Functional reconstitution of the nicotinic acetylcholine receptor by CHAPS dialysis depends on the concentrations of salt, lipid, and protein

Abstract

ABSTRACT: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, = 580 000) and monomer (M, = 290 000) species. CHAPS dialysis at NaCl concentrations C0.5 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 (5500 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 f 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 f 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 ([Owlc = 3.65 mM) was higher than in 50 mM NaCl ( [Owlc = 2.8 mM). However, it is suggested that the main effect of high salt concentrations durig 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.A necessary and essential step in the detailed characterization of intrinsic membrane proteins is the detergent solubilization and the reconstitution of the protein species in artificial lipid membranes. Impressive examples are channel t We gratefully acknowledge financial support by the Deutsche For-

Allosteric transitions of Torpedo acetylcholine receptor in lipids, detergent and amphipols: molecular interactions vs. physical constraints

“…Both receptor forms were observed to migrate slightly more slowly once trapped by A8‐35 than they did in the presence of CHAPS: s 20,w values were 9.7±0.3 s in AP vs. 10.3±0.3 s in CHAPS for the light form, and 14.7±0.3 s in AP vs. 15.4±0.3 s in CHAPS for the heavy one. These values are close to those reported for the sedimentation coefficients of the monomeric and dimeric forms of the nAChR (8.7–9.5 s and 12.5–14.4 s, respectively, depending on the nature of the detergent; see [10,26,36], and references therein).…”

“…This is comparable to the ∼90 phospholipids per dimer that have been found to be required for keeping solubilized nAChR from inactivating [39]. The presence of ∼130 tightly associated phospholipids per dimer of CHAPS‐solubilized nAChR has been reported previously [26].…”

“…After incubation for 20 min at 4°C, samples were diluted 10× with detergent‐free phosphate buffer. The CHAPS concentration thereby fell ∼7× below its CMC (∼4 mM; see [26]). Samples were centrifuged 30 min at 18 000× g as described above.…”

Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology

“…In other hand, CHAPS and BigCHAP showed instability, decaying mobile fraction and diffusion coefficients and presents a ΔFFR of 0.32 and 0.22 respectively. Is important to mention that CHAPS was considered an excellent detergent choice for the purification and reconstitution of Tc nAChRs 36 . Moreover, both detergents were shown by our group to reduce functionality of the nAChR 16 .…”

Synthesis of a Radiolabeled Zwitterionic Detergent and Its Use in Protein Purification

“…A sensitive enzymatic method of detection, using 7␣-hydroxy-steroid dehydrogenase (EC 1.1.1.59) (5), is unsuitable for the assay of low levels of Chaps in complex biological mixtures because of interference from lipids. Other methods that have been employed to assay Chaps include HPLC, TLC (6), and light scattering (7); however, there is a need for a simpler method to detect low levels of Chaps which accompany proteins during isolation and purification. For example, Chaps is added to bacterial culture supernatants during the growth of the bovine pathogenic bacterium Pasteurella haemolytica A1.…”