Removal of detergent from mixed micelles of egg yolk phosphatidylcholine and octyl glucoside leads to formation of unilamellar phospholipid vesicles with a diameter of about 230 nm. The same procedure applied to mixed micelles containing the transmembrane protein glycophorin A, in addition to lipid and detergent, produces vesicles of the same size with glycophorin incorporated into the bilayer. The pure lipid vesicles are highly impermeable to both anions and cations, and incorporation of up to 220 molecules of glycophorin per vesicle has little effect on permeability.

Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside

Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins.

Publisher Summary The reconstitution of membrane proteins into liposomes is a powerful tool that can be used to identify the mechanism of the action of membrane proteins. The prospects of achieving optimal proteoliposome reconstitution are good when reliable methods and systematic experimental analysis are used. This chapter deals with the various strategies commonly used to reconstitute proteoliposomes and focuses on approaches that have led to the production of highly functional proteoliposomes. Four basic strategies are outlined—mechanical means, freeze-thawing, organic solvents, and detergents. The chapter also introduces a new method for membrane protein reconstitution. The new reconstitution strategy proceeds in four stages: (1) preparation of large, homogeneous, and unilamellar liposomes, (2) addition of detergent to the preformed liposomes, through all the range of the solubilization process, (3) addition of solubilized protein at each well-defined step of the solubilization process, and (4) detergent removal and characterization of the reconstituted products. Besides the need for measuring the activity of the protein, any method of membrane-protein reconstitution should fulfill a number of important criteria that must be analyzed to characterize unequivocally the efficiency of the reconstitution. The first parameter to analyze to check the efficiency of a reconstitution trial is the activity of the protein after reconstitution. The most accurate method to analyze the efficiency of membrane-protein incorporation is a density gradient.

Reconstitution of membrane proteins into liposomes.

Aims: The mitochondrial respiratory chain is recognized today to be arranged in supramolecular assemblies (supercomplexes). Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. In the present study, we have directly addressed this issue by testing the ROS generation by Complex I in two experimental systems in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. Results: The results of our investigation provide experimental evidence that the production of ROS is strongly increased in either model, supporting the view that disruption or preve...

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Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I.

A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-100-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated.

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin

J. Telford

Papers

Reconstitution, a way of biochemical research; some new approaches to membrane-bound enzymes.