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Phospholipid molecular species profile and functionality of nicotinic acetylcholine receptor detergent complex from Torpedo californica solubilized with lipid‐like detergents
Author(s) -
Quesada Orestes,
Colón José,
González Carol,
Maldonado Rafael,
Rosado Irvin D.,
Acevedo Jesús A.,
LasaldeDominicci José A.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.815.13
Subject(s) - chemistry , torpedo , phospholipid , lipid bilayer , nicotinic acetylcholine receptor , acetylcholine receptor , lipid microdomain , biochemistry , chromatography , electrospray ionization , biophysics , mass spectrometry , receptor , membrane , biology
One of the limiting factors in the crystallization of membrane proteins is the process of solubilization mediated by detergents. During this process, preservation of the native protein structure and adjacent lipid environments is crucial for appropriate activity and stability. The detergent disruption of lipid–lipid and lipid‐protein interactions alters the aggregation state, stability, and functionality of the native structure. The effect is more severe when the protein activity is lipid‐dependent as is the case with the nicotinic acetylcholine receptor (nAChR) from Torpedo californica , which is required to maintain a functional conformation for crystallization. This study compares the efficiency of three families of detergents; alkylphosphocholine (FC), lysofoscholine (LFC), and Cyclo Foscholine (CF), in terms of stability, lipid composition and functionality of the nAChR‐detergent complexes (nAChR‐DCs). The molecular composition of all detected phospholipid classes from nAChR‐DCs solubilized with these detergents were analyzed by Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI‐MS/MS). To investigate the possible delipidation effect of the above‐mentioned detergents, we performed a functional assessment of these nAChR‐DCs in Xenopus oocytes using a two‐electrode voltage clamp (TEVC). The stability was monitored by fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase of the Alexa labeled nAChR. A qualitative cross‐correlation analysis of the lipid species present in each of these nAChR‐DCs cannot dissect which lipid species is essential for nAChR function in the soluble state, however, the partially functional nAChR‐FC12 detergent complex lack for lipid species (i.e. PC 18:0/20:4, PC20:4/20:5 and PC20:5/20;4) that are present in the fully functional, nAChR‐LFC16s. The nAChR‐FC16 not only produces the higher fractional mobility, but sustains the mobility throughout the whole 30 day period which is essential for protein nucleation crystallization but still contains some ATPase. In contrast, the nAChR‐FC12 complex is only partially functional but is almost free of ATPase. Support or Funding Information This work was supported by the NIH Grant #1R01GM098343 and UPR‐RP RISE Program NIH Grant #5R25GM061151 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .