Nanosized bilayer disks: Attractive model membranes for drug partition studies

Stable nanosized bilayer disks were prepared from either 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, or lipid mixtures with a composition reflecting that of the porcine brush border membrane. Two different polyethylene glycol (PEG)-grafted lipids, the negatively charged 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000] (DSPE-PEG₅₀₀₀) and the neutral N-palmitoyl-sphingosine-1-[succinyl (methoxy (polyethylene glycol) 5000] (Ceramide-PEG₅₀₀₀), were used to stabilize the disks. The disks were employed as model membranes in drug partition studies based on a fast chromatography method. Results show that the lipid composition, as well as the choice of PEG-lipid, have an important influence on the partition behavior of charged drugs. Comparative studies using multilamellar liposomes indicate that bilayer disks have the potential to generate more accurate partition data than do liposomes. Further, initial investigations using bacteriorhodopsin suggest that membrane proteins can be reconstituted into the bilayer disks. This fact further strengthens the potential of the bilayer disk as an attractive model membrane.     

Development and initial evaluation of PEG-stabilized bilayer disks as novel model membranes

We show in this study that stable dispersions dominated by flat bilayer disks may be prepared from a carefully optimized mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] [PEG-DSPE(5000)]. By varying the content of the latter component, the average diameter of the disks can be changed in the interval from about 15 to 60 nm. The disks show excellent long-term stability, and their size and structure remain unaltered in the temperature range between 25 and 37 degrees C. The utility of the disks as artificial model membranes was confirmed and compared to uni- and multilamellar liposomes in a series of drug partition studies. Data obtained by isothermal titration calorimetry and drug partition chromatography (also referred to as immobilized liposome chromatography) indicate that the bilayer disks may serve as an attractive and sometimes superior alternative to liposomes in studies aiming at the investigation of drug-membrane interactions. The disks may, in addition, hold great potential for structure/function studies of membrane-bound proteins. Furthermore, we suggest that the sterically stabilized bilayer disks may prove interesting as carriers for in vivo delivery of protein/peptide, as well as conventional amphiphilic and/or hydrophobic, drugs.     

Evaluation of bilayer disks as plant cell membrane models in partition studies

We have studied the partitioning of a set of phenolic compounds used as lignin precursor models into lipid bilayer disks and liposomes. The bilayer disks are open bilayer structures stabilized by polyethylene glycol-conjugated lipids. Our results indicate that disks generate more accurate partition data than do liposomes. Furthermore, we show that the partitioning into the membrane phase is reduced slightly if disks composed of 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol are exchanged for disks with a lipid composition mimicking that of the root tissue of Zea mays L.     

Biotinylated lipid bilayer disks as model membranes for biosensor analyses

The aim of this study was to investigate the potential of polyethylene glycol (PEG)-stabilized lipid bilayer disks as model membranes for surface plasmon resonance (SPR)-based biosensor analyses. Nanosized bilayer disks that included 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)₂₀₀₀] (DSPE-PEG₂₀₀₀-biotin) were prepared and structurally characterized by cryo-transmission electron microscopy (cryo-TEM) imaging. The biotinylated disks were immobilized via streptavidin to three different types of sensor chips (CM3, CM4, and CM5) varying in their degree of carboxymethylation and thickness of the dextran matrix. The bilayer disks were found to interact with and bind stably to the streptavidin-coated sensor surfaces. As a first step toward the use of these bilayer disks as model membranes in SPR-based studies of membrane proteins, initial investigations were carried out with cyclooxygenases 1 and 2 (COX 1 and COX 2). Bilayer disks were preincubated with the respective protein and thereafter allowed to interact with the sensor surface. The signal resulting from the interaction was, in both cases, significantly enhanced as compared with the signal obtained when disks alone were injected over the surface. The results of the study suggest that bilayer disks constitute a new and promising type of model membranes for SPR-based biosensor studies.     

Influence of preparation path on the formation of discs and threadlike micelles in DSPE-PEG(2000)/lipid systems

In a recent study we showed that the surfactant 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG(2000)) induce mixed micelles of either threadlike or discoidal shape when mixed with different types of lipids. In certain lipid systems the discoidal micelles adapt sizes large enough to be characterized as bilayer discs. The discs hold great potential for use in various biotechnical applications and may e.g. be used as model membranes in drug/membrane partition studies. Depending on the application, discs with certain characteristics, such as a particular size or size homogeneity, may be required. These factors can in our experience be influenced by the preparation method. In this study we systematically investigated three different PEG-lipid/lipid mixtures prepared by four commonly used preparation techniques. The techniques used were simple hydration, freeze-thawing, sonication and detergent depletion, and the aggregate size and structure was analyzed by cryo transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). Our results show that the type and size of the micellar structure found, as well as the structure homogeneity of the preparation, can be modified by the choice of preparation path.     

Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

To investigate drug–membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03 mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance–Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.     

Stimulated release of cholesterol from liposomal membranes by a PEGylated phospholipid

PEGylated phospholipids are commonly used to increase the blood-circulation time of liposomes by providing a steric barrier around them. This paper documents a fundamentally new property of these lipids-an ability to stimulate the release of cholesterol from phospholipid membranes. Evidence for such stimulation has been obtained by measuring the transport of dehydroergosterol (DHE), a fluorescent simulant of cholesterol, from donor liposomes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG(2000)), and DHE to acceptor liposomes made from POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and cholesterol. The potential of PEGylated lipids to serve as novel cholesterol-lowering agents is briefly discussed.     

Polyethylene glycol-stabilized lipid disks as model membranes in interaction studies based on electrokinetic capillary chromatography and quartz crystal microbalance

Distearoylphosphatidylcholine (DSPC)/cholesterol/distearoylphosphatidylethanolamine (DSPE)–polyethylene glycol 5000 [PEG(5000)] lipid disks, mimicking biological membranes, were used as pseudostationary phase in partial filling electrokinetic capillary chromatography (EKC) to study interactions between pharmaceuticals and lipid disks. Capillaries were coated either noncovalently with a poly(1-vinylpyrrolidone)-based copolymer or covalently with polyacrylamide to mask the negative charges of the fused-silica capillary wall and to minimize interactions between positively charged pharmaceuticals and capillary wall. Although the noncovalent copolymer coating method was faster, better stability of the covalent polyacrylamide coating at physiological pH 7.4 made it more reliable in partial filling EKC studies. Migration times of pharmaceuticals were proportional to the amount of lipids in the pseudostationary phase, and partition coefficients were successfully determined. Because the capillary coatings almost totally suppressed the electroosmotic flow, it was not practical to use the EKC-based method for partition studies involving large molecules with low mobilities. Hence, the applicability of the biomembrane mimicking lipid disks for interactions studies with large molecules was verified by the quartz crystal microbalance technique. Biotinylated lipid disks were then immobilized on streptavidin-coated sensor chip surface, and interactions with a high-molecular-mass molecule, lysozyme, were studied. Cryo-transmission electron microscopy and asymmetrical flow field-flow fractionation were used to clarify the sizes of lipid disks used.     

Targeting of antibody conjugated,phosphatidylserine-containing liposomes to vascular cell adhesion molecule 1 for controlled thrombogenesis

Phosphatidylserine (PS) membrane exposure plays an important role in blood coagulation, and the development of a liposome formulation containing PS may be of potential therapeutic utility if they can be designed to achieve tumor selective thrombosis. The objective of this study was to develop proof-of-principle data for a thrombogenic PS liposome targeted to vascular cell adhesion molecule 1 (VCAM-1) via the attachment of an anti-VCAM-1 monoclonal antibody (Ab). We have evaluated binding of the anti-VCAM-1 Ab-conjugated PS liposomes to VCAM-1 using two in vitro models, as well as assessing the ability of these liposomes to catalyze blood coagulation reactions. Binding of the Ab-conjugated PS liposomes containing 2 or 14 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DSPE-PEG(2000)) to interleukin 1alpha stimulated human umbilical vein endothelial cells was 8- and 16-fold higher than those without conjugated Ab, respectively, based on the percentage relative increase in cell associated lipid for these liposomes. Binding to VCAM-1-coated ELISA plates produced similar results. The VCAM-1-bound Ab-conjugated PS liposomes were capable of catalyzing blood coagulation reactions upon the exposure of the thrombogenic PS membrane surface. This control of PS surface exposure was achieved using exchangeable PEG-derivatized phosphatidylethanolamines (PE-PEG), with 97% of clotting activity recovered after PE-PEG exchanged out. Our results demonstrate the potential for considering further development of procoagulant liposomes that selectively target thrombogenesis in tumor vasculature.     

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

Abstract

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (F_s), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when F_s is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.     

Steric stabilization of lipid/polymer particle assemblies by poly(ethylene glycol)-lipids

Biocompatible and biodegradable assemblies consisting of spherical particles coated with lipid layers were prepared from sub-micrometer poly(lactic acid) particles and lipid mixtures composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-3-trimethylammonium-propane. These original colloidal assemblies, named LipoParticles, are of a great interest in biotechnology and biomedicine. Nevertheless, a major limitation of their use is their poor colloidal stability toward ionic strength. Indeed, electrostatic repulsions failed to stabilize LipoParticles in aqueous solutions containing more than 10 mM NaCl. By analogy with the extensive use of poly(ethylene glycol) (PEG)-lipid conjugates to improve the circulation lifetime of liposomes in vivo, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] with various PEG chain lengths was added to the lipid formulation. Here, we show that LipoParticle stabilization was enhanced at least up to 150 mM NaCl (for more than 1 year at 4 degrees C). To determine the structure of PEG-modified LipoParticles as a function of the PEG chain length and the PEG-lipid fraction in the lipid formulation, a thorough physicochemical characterization was carried out by means of many techniques including quasi-elastic light scattering, zeta potential measurements, transmission electron microscopy, 1H NMR spectroscopy, and small-angle X-ray scattering. Finally, an attempt was made to link the resulting structural data to the colloidal behavior of PEG-modified LipoParticles.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids

Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.     

Adhesion of Positively Charged Liposomes to Mucosal Tissues

Abstract

A series of positively charged phospholipid and cholesterol derivatives was synthesized and evaluated as membrane components for liposomes. Small unilamellar liposomes containing up to 40 mole% of the synthetic lipids were prepared by sonication. Selected liposome preparations containing these synthetic lipid materials were found to be noncytotoxic in vitro by using a cell growth inhibition assay, whereas liposomes containing more classic positively charged components (stearylamine and cetyltrimethylammonium bromide) showed considerable cytotoxicity. Using an unanesthetized rabbit eye model, we have found that inclusion of the positively charged lipid derivatives into the liposomes significantly enhanced the ocular retention compared to neutral or negatively charged liposomes, presumably by molecular association with poly anionic corneal and conjunctival surface mucoglycoproteins. the increased retention was dependent on charge density and rigidity of the lipid bilayer. An assay for primary amino groups in these liposomes suggested that the distribution of the charged molecules between the inner and outer leaflets of the bilayer could be manipulated by lipid composition. Studies of liposomes containing cholesteryl esters of amino acids of various carbon chain lengths indicated that the charged amino groups need to extend from the surface of the lipid bilayers for better adhesion and retention. the ocular surface was saturable with respect to applied liposomes, which were cleared slowly from the eye with a half-time of clearance of about 2 hr. these data suggest a specific adhesion of the cationic liposomes to the surface of mucosal tissues.     

Interactions of polymerizable phosphatidylcholine vesicles with blood components: relevance to biocompatibility

We have studied the biocompatibility properties of polymerizable phosphatidylcholine bilayer membranes, in the form of liposomes, with a view toward the eventual utilization of such polymerized lipid assemblies in drug carrier systems or as surface coatings for biomaterials. The SH-based polymerizable lipid 1,2-bis[1,2-(lipoyl)dodecanoyl]-sn-glycero-3-phosphocholine (dilipoyl lipid, DLL) and the methacryl-based lipid 1,2-bis[(methacryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (dipolymerizable lipid, DPL) were studied in comparison to ‘conventional’ zwitterionic or charged phospholipids. We examined binding of serum proteins to liposomes and effects of liposomes on fibrin clot formation and on platelet aggregation. All types of liposomes tested bound complex mixtures of serum proteins with IgG being the most abundant bound component. DPL vesicles and anionic vesicles bound substantially more protein than other vesicle types. Polymerized DPL vesicles uniquely bound a protein of about 53 kDa which was not bound to other types of phosphatidylcholine liposomes. Likewise polymerized DPL vesicles, but not other types of phosphatidylcholine vesicles, caused a marked alteration in coagulation as measured by activated partial thromboplastin time (APTT) and prothrombin time (PT) tests; this effect was shown to be due to binding and depletion of clothing factor V by the DPL polymerized vesicles. Polymerized DPL liposomes and DLL liposomes in polymerized or nonpolymerized form, were without substantial effect on platelet aggregation. However, DPL nonpolymerized vesicles, while not causing aggregation, did impair ADP-induced aggregation of platelets. These studies suggest that SH based polymerizable lipids of the DLL type may be very suitable for in vivo use in the contexts of drug delivery systems or biomaterials development. Methacryloyl-based lipids of the DPL type seem to display interactions with the hemostatic process which militate against their in vivo utilization.     

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.     

Membrane fusion by proline-rich Rz1 lipoprotein, the bacteriophage lambda Rz1 gene product.

The fusogenic properties of Rz1, the proline-rich lipoprotein that is the bacteriophage lambda Rz1 gene product, were studied. Light scattering was used to monitor Rz1-induced aggregation of artificial neutral (dipalmitoylphosphatidylcholine/cholesterol) and negatively charged (dipalmitoylphosphatidylcholine/cholesterol/dioleoylphosphatidylserin e) liposomes. Fluorescence assays [the resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)dihexadecanol-sn-glycero-3-phosphoethanolamine lipid fluorescent probes, as well as fluorescent complex formation between terbium ions and dipicolinic acid encapsulated in two liposome populations and calcein fluorescence] were used to monitor Rz1-induced lipid mixing, contents mixing and leakage of neutral and negatively charged liposomes. The results demonstrated that Rz1 caused adhesion of neutral and negatively charged liposomes with concomitant lipid mixing; membrane distortion, leading to the fusion of liposomes and hence their internal content mixing; and local destruction of the membrane accompanied by leakage of the liposome contents. The use of artificial membranes showed that Rz1 induced the fusion of membranes devoid of any proteins. This might mean that the proline stretch of Rz1 allowed interaction with membrane lipids. It is suggested that Rz1-induced liposome fusion was mediated primarily by the generation of local perturbation in the bilayer lipid membrane and to a lesser extent by electrostatic forces.     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.