The detergent CHAPS was found to be the preferable surfactant for the efficient purification and reconstitution of the Torpedo californica nicotinic acetylcholine receptor (AChR). The main result is that the incorporation of the AChR proteins into lipid vesicles by CHAPS dialysis was strongly dependent on the salt and protein concentrations. As monitored by sucrose gradients, by electron microscopy, and by agonist-induced lithium ion flux, the best reconstitution yields were obtained in 0.5 M NaCl at a protein concentration of 0.5 g/L and in 0.84 M NaCl at 0.15 g/L protein. Electron micrographs of receptor molecules, which were incorporated into vesicles, showed single, nonaggregated dimer (M(r) = 580,000) and monomer (M(r) = 290,000) species. CHAPS dialysis at NaCl concentrations less than 0.5 M largely reduced the receptor incorporation concomitant with protein aggregation. Electron micrographs of these preparations revealed large protein sheets or ribbons not incorporated into vesicles. The analysis of static and dynamic light scattering demonstrated that the detergent-solubilized AChR molecules aggregate at low lipid contents (less than or equal to 500 phospholipids/AChR dimer), independent of the salt concentration. AChR proteins eluted from an affinity column with a solution containing 8 mM CHAPS (but no added lipid) still contained 130 +/- 34 tightly bound phospholipids per dimer. The aggregates (about 10 dimers on the average) could be dissociated by readdition of lipid and, interestingly, also by increasing the CHAPS concentration up to 15 mM. This value is much higher than the CMC of CHAPS = 4.0 +/- 0.4 mM, which was determined by surface tension measurements. The data clearly suggest protein-micelle interactions in addition to the association of monomeric detergents with proteins. Furthermore, the concentration of the (free) monomeric CHAPS at the vesicle-micelle transformation in 0.5 M NaCl ([Dw]c = 3.65 mM) was higher than in 50 mM NaCl ([Dw]c = 2.8 mM). However, it is suggested that the main effect of high salt concentrations during the reconstitution process is an increase of the fusion (rate) of the ternary protein/lipid/CHAPS complexes with mixed micelles or with vesicular structures, similar to the salt-dependent fusion of vesicles.

Phospholipid phosphatidylcholines (PC) and the nonionic detergent octyl glucoside (OG) were found to form a miscibility gap in aqueous solution at a molar ratio [OG]T/[PC]T = 2.6−2.4. This ratio is just below the critical ratio for the formation of vesicles. At [PC] > 1.3 g/l the transition from micelles to vesicles is not direct but via a surfactant-rich phase (SRP). The various stability regions are summarized in a phase diagram. Though the partition coefficient Γ = [Db]/[Dw] increases markedly with temperature, the critical ratio for the phase separation Rc is biased to higher detergent concentrations. This behaviour can be explained by increased motion of the hydrocarbon moiety at higher temperatures. The different phases are investigated by light microscopy, nuclear magnetic resonance and thin-layer chromatography. The miscibility gap of PC/OG is compared to the lemmar phase of Triton X-114; the miscibility gap has implications for membrane protein reconstitution and can be used for membrane protein purification. The Torpedo californica nicotinic acetylcholine receptor (n-AcChR) was found to partition into the SRP.