platform for membrane proteins that has increased exponentially the number of crystal structures in the past decade. However, little is known about the mechanism by which membrane protein detergent complexes diffuse in the LCP to form crystals. Also, the relation between detergent solubilization and nAChR stability is still poorly understood. We used three families of lipid-like detergents with different chain length to isolates nAChR from Torpedo californica’s electroplax tissue ( Tc ET). The functionality of affinity-purified nicotinic acetylcholine receptors detergent complexes (nAChR-DCs) was assessed prior to LCP preparation using the two electrode voltage clamp (TEVC). The stability and Fractional Mobility (FM) of the nAChR-DCs in LCP was assayed for 30 days using fluorescence recovery after photobleaching (FRAP). Our results show that phospholipid-analog detergents with 16 carbon acyl chains sustain nAChR-DC mobility unchanged for the 30 day period, while in most of cholesterol-analog detergents, and CF analogs the MF decays on the first 15 to 20 days of the experiment. We found an interesting correlation between the elongation of the acyl chain on phospholipid-analog detergents and an increment on the FM, particularly observed on the LFC detergent family. The most abundant fatty acids in the Tc ET phospholipid species PC, PE, PG, PI, and PS were 16:0 and 22:6. These results are consistent with the fact the nAChR-LFC16 complex is the most stable complex among all the detergents that we have evaluated. We are currently producing nAChR-LFC16 crystals for structural studies using LCP, vapor diffusion techniques and the RMP@LMx technique (U.S. patent 10,155,221) developed in our laboratory (www.nachrs.org). This research was supported by the NIH NIGMS grants 1R01GM098343, 5R25GM061151 and COBRE P20GM103642 3 University of Puerto Rico, Physical Sciences, San Juan, Puerto Rico 4 Molecular Sciences Research Center- UPR, San Juan, Puerto Rico

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