Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers

Abstract

We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles.

Electroinsertion and activation of the C-terminal domain of Colicin A, a voltage gated bacterial toxin, into mammalian cell membranes

“…All conclusions obtained with glycophorin remained valid for colicin (El Ouagari et al 1994). The transient membrane organization had thermodynamic properties previously described to support spontaneous insertion (Scotto andZakim 1985, Jain andZakim 1987), fusogenicity (Sowers andLieber 1986, Teissie´and Rols 1986), enhanced phospholipid flip-flop (Deuticke and Schwister 1989) and leakage of polar solutes Schwister 1989, Rols andTeissie´1990a,b). Earlier studies of acidic pH, showed a loss of native tertiary structure of soluble C-terminal colicin A (Van der Goot et al 1991) or E1 channel polypeptides (Schendel and Cramer 1994), along with conservation of their secondary structure suggested the presence of 'molten globule' intermediates.…”

Insertion of Glycophorin A, A Transmembraneous Protein, in Lipid Bilayers can be Mediated by Electropermeabilization

“…It should be stressed that thermodynamics predicts that spontaneous insertion requires a membrane state in which modulation of bilayer organization is present. This should be associated with fusogenicity and leakage of polar compounds (Scotto and Zakim, 1985;Jain and Zakim, 1987). These two properties are indeed present in electropermeabilized lipid vesicles.…”

Differential effects of glycosphingolipids on the detergent-insolubility of the glycosylphosphatidylinositol-anchored membrane dipeptidase

“…Liposomes were prepared as described previously [33,34] by dissolving 35 mg of dimyristoyl phosphatidylcholine (DMPC) and the desired amount of sphingolipid (bovine brain sphingomyelin, chicken egg-yolk sphingomyelin, bovine brain Type II GalCer, human spleen GlcCer, bovine LacCer ; all from Sigma) and\or cholesterol in chloroform and the solution was then dried under nitrogen to produce a film on the interior of a glass vial. Myristic acid (15 mg) was suspended using gentle heating in 10 ml of ethanol and a 100 µl aliquot was transferred to a glass vial.…”