A New “Gel-like” Phase in Dodecyl Maltoside–Lipid Mixtures: Implications in Solubilization and Reconstitution Studies

Abstract

The interaction of dodecyl maltoside with lipids was investigated through the studies of solubilization and reconstitution processes. The solubilization of large unilamellar liposomes was analyzed through changes in turbidity and cryo-transmission electron microscopy. Solubilization was well described by the three-stage model previously reported for other detergents, and the critical detergent/phospholipid ratios at which lamellar-to-micellar transition occurred (Rsat = 1 mol/mol) and finished (Rsol = 1.6 mol/mol) were determined. The vesicle-micelle transition was further observed in the vitrified hydrated state by cryo-transmission electron microscopy. A striking feature of the solubilization process by dodecyl maltoside was the discovery of a new phase consisting of a very viscous "gel-like" sample. It is shown that this equilibrium cohesive phase is composed of long filamentous thread-like micelles, over microns in length. Similar structures were observed upon solubilization of sonicated liposomes, multilamellar liposomes, or biological Ca2+ ATPase membranes. This "gel-like" phase was also visualized during the process of liposome reconstitution after detergent removal from lipid-dodecyl maltoside micelles. The rate of detergent removal, controlled through the use of SM2 Bio-Beads, was demonstrated to drastically influence the morphology of reconstituted liposomes with a propensity for multilamellar liposome formation upon slow transition through the "gel-like" phase. Finally, on the basis of these observations, the mechanisms of dodecyl maltoside-mediated reconstitution of bacteriorhodopsin were analyzed, and optimal conditions for reconstitution were defined.

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

“…To investigate the effect of anionic phospholipids on the interaction of FXYD1 with the enzyme, in some experiments the zwitterionic DEPC was replaced with 10, 20, 35 and 50 mol% of the anionic DOPS. Lipid vesicles containing native Na,K-ATPase from rabbit kidney, purified recombinant a 1 / His 10 -b 1 or a 1 /His 10 -b 1 /FXYD1 were prepared by a method similar to the one described before for the reconstitution of bacteriorhodopsin (Lambert et al 1998). Since DDM cannot be efficiently removed by dialysis due to its low critical micelle concentration (0.01 % at 25°C), proteoliposomes were obtained by adsorption of the detergent onto SM2 Bio-Beads (BioRad, 20 50 mesh).…”

“…Since DDM cannot be efficiently removed by dialysis due to its low critical micelle concentration (0.01 % at 25°C), proteoliposomes were obtained by adsorption of the detergent onto SM2 Bio-Beads (BioRad, 20 50 mesh). In summary, the addition of an excess of SM2 Bio-Beads to a solution containing a mix of DDM-solubilized native Na,K-ATPase or purified recombinant enzyme and lipid/DDM micelles leads to fast adsorption of the detergent by the beads and the formation of active unilamellar proteoliposomes with an average diameter of 110 nm (Lambert et al 1998). Typically, half of the protein molecules are reconstituted with the ATP-binding site facing outward.…”

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

“…To this end, the first stage in our strategy was to add detergent to preformed liposomes using the entire range of detergent concentrations that cause the transformation of lamellar structures to mixed micelles (17)(18)(19). The lipid-detergent mixed amphiphilic structures that are formed during the solubilization process are similar to those pre- With respect to optimal protein incorporation, we have identified three mechanisms by which membrane proteins can associate with lipids to give proteoliposomes ( Figure 3).…”

Process of Destruction of Large Unilamellar Vesicles by a Zwitterionic Detergent, CHAPS: Partition Behavior between Membrane and Water Phases

“…31) Upon the addition of CHAPS to EggPC vesicles, the turbidity increased considerably and reached the maximum value. A further increase in the CHAPS concentration led to a precipitous decrease in turbidity.…”