S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Structure and activity of lipid membrane biosensor surfaces studied with atomic force microscopy and a resonant mirror.

Three variants of the liposome fusion (coalescence) method to produce supported lipid bilayers, containing the ganglioside GM1 on silicon nitride surfaces, were studied. The first procedure involved attachment and fusion of liposomes containing DMPC, GM1 and a small amount of biotinylated lipid (Biotin-LC-DPPE) to a streptavidin coated surface. Direct fusion of liposomes composed of a mixture of DPPC, DPPG, DPPE, GM1 and cholesterol to the surface were the second variant. The final method utilised the second type of liposomes, fused onto a streptavidin layer with a small amount of exposed hydrophobic tails. The methods produced similar lipid layers, but with different ways of attachment to the surface. The binding of cholera toxin B-subunit (CTB) towards these sensor surfaces was measured in a resonant mirror biosensor instrument and the activity and longer-term stability of the layers were examined. The prepared surfaces were also imaged by atomic force microscopy (AFM) in liquid to characterise the topography of the lipid layers. The binding efficiency of CTB towards these surfaces was discussed in terms of lipid fluidity and surface roughness.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

Non-natural cell surface receptors: synthetic peptides capped with N-cholesterylglycine efficiently deliver proteins into Mammalian cells

Protein toxins such as shiga toxin and cholera toxin penetrate into cells by binding small molecule-based cell surface receptors localized to cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. Molecular recognition between these toxins and their receptors triggers endocytic protein uptake through endogenous membrane trafficking pathways. We report herein the synthesis of functionally related non-natural cell surface receptors comprising peptides capped with N-cholesterylglycine as the plasma membrane anchor. The peptide moieties of these receptors were based on high-affinity epitopes of anti-hemaglutinin antibodies (anti-HA), anti-Flag antibodies, and a moderate-affinity Strep Tag II peptide ligand of the streptavidin protein from Streptomyces avidini. These non-natural receptors were directly loaded into plasma membranes of Jurkat lymphocytes to display peptides from lipid rafts on the cell surface. Molecular recognition between these receptors and added cognate anti-HA, anti-Flag, or streptavidin proteins resulted in rapid clathrin-mediated endocytosis; fluorescent target proteins were completely internalized within 4-12 h of protein addition. Analysis of protein uptake by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of 100-fold to 200-fold (10 microM non-natural receptor) with typically >99% efficiency. This method enabled intracellular delivery of a functional Escherichia coli beta-galactosidase enzyme conjugated to Protein A from Staphylococcus aureus. We termed this novel delivery strategy "synthetic receptor targeting", which is an efficient method to enhance macromolecular uptake by decorating mammalian cells with chemically defined synthetic receptors that access the molecular machinery controlling the organization of cellular plasma membranes.     

Interaction of S-layer proteins of Lactobacillus kefir with model membranes and cells

In previous works, it was shown that S-layer proteins from Lactobacillus kefir were able to recrystallize and stabilize liposomes, this feature reveling a great potential for developing liposomal-based carriers. Despite previous studies on this subject are important milestones, a number of questions remain unanswered. In this context, the feasibility of S-layer proteins as a biomaterial for drug delivery was evaluated in this work. First, S-layer proteins were fully characterized by electron microscopy, 2D-electrophoresis, and anionic exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Afterward, interactions of S-layer proteins with model lipid membranes were evaluated, showing that proteins adsorb to the lipid surface following a non-fickean or anomalous diffusion, when positively charged lipid were employed, suggesting that electrostatic interaction is a key factor in the recrystallization process on these proteins. Finally, the interaction of S-layer coated liposomes with Caco-2 cell line was assessed: First, cytotoxicity of formulations was tested showing no cytotoxic effects in S-layer coated vesicles. Second, by flow cytometry, it was observed an increased ability to transfer cargo molecules into Caco-2 cells from S-layer coated liposomes in comparison to control ones. All data put together, supports the idea that a combination of adhesive properties of S-layer proteins concomitant with higher stability of S-layer coated liposomes represents an exciting starting point in the development of new drug carriers.     

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir–Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM).The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.     

Synthesis of the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) and its use with the IAsys biosensor to study receptor-ligand interactions on model membranes

This work describes the synthesis and use of the chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). This lipid is readily dispersed in aqueous media, both alone and when mixed with carrier lipids like dimyristoylphosphatidylcholine (DMPC). Fluorescence microscopic examination of membranes deposited from NTA-DTDA-containing liposomes shows that NTA-DTDA mixes uniformly with the carrier lipid, and does not phase separate. NTA-DTDA-membranes deposited onto the sensing surface of IAsys biosensor cuvettes show good stability, permitting use of the biosensor to study protein interactions. Hexahistidine-tagged proteins including recombinant forms of the extracellular regions of murine B7.1 (B7.1-6H) and of the human erythropoietin receptor (EPOR-6H) bind to NTA-DTDA-membranes; the stability of binding is dependent on both protein concentration, and density of NTA-DTDA. Kinetic measurements show that high stability of anchored proteins (t(1/2) approximately 10-20 h, apparent K(d) approximately 1 nM) can be achieved using membranes containing 25 mol% NTA-DTDA, but low levels of bound protein (<200 arc seconds). The system is used to study the interaction of human EPO with the EPOR anchored onto NTA-DTDA-membranes. In addition to the biological applications reported recently, the results show that NTA-DTDA can be a useful reagent in the study of receptor-ligand interactions.     

Artificially lipid-anchored proteins can elicit clustering-induced intracellular signaling events in Jurkat T-lymphocytes independent of lipid raft association

We have incorporated artificial lipid-anchored streptavidin conjugates with fully saturated or polyunsaturated lipid anchors into the plasma membranes of Jurkat T-lymphocytes to assess previous conclusions that the activation of signaling processes induced in these cells by clustering of endogenous glycosylphosphatidylinositol-anchored proteins or ganglioside GM1 depends specifically on the association of these membrane components with lipid rafts. Lipid-anchored streptavidin conjugates could be incorporated into Jurkat or other mammalian cell surfaces by inserting biotinylated phosphatidylethanolamine-polyethyleneglycols (PE-PEGs) and subsequently binding streptavidin to the cell-incorporated PE-PEGs. Saturated dipalmitoyl-PE-PEG-streptavidin conjugates prepared in this manner partitioned substantially into the detergent-insoluble membrane fraction isolated from Jurkat or fibroblast cells, whereas polyunsaturated dilinoleoyl-PE-PEG-anchored conjugates were wholly excluded from this fraction, consistent with the differences in the affinities of the two types of lipid anchors for liquid-ordered membrane domains. Remarkably, however, antibody-mediated cross-linking of either dipalmitoyl- or dilinoleoyl-PE-PEG-anchored streptavidin conjugates in Jurkat cells induced elevation of cytoplasmic calcium levels and tyrosine phosphorylation of the scaf-folding protein linker of T-cell activation in a manner similar to that observed upon cross-linking of endogenous CD59 or ganglioside GM1. The amplitude of the cross-linking-stimulated elevation of cytoplasmic calcium moreover showed an essentially identical dependence on the level of incorporated streptavidin conjugate for either type of lipid anchor. Confocal fluorescence microscopy revealed that PE-PEG-streptavidin conjugates with saturated versus polyunsaturated anchors showed very similar surface distributions vis à vis GM1 or CD59 under conditions where one or both species were cross-linked. These results indicate that cross-linking of diverse proteins anchored only to the outer leaflet of the plasma membrane can induce activation of Jurkat T-cell-signaling responses, but they appear to contradict previous suggestions that this phenomenon rests specifically on the association of such species with lipid rafts.     

Structure of planar membrane formed from liposomes

Lipid vesicles with incorporated ion channels from polyene antibiotic amphotericin B were used to investigate structures of planar membranes formed by Shindler's techniques. A planar membrane assembled on the aperture in a lavsan film from two layers generated at the air-aqueous liposome suspension interface is not a simple bilayer but a bimolecular membrane containing numerous partly fused liposomes. A complete fusion of liposomal membranes with the planar bilayer is an unlikely event during membrane formation. A planar bimolecular lipid membrane without incorporated liposomes can be made by a method consisting of three stages: formation of a lipid layer on the air-water interface of a suspension containing liposomes, transfer of this layer along the surface of the solution into a chamber containing a solution without liposomes where a lipid monomolecular layer forms gradually (within about 20 min) at the air-water interface, assembling of the planar bilayer membrane from this monolayer. The knowledge of the planar membrane structure may be useful in experiments on incorporation of membrane proteins into a planar lipid bilayer.     

Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system. By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic uptake, deliver their contents to the cytosol through fusion from within acidic endosomes. Here, we show that binding to sialic acid is not necessary for fusion. In the presence of streptavidin, virosomes containing a biotinylated lipid fused with liposomes lacking sialic acid if these liposomes also had a biotinylated lipid in their membranes. Moreover, fusion characteristics corresponded well with fusion of virosomes with ganglioside-containing liposomes.     

Stabilized lipid film based biosensor for atenolol

This work reports a technique for the stabilization after storage in air of a lipid film based biosensor for atenolol. Microporous filters composed of glass fibers (nominal pore sizes 0.7 and 1.0 microm) were used as supports for the formation and stabilization of these devices. The lipid film is formed on the filter by polymerization prior to its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. The method for preparation of stabilized lipid film biosensor is studied throughout this work. The response towards atenolol of these stabilized lipid membrane biosensor, for repetitive use, composed of phosphatidylcholine was compared with planar freely suspended bilayer lipid membranes (BLMs). The stabilized lipid membranes provided similar artificial ion gating events as BLMs in the form of transient signals and can function for repetitive uses after storage in air. This will allow the practical use of the techniques for chemical sensing based on lipid films and commercialization of these devices.     

Liposome filtration. Dependence on transition temperature

Liposomes formed by vortexing and passed through polycarbonate surface retention membranes showed appreciable differences in filtration behavior depending on the temperature of filtration relative to the liposome gel-liquid crystal transition temperature. Below transition, liposomes were filterable and size distributions could be determined; the cumulative volume distributions were log-normal. Above transition, liposomes were not filterable: smaller liposomes were formed until a limiting size was reached. These results suggest that liquid crystal liposome size distributions cannot be determined by filtration. This filtration behavior is a physical property of liposomes, related to the gel-liquid crystal transition, not previously reported. This property could be exploited as a new method for controlling liposome size distributions, but the implications for lipid membranes, including biological membranes, are general.     

Molecular organization of glutamate-sensitive chemoexcitable membranes of nerve cells. Function of glutamate-binding proteins of the central nervous system when incorporated into liposomes

The functioning of the glutamate-binding protein of rat brain cortex synaptic membranes was studied by its incorporation into liposomes. The optimal conditions for the receptor protein incorporation were established and the kinetics of 22Na+ and 86Rb+ incorporation into the liposomes in the presence of L-glutamate were analyzed. Modelling of the CNS glutamate receptor functions was found to be dependent on the lipid composition and amount of the incorporated membrane protein. The selective transport of 22Na+ into the liposomes was stimulated in the presence of 10(-4) M glutamate. Addition of monoclonal antibodies against glutamate-binding proteins blocked the incorporation of Na+ into the liposomes. The experimental results are suggestive of the nativity of the liposome-incorporated membrane protein, which is capable of binding glutamate and regulating selective transport of Na+. It was assumed that the glutamate receptor macromolecule represents an integral complex made up of several low molecular weight subunits of glucoprotein nature that form a selective ionic channel.     

The Gbetagamma dimer drives the interaction of heterotrimeric Gi proteins with nonlamellar membrane structures

Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Galphabetagamma) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Galpha and Gbetagamma). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal H(II)) membrane structures. We demonstrated that although heterotrimeric G(i) proteins and Gbetagamma dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein alpha subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gbetagamma dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gbetagamma dimers, and Galpha subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors.     

Functional reconstitution of the integral membrane proteins of influenza virus into phospholipid liposomes

The integral membrane proteins of influenza virus, a hemagglutinin and a neuraminidase, have been incorporated into liposomes composed of either phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylethanolamine (2:1 w/w) using detergent dialysis. The virus spike glycoproteins for reconstitution were selectively solubilized by using cetyltrimethylammonium bromide to leave a "core particle", which lacked a lipid bilayer but possessed quaternary structure as observed by electron microscopy. The viral spike proteins were combined with exogenous phospholipid in excess sodium cholate followed by exhaustive dialysis for 150 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that only the viral glycoproteins were associated with all the complexes formed. The level of sodium cholate remaining after dialysis was shown to be reduced to less than 1 molecule per 80 protein molecules. Viral proteins reconstituted into dimyristoylphosphatidylcholine liposomes were shown to have retained hemagglutination, low-pH-dependent hemolysis, and neuraminidase activities and were associated with a lipid bilayer in two types of complexes with average lipid to protein mole ratios after sucrose density gradient purification of either 590:1 or 970:1. The bilayer vesicles formed were of similar sizes and were shown by negative-stain electron microscopy to be 150-300 nm in diameter with well-defined spikes on their surface. Reconstituted liposomes of dimyristoylphosphatidylcholine were found to be unstable with respect to their trapped volume and therefore were unsuitable for fusion studies, unlike complexes formed with phosphatidylcholine or a mixture of phosphatidylcholine/phosphatidylethanolamine derived from hen eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

Sorting of streptavidin protein coats on phase-separating model membranes

Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.     

Liposome-associated tumor necrosis factor retains bioactivity in the presence of neutralizing anti-tumor necrosis factor antibodies

Cell-associated TNF-alpha, either bound to its receptor on monocyte membranes or expressed as an integral membrane protein, can exert potent tumor cytolytic activity. We assessed the interaction of TNF with the lipid bilayer membrane system, liposomes, and the effects of membrane association on TNF bioactivity. High levels of TNF can be encapsulated within liposomes. At neutral pH, TNF binds to the surface of preformed liposomes (liposome-associated TNF), but does not partition into the lipid bilayer. TNF appears to bind to negatively charged phospholipid head groups of the outer membrane leaflet. Free TNF, liposome-associated TNF, and liposome-encapsulated TNF display comparable abilities to activate human peripheral blood monocytes and to lyse tumor cells. However, liposome-encapsulated TNF, as well as TNF bound to the outer surface of preformed liposomes, retains bioactivity in the presence of anti-TNF antibodies that neutralize free TNF. The interaction of liposomal TNF with cell surface TNF receptors thus appears to be preserved in the presence of neutralizing antibodies.     

Two-dimensional crystallization of streptavidin by nonspecific binding to a surface film: study with a scanning electron microscope.

A two-dimensional (2D) crystal of streptavidin has been obtained by a nonspecific binding method. The protein molecules were bound and formed a dense packing on the film of poly(1-benzyl-L-histidine) spread at the surface of protein solution. The surface film was moderately heated to stimulate crystallization of bound streptavidin. A potential of this method for obtaining 2D crystals of soluble proteins is demonstrated. The present 2D crystal structure of streptavidin resembles that previously obtained by specific binding to biotinylated lipid. We show in addition that the 2D array of protein with usual size approximately 50 A can be imaged using a high resolution scanning electron microscope (HR-SEM) and subject to structural analysis at low resolution. Various limitations in HR-SEM degrade considerably the image quality. However, the usability of a bulk plate as specimen support would make HR-SEM a convenient tool, when such a substrate must be considered in application of protein arrays, and if an intrinsic low resolution is acceptable.