Asymmetry of lipid bilayers induced by monovalent salt: atomistic molecular-dynamics study

Interactions between salt ions and lipid components of biological membranes are essential for the structure, stability, and functions of the membranes. The specific ionic composition of aqueous buffers inside and outside of the cell is known to differ considerably. To model such a situation we perform atomistic molecular-dynamics (MD) simulations of a single-component phosphatidylcholine lipid bilayer which separates two aqueous reservoirs with and without NaCl salt. To implement the difference in electrolyte composition near two membrane sides, a double bilayer setup (i.e., two bilayers in a simulation box) is employed. It turns out that monovalent salt, being in contact with one leaflet only, induces a pronounced asymmetry in the structural, electrostatic, and dynamical properties of bilayer leaflets after 50 ns of MD simulations. Binding of sodium ions to the carbonyl region of the leaflet which is in contact with salt results in the formation of "Na-lipids" complexes and, correspondingly, reduces mobility of lipids of this leaflet. In turn, attractive interactions of chloride ions (mainly located in the aqueous phase close to the water-lipid interface) with choline lipid groups lead to a substantial (more vertical) reorientation of postphatidylcholine headgroups of the leaflet adjoined to salt. The difference in headgroup orientation on two sides of a bilayer, being coupled with salt-induced reorientation of water dipoles, leads to a notable asymmetry in the charge-density profiles and electrostatic potentials of bilayer constitutes of the two leaflets. Although the overall charge density of the bilayer is found to be almost insensitive to the presence of salt, a slight asymmetry in the charge distribution between the two bilayer leaflets results in a nonzero potential difference of about 85 mV between the two water phases. Thus, a transmembrane potential of the order of the membrane potential in a cell can arise without ionic charge imbalance between two aqueous compartments.     

Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy

There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103, 307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never been experimentally examined in any of these systems. In the present work, we developed a technique that is based on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supported bilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and without cholesterol in liquid-ordered (l(o)) and liquid-disordered (l(d)) phases. Of five different fluorescent lipid probes that were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine B] was the best for studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetrically labeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed. In LB/LS bilayers of either l(o) or l(d) phase, cholesterol increased the degree of lipid mixing between the opposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipid asymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetric label, with or without the use of a tethered polymer support. Finally, lipid mixing across the center of LB/LS bilayers was found to have drastic effects on the appearance of l(d)-l(o) phase coexistence as shown by epifluorescence microscopy.     

Nonintercalating nanosubstrates create asymmetry between bilayer leaflets

The physical properties of lipid bilayers can be remodeled by a variety of environmental factors. Here we investigate using molecular dynamics simulations the specific effects of nanoscopic substrates or external contact points on lipid membranes. We expose palmitoyl-oleoyl phosphatidylcholine bilayers unilaterally and separately to various model nanosized substrates differing in surface hydroxyl densities. We find that a surface hydroxyl density as low as 10% is sufficient to keep the bilayer juxtaposed to the substrate. The bilayer interacts with the substrate indirectly through multiple layers of water molecules; however, despite such buffered interaction, the bilayers exhibit certain properties different from unsupported bilayers. The substrates modify transverse lipid fluctuations, charge density profiles, and lipid diffusion rates, although differently in the two leaflets, which creates an asymmetry between bilayer leaflets. Other properties that include lipid cross-sectional areas, component volumes, and order parameters are minimally affected. The extent of asymmetry that we observe between bilayer leaflets is well beyond what has been reported for bilayers adsorbed on infinite solid supports. This is perhaps because the bilayers are much closer to our nanosized finite supports than to infinite solid supports, resulting in a stronger support-bilayer electrostatic coupling. The exposure of membranes to nanoscopic contact points, therefore, cannot be considered as a simple linear interpolation between unsupported membranes and membranes supported on infinite supports. In the biological context, this suggests that the exposure of membranes to nonintercalating proteins, such as those belonging to the cytoskeleton, should not always be considered as passive nonconsequential interactions.     

Membrane potential and electrostatics of phospholipid bilayers with asymmetric transmembrane distribution of anionic lipids

It is well-established that native plasma membranes are characterized by an asymmetric distribution of charged (anionic) lipids across the membrane. To clarify how the asymmetry can affect membrane electrostatics, we have performed extensive atomic-scale molecular dynamics simulations of asymmetric lipid membranes composed of zwitterionic (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and anionic (phosphatidylserine (PS)) leaflets. It turns out that the asymmetry in transmembrane distribution of anionic lipids gives rise to a nonzero potential difference between the two sides of the membrane. This potential arises from the difference in surface charges of the two leaflets. The magnitude of the intrinsic membrane potential was found to be 238 mV and 198 mV for PS/PC and PS/PE membranes, respectively. Remarkably, this potential is of the same sign as the membrane potential in cells. Our findings, being in reasonable agreement with available experimental data, lend support to the idea that the transmembrane lipid asymmetry typical of most living cells contributes to the membrane potential.     

Structure of docosahexaenoic acid-containing phospholipid bilayers as studied by (2)H NMR and molecular dynamics simulations.

Polyunsaturated phospholipids are known to be important with regard to the biological functions of essential fatty acids, for example, involving neural tissues such as the brain and retina. Here we have employed two complementary structural methods for the study of polyunsaturated bilayer lipids, viz. deuterium ((2)H) NMR spectroscopy and molecular dynamics (MD) computer simulations. Our research constitutes one of the first applications of all-atom MD simulations to polyunsaturated lipids containing docosahexaenoic acid (DHA; 22:6 cis-Delta(4,7,10,13,16,19)). Structural features of the highly unsaturated, mixed-chain phospholipid, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), have been studied in the liquid-crystalline (L(alpha)) state and compared to the less unsaturated homolog, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The (2)H NMR spectra of polyunsaturated bilayers are dramatically different from those of less unsaturated phospholipid bilayers. We show how use of MD simulations can aid in interpreting the complex (2)H NMR spectra of polyunsaturated bilayers, in conjunction with electron density profiles determined from small-angle X-ray diffraction studies. This work clearly demonstrates preferred helical and angle-iron conformations of the polyunsaturated chains in liquid-crystalline bilayers, which favor chain extension while maintaining bilayer flexibility. The presence of relatively long, extended fatty acyl chains may be important for solvating the hydrophobic surfaces of integral membrane proteins, such as rhodopsin. In addition, the polyallylic DHA chains have a tendency to adopt back-bended (hairpin-like) structures, which increase the interfacial area per lipid. Finally, the material properties have been analyzed in terms of the response of the bilayer to mechanical stress. Simulated bilayers of phospholipids containing docosahexaenoic acid were less sensitive to the applied surface tension than were saturated phospholipids, possibly implying a decrease in membrane elasticity (area elastic modulus, bending rigidity). The above features distinguish DHA-containing lipids from saturated or monounsaturated lipids and may be important for their biological modes of action.     

Engineered lipids that cross-link the inner and outer leaflets of lipid bilayers

The application of supported lipid bilayer systems as molecular sensors, diagnostic devices, and medical implants is limited by their lack of stability. In an effort to enhance the stability of supported lipid bilayers, three pairs of phosphatidylcholine lipids were designed to cross-link at the termini of their 2-position acyl chain upon the formation of lipid bilayers. The cross-linked lipids span the lipid bilayer, resembling naturally occurring bolaamphiphiles that stabilize archaebacterial membranes against high temperatures. The three reactions investigated here include the acyl chain cross-linking between thiol and bromine groups, thiol and acryloyl groups, and cyclopentadiene and acryloyl groups. All three reactive lipid pairs were found to cross-link in liposomal membranes, as determined by thin-layer chromatography, ion-spray mass spectrometry, and 1H NMR. The monolayer film properties of the reactive amphiphiles were characterized by surface pressure-area isotherms and showed that stable monolayers formed at the air-water interface with limiting molecular areas comparable to that of pure saturated phosphatidylcholine lipids. Langmuir-Blodgett bilayers of dimyristoylphosphatidylcholine incorporating 15 mol % of the reactive thiol and acryloyl lipids had diffusion coefficients comparable with pure dimyristoylphosphatidylcholine, while bilayers with more than 25 mol % of the reactive lipids were immobile, suggesting that interleaflet cross-linking of the lipids inhibited membrane diffusion. Our results show that the reactive lipids can cross-link within a lipid bilayer and are suitable for assembling supported lipid bilayers using Langmuir-Blodgett deposition. By using terminally reactive amphiphiles to build up supported lipid bilayers with cross-linked leaflets, bolaamphiphiles can be incorporated into asymmetric solid supported membranes to increase their stability in biosensor and medical implant applications.     

Asymmetric insertion of membrane proteins in lipid bilayers by solid-state NMR paramagnetic relaxation enhancement: a cell-penetrating Peptide example

A novel solid-state NMR technique for identifying the asymmetric insertion depths of membrane proteins in lipid bilayers is introduced. By applying Mn (2+) ions on the outer but not the inner leaflet of lipid bilayers, the sidedness of protein residues in the lipid bilayer can be determined through paramagnetic relaxation enhancement (PRE) effects. Protein-free lipid membranes with one-side Mn (2+)-bound surfaces exhibit significant residual (31)P and lipid headgroup (13)C intensities, in contrast to two-side Mn (2+)-bound membranes, where lipid headgroup signals are mostly suppressed. Applying this method to a cell-penetrating peptide, penetratin, we found that at low peptide concentrations, penetratin is distributed in both leaflets of the bilayer, in contrast to the prediction of the electroporation model, which predicts that penetratin binds to only the outer lipid leaflet at low peptide concentrations to cause an electric field that drives subsequent peptide translocation. The invalidation of the electroporation model suggests an alternative mechanism for intracellular import of penetratin, which may involve guanidinium-phosphate complexation between the peptide and the lipids.     

Effects of isoflurane, halothane, and chloroform on the interactions and lateral organization of lipids in the liquid-ordered phase

The first quantitative insight has been obtained into the effects that volatile anesthetics have on the interactions and lateral organization of lipids in model membranes that mimic "lipid rafts". Specifically, nearest-neighbor recogntion measurements, in combination with Monte Carlo simulations, have been used to investigate the action of isoflurane, halothane, and chloroform on the compactness and lateral organization of cholesterol-rich bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the liquid-ordered (l(o)) phase. All three anesthetics induce a similar weakening of sterol-phospholipid association, corresponding to ca. 30 cal/mol of lipid at clinically relevant concentrations. Monte Carlo lattice simulations show that the lateral organization of the l(o) phase, under such conditions, remains virtually unchanged. In sharp contrast to their action on the l(o) phase, these anesthetics have been found to have a similar strengthening effect on sterol-phospholipid association in the liquid-disordered (l(d)) phase. The possibility of discrete complexes being formed between DPPC and these anesthetics and the biological relevance of these findings are discussed.     

Lipid diffusion compared in outer and inner leaflets of planar supported bilayers

The translational diffusion coefficient (D) of lipids located in the outer and inner leaflets of planar supported DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) bilayers in the fluid phase was measured using fluorescence correlation spectroscopy of dye-labeled lipids at the low concentration of 0.001% and using iodide quenching of dyes in the outer leaflet to distinguish diffusion in the inner leaflet from that in the outer leaflet. To confirm the generality of these findings, the bilayers were prepared not only by vesicle fusion but also by Langmuir-Blodgett deposition. We conclude that regardless of whether the bilayers were supported on quartz or on a polymer cushion, D in the inner and outer leaflets was the same within an experimental uncertainty of +/-10% but with a small systematic tendency to be slower (by <5%) within the inner leaflet.     

Lipid-based mechanisms for vesicle fission

Shape transformations and topological changes of lipid vesicles, such as fusion, budding, and fission, have important chemical physical and biological significance. In this paper, we study the fission process of lipid vesicles. Two distinct routes are considered that are both based on an asymmetry of the lipid distribution within the membrane. This asymmetry consists of a nonuniform distribution of two types of lipids. In the first mechanism, the two types of lipids are equally distributed over both leaflets of the membrane. Phase separation of the lipids within both leaflets, however, results in the formation of rafts, which form buds that can split off. In the second mechanism, the asymmetry consists of a difference in composition between the two monolayers of the membrane. This difference in composition yields a spontaneous curvature, reshaping the vesicle into a dumbbell such that it can split. Both pathways are studied with molecular dynamics simulations using a coarse-grained lipid model. For each of the pathways, the conditions required to obtain complete fission are investigated, and it is shown that for the second pathway, much smaller differences between the lipids are needed to obtain fission than for the first pathway. Furthermore, the lipid composition of the resulting split vesicles is shown to be completely different for both pathways, and essential differences between the fission pathway and the pathway of the inverse process, i.e., fusion, are shown to exist.     

Effect of low amounts of cholesterol on the swelling behavior of floating bilayers

The effect of the addition of 1, 2, 4, and 6 mol % cholesterol to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) floating bilayers has been investigated by neutron reflectivity. All samples exhibited fully stable and reversible gel and fluid phases. Around the main lipid phase transition temperature, DPPC double bilayers exhibit large increases in the water layer separating the bilayers and the upper bilayer roughness. The inclusion of low amounts of cholesterol reduced the swelling of the water layer between the bilayers and the upper bilayer roughness and progressively widened the temperature range over which swelling occurs. Results from asymmetric bilayers are also reported. A higher amount of cholesterol in the lower bilayer induces a smaller swelling of the water layer between the bilayers than in the symmetric case. Finally, the effect of the inclusion of a leaflet of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was investigated. The presence of a leaflet with a higher gel-transition temperature (T(m)) modifies the phase behavior of the lower T(m) leaflet.     

Interactions of lipid bilayers with supports: a coarse-grained molecular simulation study

The study of lipid structure and phase behavior at the nanoscale is of utmost importance due to implications in understanding the role of the lipids in biochemical membrane processes. Supported lipid bilayers play a key role in understanding real biological systems, but they are vastly underrepresented in computational studies. In this paper, we discuss molecular dynamics simulations of supported lipid bilayers using a coarse-grained model. We first focus on the technical implications of modeling solid supports for biomembrane simulations. We then describe noticeable influences of the support on the systems. We are able to demonstrate that the bilayer system behavior changes when supported by a hydrophilic surface. We find that the thickness of the water layer between the support and the bilayer (the inner-water region in the latter part of this paper) adapts through water permeation on the microsecond time scale. Additionally, we discuss how different surface topologies affect the bilayer. Finally, we point out the differences between the two leaflets induced by the support.     

Characterization of symmetric and asymmetric lipid bilayers composed of varying concentrations of ganglioside GM1 and DPPC

Gangliosides are a group of structurally diverse, sialic acid containing glycosphingolipids embedded into the membrane via their hydrophobic ceramide moiety. To gain atomic level insights into the structural perturbations caused by Galbeta3GalNAcbeta4(NeuAcalpha3)Galbeta4Glc1Cer (GM1), molecular dynamics (MD) simulations of a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayer containing GM1 at five different concentrations have been performed. Biological membranes contain GM1 only on the exoplasmic leaflet. However, vesicles prepared in the laboratory contain GM1 in both the leaflets albeit unequally. Hence, simulations were performed with GM1 present in only one (asymmetric bilayers) or in both of the leaflets (symmetric bilayers) of the bilayer. In symmetric bilayers, there is a decrease in surface area, an increase in deuterium order parameter, and an increase in peak-to-peak distance of DPPC with increasing concentration of GM1. Thus, the overall area of the lipid bilayer decreases (condensation effect) and the thickness increases with increasing concentrations of GM1. Even in asymmetric systems, decrease in surface area and increase in deuterium order parameter of hydrocarbon chains of DPPC are observed. However, the decrease in bilayer area and the increase in bilayer thickness are not as much as in the symmetric bilayer.     

Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption

In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tapping-mode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, which-depending on the concentration of the constituents-separates into liquid-disordered (l(d)), liquid-ordered (l(o)), and solid-ordered (s(o)) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is l(d) > l(o) > s(o). Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the l(d)/l(o) phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.     

Model of an asymmetric DPPC/DPPS membrane: effect of asymmetry on the lipid properties. A molecular dynamics simulation study

The study of asymmetric lipid bilayers is of a crucial importance due to the great number of biological process in which they are involved such as exocytosis, intracellular fusion processes, phospholipid-protein interactions, and signal transduction pathway. In addition, the loss of this asymmetry is a hallmark of the early stages of apoptosis. In this regard, a model of an asymmetric lipid bilayer composed of DPPC and DPPS was simulated by molecular dynamics simulation. Thus, the asymmetric membrane was modeled by 264 lipids, of which 48 corresponded to DPPS- randomly distributed in the same leaflet with 96 DPPC. In the other leaflet, 120 DPPC were placed without DPPS-. Due to the presence of a net charge of -1 for the DPPS- in physiological conditions, 48 Na+ were introduced into the system to balance the charge. To ascertain whether the presence of the DPPS- in only one of the two leaflets perturbs the properties of the DPPC in the other leaflet composed only of DPPC, different properties were studied, such as the atomic density of the different components across the membrane, the electrostatic potential across the membrane, the translational diffusion of DPPC and DPPS, the deuterium order parameters, lipid hydration, and lipid-lipid charge bridges. Thus, we obtained that certain properties such as the surface area lipid molecule, lipid head orientation, order parameter, translational diffusion coefficient, or lipid hydration of DPPC in the leaflet without DPPS remain unperturbed by the presence of DPPS in the other leaflet, compared with a DPPC bilayer. On the other hand, in the leaflet containing DPPS, some of the DPPC properties were strongly affected by the presence of DPPS such as the order parameter or electrostatic potential.     

Asymmetric droplet interface bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.     

Molecular dynamics simulations of bilayers containing mixtures of sphingomyelin with cholesterol and phosphatidylcholine with cholesterol

We performed six molecular dynamics simulations: three on hydrated bilayers containing pure phospholipids and three on hydrated bilayers containing mixtures of these phospholipids with cholesterol. The phospholipids in our simulations were SSM (sphingomyelin containing a saturated 18:0 acyl chain), OSM (sphingomyelin with an unsaturated 18:1 acyl chain), and POPC (palmitoyloleoylphosphatidylcholine containing one saturated and one unsaturated chain). Data from our simulations were used to study systematically the effect of cholesterol on phospholipids that differed in their headgroup and tail composition. In addition to the structural analysis, we performed an energetic analysis and observed that energies of interaction between cholesterol and neighboring SM molecules are similar to the energies of interaction between cholesterol and POPC. We also observed that the interaction energy between cholesterol and neighboring lipids cannot be used for the determination of which lipids are involved in the creation of a complex.     

Layer-by-layer PMIRRAS characterization of DMPC bilayers deposited on a Au111 electrode surface

A combination of Langmuir-Blodgett and Langmuir-Schaefer techniques was employed to deposit 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at a gold electrode surface. One leaflet consisted of hydrogen-substituted acyl chains, and the second leaflet was composed of molecules with deuterium-substituted acyl chains. This architecture allowed for layer-by-layer analysis of the structure of the bilayer. Photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to determine the conformation and orientation of the acyl chains of DMPC molecules in the individual leaflets as a function of the potential applied to the gold electrode. The bilayer is adsorbed onto the metal surface when the field applied to the membrane does not exceed approximately 108 V/m. When adsorbed, the bottom leaflet is in contact with a hydrophobic metal surface, and the top leaflet is interacting with the aqueous solution. The asymmetry of the environment has an effect on the orientation of the DMPC molecules in each leaflet. The tilt angle of the acyl chains of the DMPC molecules in the bottom leaflet that is in contact with the gold is approximately 10 degrees smaller than that observed for the top leaflet that is exposed to the solution. These studies provide direct evidence that the structure of a phospholipid bilayer deposited at an electrode surface is affected by interaction with the metal.     

Composition and asymmetry in supported membranes formed by vesicle fusion

The structure and formation of supported membranes at silica surfaces by vesicle fusion was investigated by neutron reflectivity and quartz crystal microbalance (QCM-D) measurements. The structure of equimolar phospholipid mixtures of DLPC-DPPC, DMPC-DPPC, and DOPC-DPPC depends intricately on the vesicle deposition conditions. The supported bilayer membranes exhibit varying degrees of compositional asymmetry between the monolayer leaflets, which can be modified by the deposition temperature as well as the salt concentration of the vesicle solution. The total lipid composition of the supported bilayers differs from the composition of the vesicles in solution, and the monolayer proximal to the silica surface is always enriched in DPPC compared to the distal monolayer. The results, which show unambiguougsly that some exchange and rearrangement of lipids occur during vesicle deposition, can be rationalized by considering the effects of salt screening and temperature on the rates of lipid exchange, rearrangement, and vesicle adsorption, but there is also an intricate dependence on the lipid-lipid interactions. Thus, although both symmetric and asymmetric supported bilayers can be prepared from vesicles, the optimal conditions are sensitive to the lipid composition of the system.     

Regulating the size and stabilization of lipid raft-like domains and using calcium ions as their probe

We apply a means to probe, stabilize, and control the size of lipid raft-like domains in vitro. In biomembranes the size of lipid rafts is ca. 10-30 nm. In vitro, mixing saturated and unsaturated lipids results in microdomains, which are unstable and coalesce. This inconsistency is puzzling. It has been hypothesized that biological line-active surfactants reduce the line tension between saturated and unsaturated lipids and stabilize small domains in vivo. Using solution X-ray scattering, we studied the structure of binary and ternary lipid mixtures in the presence of calcium ions. Three lipids were used: saturated, unsaturated, and a hybrid (1-saturated-2-unsaturated) lipid that is predominant in the phospholipids of cellular membranes. Only membranes composed of the saturated lipid can adsorb calcium ions, become charged, and therefore considerably swell. The selective calcium affinity was used to show that binary mixtures, containing the saturated lipid, phase separated into large-scale domains. Our data suggests that by introducing the hybrid lipid to a mixture of the saturated and unsaturated lipids, the size of the domains decreased with the concentration of the hybrid lipid, until the three lipids could completely mix. We attribute this behavior to the tendency of the hybrid lipid to act as a line-active cosurfactant that can easily reside at the interface between the saturated and the unsaturated lipids and reduce the line tension between them. These findings are consistent with a recent theory and provide insight into the self-organization of lipid rafts, their stabilization, and size regulation in biomembranes.