Supporting reconstituted membrane

FIG. 14 Schematic illustration of an archaeal cell envelope structure (a) composed of the cytoplasmic membrane with associated and integral membrane proteins and an S-layer lattice, integrated into the cytoplasmic membrane, (b) Using this supramolecular construction principle, biomimetic membranes can be generated. The cytoplasmic membrane is replaced by a phospholipid or tetraether hpid monolayer, and bacterial S-layer proteins are crystallized to form a coherent lattice on the lipid film. Subsequently, integral model membrane proteins can be reconstituted in the composite S-layer-supported lipid membrane. (Modified from Ref. 124.)... 

Supported lipid bilayers with or without reconstituted membrane proteins are prepared by one of three methods described below and illustrated in Fig. 3. Methods details can be found in Reference 86. 

This method is a combination of the other two methods. In our opinion, it is the most gentle method to reconstitute membrane proteins into supported bilayers and to prepare supported bilayers with fragile coexisting liquid phases of lipids. A LB monolayer is prepared on a hydrophilic substrate as described above. To prepare tethered-polymer supported bilayers, a suitable lipopolymer may be included at a concentration of a few... 

The protein-functionalization of a decylamine modified MAH-PP supported biomimetic membrane was tested by the incorporation of cytochrome c oxidase (CcOX) from Paracoccus denitriflcans. CcOX is part of the respiratory chain and is responsible for the active transport of protons against the gradient of the electrochemical potential across the lipid membrane. This reaction is driven by the enzymatic reduction of dioxygen where electrons are derived from the coenzyme cytochrome c. This process has been studied using mitochondria or CcOX reconstituted in liposomes and by the incorporation into tethered lipid membranes. 

Whatever the mode of attachment of SIg to cell membranes, the major portion of the Fc part of the molecule is not embedded in the phospholipid bilayer. This is also supported by the fact that CD spectra of all membrane proteins (not lymphocytes specifically) show little conformation, which, as previously mentioned, is the conformation of immunoglobulin domains. Experiments with reconstituted lymphocyte plasma membranes (Chavin and Holliman, 1975) and model membrane systems (Weissman et al., 1974) have not, in general, been very revealing. With reconstituted membranes, very little of the original membrane immunoglobulin becomes reincorporated. Liposome models were tested by... 

Poglitsch, C.L., Sumner, M.T., Thompson, N.L. Binding ofIgG to MoFc gamma RE purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy. Biochemistry 30, 6662-6671 (1991)... 

In reconstitution experiments, the self-assembly of the pore-forming protein a-hemolysin of Staphylococcus aureus (aHL) [181-183] was examined in plain and S-layer-supported lipid bilayers. Staphylococcal aHL formed lytic pores when added to the lipid-exposed side of the DPhPC bilayer with or without an attached S-layer from B coagulans E38/vl. The assembly of aHL pores was slower at S-layer-supported compared to unsupported folded membranes. No assembly could be detected upon adding aHL monomers to the S-layer face of the composite membrane. Therefore, the intrinsic molecular sieving properties of the S-layer lattice did not allow passage of aHL monomers through the S-layer pores to the lipid bilayer [142]. 

These reconstitution experiments supported the model for electron transfer shown in figure 14.8. In this model the complexes do not bind to each other directly. Instead, movement of electrons from complexes I and II to complex III is mediated by diffusion of UQH2 from one complex to the other within the phospholipid bilayer. Similarly, electrons move from complex III to complex IV by the diffusion of reduced cytochrome c along the surface of the membrane. Remember that cytochrome c differs from the other cytochromes in being a water-soluble protein. It is attached loosely to the membrane surface by electrostatic interactions. 

Wagner ML, Tamm LK (2000) Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins Silane-polyethyleneglycol-lipid as a cushion and covalent linker. Biophys J 79 1400-1414... 

The idea of using polymer-supported bilayers has been around for more than a decade (34), but it became practical for chemical and biological applications only more recently. Early versions have used relatively short tethers to link the membranes to the solid substrate and thereby increase their durability for practical applications (25, 35). Because these approaches do not increase the gap distance between substrate and membrane and therefore have not been used to reconstitute integral membrane proteins functionally, they will not be discussed here. 

Adhesion of different immune cells to one another or to epithelial cells has also been studied using planar bilayer models. For example, lymphocyte function-associated protein-1 (LFA-1) promotes cell adhesion in inflammation [i.e., a reaction that can be mimicked by binding to purified ICAM-1 in supported membranes (70)]. Similarly, purified LFA-3 reconstituted into supported bilayers mediates efficient CD2-dependent adhesion and differentiation of lymphoblasts (71). This work was followed by a study in which transmembrane domain-anchored and GPl-anchored isoforms of LFA-3 were compared (72). Because this research occurred before the introduction of polymer cushions and because the bilayers were formed by the simple vesicle fusion technique, the transmembrane domain isoform was immobile, whereas the GPl isoform was partially mobile. By comparing results with these two isoforms at different protein densities in the supported bilayer, the authors showed that diffusible proteins at a sufficient minimal density in the supported membrane were required to form strong cell adhesion contacts in this system. 

Figure 3 Methods for supported bilayer formation and membrane protein reconstitution, (a) and (b) LB/LS method. A lipid monolayer is spread at the air-water interface of a Langmuir trough and transferred to a solid substrate while keeping the surface pressure constant. A second monolayer is transferred by horizontal apposition of the first transferred monolayer and collection of a counter-piece with spacers, (c) Direct VF method. Membrane vesicles are flown into a chamber with a clean surface substrate on top. After about an hour of incubation, they form a supported bilayer on the substrate and excess vesicles are flushed out. (d) LB/VF method. The procedures depicted in panels (a) and (c) are combined leading to an asymmetric bilayer with an asymmetric protein distribution. Although this method can also be performed without a polymer, it is shown here with the polymer transferred during the LB step.

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