LAUSR

The first step for reconstitution in attempts to membrane protein crystallization is the solubilization process by detergents. During this process, preservation of the native protein structure and adjacent lipid environments is crucial for activity and stability. The detergent disruption of lipid-lipid, lipid-protein interactions alter the aggregation state, stability, and functionality of the native structure. The effect is more severe when protein activity is lipid dependent. The molecular composition of all detected phospholipid classes from affinity-purified acetylcholine receptors detergent complexes (nAChR-DCs) from Torpedo californica (Tc) solubilized using a homologous series of Cyclo Foscholine (CF) detergents were analyzed by Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). We took advantage of Xenopus laevis oocytes expression system to access the nAChR-DCs activity by means of two electrode voltage clamp (TEVC) technique. UPLC/ESI/MS/MS results shown a phospholipid composition favored by phosphatidylcholine and sphingomyelin species for the three nAChR-DC studied. However, the molecular species in each nAChr-DC and cholesterol content were in some degree, quite different despite that the only detergent structural difference is 1 or 2 methylene groups. Injection of oocytes with Tc nAChRs-DCs solubilized with CF family, differing in the length of their acyl chain, resulted in responses with amplitudes that inversely depend on acyl chain length. CF-4 displayed the highest mean amplitudes in this family (−200 ± 60 nA) which is about 80% of the current produced when normalized to the mean current of the crude membrane. The longer side chain, CF-6 and CF-7 produced an insignificant current compared to the crude membrane. These findings will provide a lipidomic strategy to prepare nAChR-DCs suitable for structural studies.