Asymmetric Model Membranes Research Articles

Conformational ensemble-dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

Passive Translocation of Phospholipids in Asymmetric Model Membranes: Solid-State 1H NMR Characterization of Flip-Flop Kinetics Using Deuterated Sphingomyelin and Phosphatidylcholine.

Although lateral and inter-leaflet lipid-lipid interactions in cell membranes play roles in maintaining asymmetric lipid bilayers, the molecular basis of these interactions is largely unknown. Here, we established a method to determine the distribution ratio of phospholipids between the outer and inner leaflets of asymmetric large unilamellar vesicles (aLUVs). The trimethylammonium group, (CH3)3N+, in the choline headgroup of N-palmitoyl-sphingomyelin (PSM) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) gave rise to a relatively sharp signal in magic-angle spinning solid-state 1H NMR (MAS-ss-1H NMR). PSM and DOPC have the same headgroup structure, but one phospholipid was selectively observed by deuterating the trimethylammonium group of the other phospholipid. The addition of Pr3+ to the medium surrounding aLUVs selectively shifted the chemical shift of the (CH3)3N+ group in the outer leaflet from that in the inner leaflet, which allowed estimation of the inter-leaflet distribution ratio of the unlabeled lipid in aLUVs. Using this method, we evaluated the translocation of PSM and DOPC between the outer and inner leaflets of the cholesterol-containing aLUVs, with PSM and DOPC mostly distributed in the outer and inner leaflets, respectively, immediately after aLUV preparation; their flip and flop rates were approximately 2.7 and 6.4 × 10-6 s-1, respectively. During the passive symmetrization of aLUVs, the lipid translocation rate was decreased due to changes in the membrane order, probably through the formation of the registered liquid-ordered domains. Comparison of the result with that of symmetric LUVs revealed that lipid asymmetry may not significantly affect the lipid translocation rates, while the lateral lipid-lipid interaction may be a dominant factor in lipid translocation under these conditions. These findings highlight the importance of considering the effects of lateral lipid interactions within the same leaflet on lipid flip-flop rates when evaluating the asymmetry of phospholipids in the cell membrane.

Phosphatidylinositol phase behavior in symmetric and asymmetric model membranes.

Phosphatidylinositol (PI) is a precursor to phosphoinositides (PIPs) which are important cellular signaling molecules. While the phase behavior of PI in simple model membranes has been investigated, little is known about its phase behavior in the presence of PIPs. Furthermore, the effect of divalent cations on PI/PIP interaction has not been detailed. Determining PI/PIP phase behavior is of physiological relevance because it potentially affects the lateral distribution of PIPs in cellular membranes and hence the spatiotemporal control of protein function. For diplamitoylphosphatidylinositol (DPPI) we find in pH 7.2 buffer a phase transition temperature of 43.6°C, while in the presence of Ca2+ and Mg2+ the phase transition temperature shifts to 55.8°C and 51.6°C, respectively. In addition, we investigate divalent cation dependent PI/PI(4,5)P2 phase behavior. To study PI phase behavior in asymmetric lipid bilayers, we fabricate asymmetric giant unilamellar vesicles (aGUVs) using the hemifusion method. We describe the conditions to obtain aGUVS with PI in the outer leaflet and characterize their domain morphology.

Insights on plasma membrane asymmetry from studies of asymmetric model membranes.

Cells spend a lot of energy to maintain the plasma membrane as asymmetric, with distinct leaflet compositions. In contrast, unhealthy cells, or cells with a signal to die, typically lose this asymmetry. Certainly, cells would evolve to such high complexity for a purpose. Studies in model membranes with rationally simplified lipid composition point to an exoplasmic model leaflet with coexistence of liquid disordered (Ld) and liquid ordered (Lo) phases. The apposed cytoplasmic leaflet lacks the large fraction of high melting lipids needed to sustain phase separation. To investigate properties of the plasma membrane asymmetry, we use a new method to engineer asymmetric bilayers by hemifusion between giant unilamellar vesicles (GUVs) and a supported lipid bilayer (SLB). The newly exchanged lipids from the SLB replace the lipids on the GUV outer leaflet, forming asymmetric vesicles (aGUVs). Little perturbation was observed in the vesicle content of these aGUVs. We assemble a phase-separated Ld+Lo leaflet apposed to a leaflet with a uniform Ld composition. For several different lipid mixtures, we observe an ordered phase inducing exactly apposed order on the fluid leaflet. Even with the challenge to track cholesterol movements, our results suggest a composition change, in which induced order domains become enriched with cholesterol. This result brings a new picture and a significant implication for the plasma membrane: The cytoplasmic leaflet has “raft-like” domains induced by the exoplasmic leaflet. Membrane-bound molecules in the cytoplasmic leaflet that can move laterally must partition between disordered regions and induced order regions, as we observe with lipid dyes. Based on line tension estimates, we explain why the induced order domains would be macroscopic domains, or modulated phases, or else nanodomains.

Investigating the influence of low-melting phospholipids on interleaflet coupling in asymmetric giant unilamellar vesicles

Compositional asymmetry is a characteristic of the plasma membrane whose biological importance is still mostly unknown. Asymmetric model membranes mimicking cell membrane composition offer a useful, highly controlled system for studying the coupling between lateral and transverse lipid organization. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were prepared by electroformation.

Model Membrane Systems Used to Study Plasma Membrane Lipid Asymmetry.

It is well known that the lipid distribution in the bilayer leaflets of mammalian plasma membranes (PMs) is not symmetric. Despite this, model membrane studies have largely relied on chemically symmetric model membranes for the study of lipid–lipid and lipid–protein interactions. This is primarily due to the difficulty in preparing stable, asymmetric model membranes that are amenable to biophysical studies. However, in the last 20 years, efforts have been made in producing more biologically faithful model membranes. Here, we review several recently developed experimental and computational techniques for the robust generation of asymmetric model membranes and highlight a new and particularly promising technique to study membrane asymmetry.

Investigation of the domain line tension in asymmetric vesicles prepared via hemifusion

The plasma membrane (PM) is asymmetric in lipid composition. The distinct and characteristic lipid compositions of the exoplasmic and cytoplasmic leaflets lead to different lipid-lipid interactions and physical-chemical properties in each leaflet. The exoplasmic leaflet possesses an intrinsic ability to form coexisting ordered and disordered fluid domains, whereas the cytoplasmic leaflet seems to form a single fluid phase. To better understand the interleaflet interactions that influence domains, we compared asymmetric model membranes that capture salient properties of the PM with simpler symmetric membranes. Using asymmetric giant unilamellar vesicles (aGUVs) prepared by hemifusion with a supported lipid bilayer, we investigate the domain line tension that characterizes the behavior of coexisting ordered + disordered domains. The line tension can be related to the contact perimeter of the different phases. Compared to macroscopic phase separation, the appearance of modulated phases was found to be a robust indicator of a decrease in domain line tension. Symmetric GUVs of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)/cholesterol (chol) were formed into aGUVs by replacing the GUV outer leaflet with DOPC/chol = 0.8/0.2 in order to create a cytoplasmic leaflet model. These aGUVs revealed lower line tension for the ordered + disordered domains of the exoplasmic model leaflet.

Coupling between Lateral and Transverse Organization in Three-Component Lipid Mixtures Investigated with Asymmetric GUVs Prepared by Hemifusion

Asymmetry is a ubiquitous feature of biological plasma membranes. Asymmetric model membranes allow a means of investigating molecular determinants of coupling between the inner and outer leaflets of the lipid bilayer. Here, we report a partial phase diagram for asymmetric bilayers composed of the ternary mixture DPPC, DOPC, and cholesterol. Symmetric giant unilamellar vesicles (GUVs) of DPPC/DOPC/Cholesterol with compositions along a tie-line in the symmetric composition space were prepared by electroformation.

Bilayer compositional asymmetry influences the nanoscopic to macroscopic phase domain size transition

The eukaryotic plasma membrane (PM) exhibits lipid mixing heterogeneities known as lipid rafts. These lipid rafts, the result of liquid-liquid phase separation, can be modeled by coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. Four-lipid component systems with a high-melting lipid, a nanodomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol) can give rise to domains of different sizes. These four-component systems have been characterized in experiments, yet there are few studies that model the asymmetric distribution of lipids actually found in the PM. We used molecular dynamics (MD) simulations to analyze the transition from nanoscopic to macroscopic domains in symmetric and in asymmetric model membranes. Using coarse-grained MD simulations, we found that asymmetry promotes macroscopic domain growth in a case where symmetric systems exhibit nanoscopic domains. Also, macroscopic domain formation in symmetric systems is highly dependent on registration of like phases in the cytoplasmic and exoplasmic leaflets. Using united-atom MD simulations, we found that symmetric Lo domains are only slightly more ordered than asymmetric Lo domains. We also found that large Lo domains in our asymmetric systems induce a slight chain ordering in the apposed cytoplasmic regions. The chol fractions of phase-separated Lo and Ld domains of the exoplasmic leaflet were unchanged whether the system was symmetric or asymmetric.

Lipid asymmetry of a model mitochondrial outer membrane affects Bax-dependent permeabilization

The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-β-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.

Translocation of the Cell Penetrating Peptide Penetratin through Asymmetric Model Membranes Formed by a Microfluidic Device: Role of the Lipids and Transmembrane Potential

Translocation of the Cell Penetrating Peptide Penetratin through Asymmetric Model Membranes Formed by a Microfluidic Device: Role of the Lipids and Transmembrane Potential

Peptide-Induced Lipid Flip-Flop in Asymmetric Liposomes Measured by Small Angle Neutron Scattering.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes in vitro, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with time-resolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) displayed slow spontaneous flip-flop at 37 ◦C (half-time, t1/2 = 140 h). However, inclusion of peptides, namely, gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide), accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to preformed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 h. Gramicidin, on the other hand, was preincorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g., adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.

Effect of Sterol Structure on Ordered Lipid Domains in Symmetric and Asymmetric Model Membranes

The packing of ordered sterol-sphingolipid-rich liquid ordered domains in biomembranes is greatly impacted by sterol structure. For this reason, the dependence of biological functions upon sterol structure can be used to identify processes likely to be dependent upon ordered domain formation. In this study we compared the effect of sterol structure upon ordered domain formation in symmetric vesicles composed of a mixture of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, to that in asymmetric vesicles in which sphingomyelin was introduced into only the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains being most strongly stabilized by 7-dehydrocholesterol and cholesterol, to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, we found that in asymmetric vesicles desmosterol stabilizes ordered domain almost as well as cholesterol, and stabilizes ordered domains to a much greater degree than epicholesterol. From previous studies using symmetric vesicles and a different lipid composition, we had come to the opposite conclusion: that epicholesterol stabilizes ordered domains similarly to cholesterol and to a greater degree than desmosterol. Based on behavior in asymmetric membranes, which mimic cell membranes more closely than symmetric membranes, we have re-evaluated our prior endocytosis and bacterial uptake studies and now conclude that sterol raft-forming ability may be the sole structural property of sterol that is necessary for these functions.

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition.

The double-membrane cellular envelope of Gram-negative bacteria enables them to endure harsh environments and represents a barrier to many clinically available antibiotics. The outer membrane (OM) is exposed to the environment and is the first point of contact involved in bacterial processes such as signaling, pathogenesis, and motility. As in the cytoplasmic membrane, the OM in Gram-negative bacteria has a phospholipid-rich inner leaflet and an outer leaflet that is predominantly composed of lipopolysaccharide (LPS). We report on a microfluidic technique for fabricating monodisperse asymmetric giant unilamellar vesicles (GUVs) possessing the Gram-negative bacterial OM lipid composition. Our continuous microfluidic fabrication technique generates 50-150 μm diameter water-in-oil-in-water double emulsions at high-throughput. The water-oil and oil-water interfaces facilitate the self-assembly of phospholipid and LPS molecules to create the inner and outer leaflets of the lipid bilayer, respectively. The double emulsions have ultrathin oil shells, which minimizes the amount of residual organic solvent that remains trapped between the leaflets of the GUV membrane. An extraction process by ethanol and micropipette aspiration of the ultrathin oil shells triggers an adhesive interaction between the two lipid monolayers assembled on the water-oil and oil-water interfaces (i.e., dewetting transition), forcing them to contact and form a lipid bilayer membrane. The effect of different inner-leaflet lipid compositions on the emulsion/vesicle stability and the dewetting transition is investigated. We also report on the values for bending and area expansion moduli of synthetic asymmetric model membranes with lipid composition/architecture that is physiologically relevant to the OM in Pseudomonas aeruginosa bacteria.

Accurate In Silico Modeling of Asymmetric Bilayers Based on Biophysical Principles

Technological advances in the last decade have enabled the study of ever more complex and physiologically relevant model membranes to help dispel the mystery surrounding the role of plasma membrane asymmetry in various cellular processes. The slowly accumulating body of experimental data is fueling renewed interest in and the need for computational methods to support interpretations and address a wide range of problems that are still not amenable to direct experimental study. The specific appeal of molecular dynamics simulations for this purpose lies in their ability to access information at atomic resolution, which is useful for the formulation of testable mechanistic hypotheses. But, the range of questions that can be addressed reliably with such simulations is determined by the appropriate construction and simulation of asymmetric bilayer models. One essential way to achieve this goal is to follow rigorous biophysical criteria and principles. In this context, we show that the requirement for a robust comparison between the properties of simulated asymmetric and symmetric model membranes is for the tension in each bilayer leaflet to be zero. Commonly used methods for constructing asymmetric bilayers, including matching the average areas of the leaflets from the corresponding symmetric systems, do not ensure zero leaflet tension, thus precluding physically realistic changes in the areas of the two leaflets. We present, to our knowledge, a new method for identifying the ideal lipid packing in bilayers with different leaflet compositions that achieves the zero-tension goal, and discuss the basic principles underlying the biophysically correct computational study of asymmetric membranes.

Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition.

The asymmetric distribution of lipids between the two bilayer leaflets represents a typical feature of biological membranes. The loss of this asymmetry, for example the exposure of negatively charged lipids on the extracellular membrane leaflet of mammalian cells, is involved in apoptosis and occurs in tumor cells. Thus, the controlled production of asymmetric liposomes helps to better understand such crucial cellular processes. Here, we present an approach that allows us to design asymmetric model-membrane experiments on a rational basis and predict the fraction of exchanged lipid. In addition, we developed a label-free and nondestructive assay to quantify the asymmetric uptake of negatively charged lipids in terms of the zeta potential. This significantly enhances the applicability, impact, and predictive power of model membranes.

Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.

Translocase Activity and Asymmetric Model Membranes Probed by Neutron Scattering

Biological membranes are almost universally asymmetric with individual leaflets containing different lipid species at varying concentrations. This asymmetry may play a key role in biological functions such as protein-membrane interactions, membrane trafficking, and cellular signaling. Traditional model membrane systems are comprised of symmetric membrane leaflets and though progress has been made in developing asymmetric model membranes, these systems are unstable and easily contaminated. We have developed an actively asymmetric biomembrane model system by purifying and reconstituting the well-characterized E. coli phospholipid ABC transporter MsbA into proteoliposomes that mimic the phospholipid composition of a physiological bacterial membrane. Subsequent MsbA-mediated phospholipid translocation and resultant membrane asymmetry was studied using small-angle neutron scattering (SANS). SANS is exceptionally well suited for studying the phospholipid distribution perpendicular to the bilayer plane due to its nondestructive nature and sub-nanometer resolution. Additionally, the hydrogen/deuterium (H/D) neutron scattering contrast variation from isotopic phospholipid labeling enables us to track the translocation of lipid membrane components without introducing bulky fluorescence or spin labels. In this study, the MsbA-mediated translocation of the phospholipid phosphatidylethamolamine (PE) between proteoliposome leaflets and the formation of asymmetric bilayers was studied using SANS for the first time. The sustained asymmetry in these model protocells will be central for future studies on biomimetic membrane systems and their role in diverse membrane-directed biological processes.

Structure and Correlation Functions of an Asymmetric Model Membrane of Five Components Display No Rafts but Rather the Propensity to Create them

We consider a model of an asymmetric lipid bilayer, one that consists of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (Co) in the outer leaf and phosphatidyletanolamine (PE), phosphatidylserine (PS), and cholesterol (Ci) in the inner leaf. As cholesterol can interchange between leaves, its chemical potential is the same in each leaf.The spontaneous curvature of the PE depends upon the cholesterol concentration and decreases with sufficient cholesterol concentration. Height fluctuations couple to the local concentration of PE which has the largest magnitude of spontaneous curvature. At physiological temperatures, the membrane is in a disordered phase with somewhat more than half of the total cholesterol in the inner leaf. We calculate all independent structure functions. Many, but not all of them, display a peak at a non-zero value of the wavenumber, a characteristic of a microemulsion. These include the intraleaf PE-PE, PE-PS, Ci-Ci, and SM-SM, PC-PC, Co-Co, and SM-Co structure functions. The interleaf Ci-Co and SM-Ci structure functions have the same behavior. The peak occurs at a wavenumber corresponding to a wavelength on the order of 80 nm indicating that the system is most susceptible to disturbances of this size. However the correlation functions corresponding to these structure functions, modeled as exponentially damped oscillations, display a correlation length which is only a few nanometers, and shorter than the wavelength. Taken together, these results indicate that the bilayer evidences no “rafts”, but is primed to create them in response to an external perturbation.