Functional reconstitution of the nicotinic acetylcholine receptor by CHAPS dialysis depends on the concentrations of salt, lipid, and protein.
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.
Schürholz
T
T
Physikalische und Biophysikalische Chemie, Fakultät für Chemie, Universität Bielefeld, Germany.
Kehne
J
J
Gieselmann
A
A
Neumann
E
E
D000818:Animals; D002793:Cholic Acids / Q000737:chemistry*; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000737:chemistry*; D008027:Light; D008055:Lipids / Q000737:chemistry*; D008823:Micelles; D008854:Microscopy, Electron; D011506:Proteins / Q000737:chemistry*; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000302:isolation & purification; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014101:Torpedo
D002793:Cholic Acids; D003902:Detergents; D008055:Lipids; D008823:Micelles; D011506:Proteins; D011978:Receptors, Nicotinic; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate