The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure

Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains.     

Transmembrane asymmetry and lateral domains in biological membranes

It is generally assumed that rafts exist in both the external and internal leaflets of the membrane, and that they overlap so that they are coupled functionally and structurally. However, the two monolayers of the plasma membrane of eukaryotic cells have different chemical compositions. This out-of-equilibrium situation is maintained by the activity of lipid translocases, which compensate for the slow spontaneous transverse diffusion of lipids. Thus rafts in the outer leaflet, corresponding to domains enriched in sphingomyelin and cholesterol, cannot be mirrored in the inner cytoplasmic leaflet. The extent to which lipids contribute to raft properties can be conveniently studied in giant unilamellar vesicles. In these, cholesterol can be seen to condense with saturated sphingolipids or phosphatidylcholine to form μm scale domains. However, such rafts fail to model biological rafts because they are symmetric, and because their membranes lack the mechanism that establishes this asymmetry, namely proteins. Biological rafts are in general of nm scale, and almost certainly differ in size and stability in inner and outer monolayers. Any coupling between rafts in the two leaflets, should it occur, is probably transient and dependent not upon the properties of lipids, but on transmembrane proteins within the rafts.     

Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation

In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ～37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting intact PMVs exhibit some degree of lipid asymmetry. This was supported by phosphatidylserine mislocalization on PMV outer leaflets as judged by annexin binding, which indicated NEM-induced PMVs are much more asymmetric than PMVs formed by dithiothreitol/paraformaldehyde treatment. Destroying asymmetry by reconstitution of PMVs using detergent dilution also showed stabilization of domain formation, even though membrane proteins remained associated with reconstituted vesicles. Similar domain stabilization was observed in artificial asymmetric lipid vesicles after destroying asymmetry via detergent reconstitution. Proteinase K digestion of proteins had little effect on domain stability in NEM PMVs. We conclude that loss of PMV lipid asymmetry can induce ordered domain formation. The dynamic control of lipid asymmetry in cells may regulate domain formation in plasma membranes.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.     

Improving our picture of the plasma membrane: Rafts induce ordered domains in a simplified model cytoplasmic leaflet

By study of asymmetric membranes, models of the cell plasma membrane (PM) have improved, with more realistic properties of the asymmetric lipid composition of the membrane being explored. We used hemifusion of symmetric giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB) to engineer bilayer leaflets of different composition. During hemifusion, only the outer leaflets of GUV and SLB are connected, exchanging lipids by simple diffusion. aGUVs were detached from the SLB for study. In general these aGUVs are formed with one leaflet that phase-separates into Ld (liquid disordered) + Lo (liquid ordered) phases, and another leaflet with lipid composition that would form a single fluid phase in a symmetric bilayer. We observed that ordered phases of either Lo or Lβ (gel phase) induce an ordered domain in the apposed fluid leaflet that lacks high melting lipids. Results suggest both an inter-leaflet and an intra-leaflet redistribution of cholesterol. We used C-Laurdan spectral images to investigate the lipid packing/order of aGUVs, finding that cholesterol partitions into the induced ordered domains. We suggest this behavior to be commonplace, that when Ld + Lo phase separation occurs in a cell PM exoplasmic leaflet, an induced order domain forms in the cytoplasmic leaflet.     

A Coarse Grained Model for a Lipid Membrane with Physiological Composition and Leaflet Asymmetry

The resemblance of lipid membrane models to physiological membranes determines how well molecular dynamics (MD) simulations imitate the dynamic behavior of cell membranes and membrane proteins. Physiological lipid membranes are composed of multiple types of phospholipids, and the leaflet compositions are generally asymmetric. Here we describe an approach for self-assembly of a Coarse-Grained (CG) membrane model with physiological composition and leaflet asymmetry using the MARTINI force field. An initial set-up of two boxes with different types of lipids according to the leaflet asymmetry of mammalian cell membranes stacked with 0.5 nm overlap, reliably resulted in the self-assembly of bilayer membranes with leaflet asymmetry resembling that of physiological mammalian cell membranes. Self-assembly in the presence of a fragment of the plasma membrane protein syntaxin 1A led to spontaneous specific positioning of phosphatidylionositol(4,5)bisphosphate at a positively charged stretch of syntaxin consistent with experimental data. An analogous approach choosing an initial set-up with two concentric shells filled with different lipid types results in successful assembly of a spherical vesicle with asymmetric leaflet composition. Self-assembly of the vesicle in the presence of the synaptic vesicle protein synaptobrevin 2 revealed the correct position of the synaptobrevin transmembrane domain. This is the first CG MD method to form a membrane with physiological lipid composition as well as leaflet asymmetry by self-assembly and will enable unbiased studies of the incorporation and dynamics of membrane proteins in more realistic CG membrane models.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

The dependence of lipid asymmetry upon polar headgroup structure

The effect of lipid headgroup structure upon the stability of lipid asymmetry was investigated. Using methyl-β-cyclodextrin -induced lipid exchange, sphingomyelin (SM) was introduced into the outer leaflets of lipid vesicles composed of phosphatidylglycerol, phosphatidylserine (PS), phosphatidylinositol, or cardiolipin, in mixtures of all of these lipids with phosphatidylethanolamine (PE), and in a phosphatidylcholine/phosphatidic acid mixture. Efficient SM exchange (>85% of that expected for complete replacement of the outer leaflet) was obtained for every lipid composition studied. Vesicles containing PE mixed with anionic lipids showed nearly complete asymmetry which did not decay after 1 day of incubation. However, vesicles containing anionic lipids without PE generally only exhibited partial asymmetry, which further decayed after 1 day of incubation. Vesicles containing the anionic lipid PS were an exception, showing nearly complete and stable asymmetry. It is likely that the combination of multiple charged groups on PE and PS inhibit transverse diffusion of these lipids across membranes relative to those lipids that only have one anionic group. Possible explanations of this behavior are discussed. The asymmetry properties of PE and PS may explain some of their functions in plasma membranes.     

Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.     

Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l(o)) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l(o) phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l(o) phase domains. We show here that l(o) phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l(o) and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.     

Coupling of cholesterol-rich lipid phases in asymmetric bilayers

We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typically found in the outer leaflet of plasma membranes induce liquid-ordered domains in adjacent regions of asymmetric lipid bilayers with apposed leaflets composed of typical inner leaflet lipid mixtures [Kiessling, V., Crane, J. M., and Tamm, L. K. (2006) Biophys. J. 91, 3313-26]. To further examine the nature of transbilayer couplings in asymmetric cholesterol-rich lipid bilayers, the effects on the lipid phase behavior in asymmetric bilayers of different lipid compositions were investigated. We established systems containing several combinations of natural extracted and synthetic lipids that exhibited coexisting liquid-ordered (lo) and liquid-disordered (ld) domains in a supported bilayer format. We find that lo phase domains are induced in all quaternary inner leaflet combinations composed of PCs, PEs, PSs, and cholesterol. Ternary mixtures of PCs/PEs/Chol, PCs/PSs/Chol also exhibit lo phases adjacent to outer leaflet lo phases. However, with the exception of brain PC extracts, binary PC/Chol mixtures are not induced to form lo phases by adjacent outer leaflet lo phases. Higher melting lipid ad-mixtures of PEs and PSs are needed for lo phase induction in the inner leaflet. It appears that the phase behavior of the inner leaflet mixtures is dominated by the intrinsic chain melting temperatures of the lipid components, rather than by their specific headgroup classes. In addition, similar studies with synthetic, completely saturated lipids and cholesterol show that lipid oxidation is not a factor in the observed phase behavior.     

Randomly organized lipids and marginally stable proteins: A coupling of weak interactions to optimize membrane signaling

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6–5.8 bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP₂), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP₂, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.     

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.     

Transbilayer lipid diffusion promoted by Bax: implications for apoptosis

It is known that the proapoptotic protein Bax facilitates the formation of pores in bilayers, resulting in the release of proteins from the intermitochondrial space. We demonstrate that another consequence of the interaction of Bax with membranes is an increase in the rate of lipid transbilayer diffusion. We use two independent assays for transbilayer diffusion, one involving the formation of asymmetric liposomes by placing a pyrene-labeled lipid into the outer monolayer of preformed vesicles and another assay based on the initial preparation of liposomes having an asymmetric transbilayer distribution of lipids. With both methods we find that oligomeric BaxDeltaC or full-length Bax in the presence of tBid, but not monomeric full-length Bax, strongly promotes the rate of transbilayer diffusion. Although biological membranes exhibit rates of lipid transbilayer diffusion of minutes or less, they are able to maintain an asymmetric distribution of lipids across the bilayer. In the case of mitochondria, cardiolipin is sequestered on the inner leaflet of the inner mitochondrial membrane. However, during apoptosis this lipid translocates to the outer surface of the outer mitochondrial membrane. This phenomenon must involve an increase in the rate of transbilayer diffusion. The results of the present paper demonstrate that an activated form of Bax can cause this increased rate.     

Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation

Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7 M serine and 0.3 M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.     

Physical Properties of Bacterial Outer Membrane Models: Neutron Reflectometry & Molecular Simulation

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM.     

How interaction of perfringolysin O with membranes is controlled by sterol structure, lipid structure, and physiological low pH: insights into the origin of perfringolysin O-lipid raft interaction

Perfringolysin O (PFO) is a sterol-dependent, pore-forming cytolysin. To understand the molecular basis of PFO membrane interaction, we studied its dependence upon sterol and lipid structure and aqueous environment. PFO interacted with diverse sterols, although binding was affected by double bond location in the sterol rings, sterol side chain structure, and sterol polar group structure. Importantly, a sterol structure promoting formation of ordered membrane domains (lipid rafts) was not critical for interaction. PFO membrane interaction was also affected by phospholipid acyl chain structure, being inversely related to tight acyl chain packing with cholesterol. Experiments using the pre-pore Y181A mutant demonstrated that sterol binding strength and specificity was not affected by whether PFO forms a transmembrane beta-barrel. Combined, these observations are consistent with a model in which the strength and specificity of sterol interaction arises from both sterol interactions with domain 4 and sterol chemical activity within membranes. The lipid raft-binding portions of sterol bound to PFO may remain largely exposed to the lipid bilayer. These results place important constraints upon the origin of PFO raft affinity. Additional experiments demonstrated that the structure of membrane-inserted PFO at low and neutral pH was similar as judged by the effect of phospholipid and sterol structure upon PFO properties and membrane interaction. However, low pH enhanced PFO membrane binding, oligomerization, and pore formation. In lipid vesicles mimicking the exofacial (outer) membrane leaflet, PFO-membrane binding was maximal at pH 5.5-6. This is consistent with the hypothesis that PFO function involves acidic vacuoles.