Multi-scale modeling of mycosubtilin lipopeptides at the air/water interface: structure and optical second harmonic generation.

Monolayers of the lipopeptide mycosubtilin are studied at the air/water interface. Their structure is investigated using molecular dynamics simulations. All-atom models suggest that the lipopeptide is flexible and aggregates at the interface. To achieve simulation times of several microseconds, a coarse-grained (CG) model based on the MARTINI force field was also used. These CG simulations describe the formation of half-micelles at the interface for surface densities up to 1 lipopeptide per nm(2). In these aggregates, the tyrosine side chain orientation is found to be constrained: on average, its main axis, as defined along the C-OH bond, aligns along the interface normal and points towards the air side. The origin of the optical second harmonic generation (SHG) from mycosubtilin monolayers at the air/water interface is also investigated. The molecular hyperpolarizability of the lipopeptide is obtained from quantum chemistry calculations. The tyrosine side chain contribution to the hyperpolarizability is found to be dominant. The orientation distribution of tyrosine, associated with a dominant hyperpolarizability component along the C-OH bond of the tyrosine, yields a ratio of the susceptibility elements χ((2))(ZZZ)/χ((2))(ZXX) consistent with the experimental measurements recently reported by M. N. Nasir et al. [Phys. Chem. Chem. Phys., 2013, 15, 19919].

Reorientation of the helix of the tryptophan-rich gp41W peptide from HIV-1 at interfaces.

The glycoprotein gp41 from the Human Immunodeficiency Virus type 1 (HIV-1) has an amino acid sequence enriched in tryptophan residues, the so-called gp41W peptide (i.e., KWASLWNWFNITNWLWYIK) and plays a crucial role in HIV-1 host cell infection. Using the coupling of Second Harmonic Generation targeting the tryptophan residues with lateral surface tension measurements, we investigate the interaction of gp41W with a neat air∕water and a lipid∕water interfaces. At the air∕water interface, gp41W presents a well-defined orientation and this orientation is strongly modified at the lipid∕water interface, depending on the surface pressure. These results show that this strategy is well suited to monitor tryptophan containing α-helices orientation at lipid∕water interfaces.

Influence of the tyrosine environment on the second harmonic generation of iturinic antimicrobial lipopeptides at the air-water interface.

The second harmonic generation (SHG) response at the air-water interface from the tyrosine-containing natural iturinic cyclo-lipopeptides mycosubtilin, iturin A and bacillomycin D is reported. It is shown that this response is dominated by the single tyrosine residue present in these molecules owing to the large first hyperpolarizability arising from the non-centrosymmetric aromatic ring structure of this amino acid. The SHG response of these iturinic antibiotics is also compared to the response of surfactin, a cyclo-lipopeptide with a similar l,d-amino acid sequence but lacking a tyrosine residue, and PalmATA, a synthetic linear lipopeptide possessing a single tyrosine residue but lacking the amino acid sequence structuring the cycle of the iturinic antibiotics. From the light polarization analysis of the SHG response, it is shown that the tyrosine local environment is critical in defining the SHG response of these peptides at the air-water interface. Our results demonstrate that tyrosine, similar to tryptophan, can be used as an endogenous molecular probe of peptides and proteins for SHG at the air-water interface, paving the way for SHG studies of other tyrosine-containing bioactive molecules.

Influence of the 524-VAAEIL-529 sequence of annexins A6 in their interfacial behavior and interaction with lipid monolayers.

Annexin A6 (AnxA6), a calcium- and membrane-binding protein, is expressed in mammalian cells in two isoforms: AnxA6-1 and AnxA6-2, the latter lacking the 524-VAAEIL-529 sequence at the start of repeat 7. The different intracellular localization of these two isoforms suggests distinct function in membrane dynamics. The aim of this work was to analyze the behavior of AnxA6 isoforms at the air/water interface alone and in the presence of membrane mimicking lipid monolayers. Using Langmuir technique showed that AnxA6-2 was less adsorbed to the neat air-water interface than AnxA6-1 at acidic pH and minor differences in their PM-IRRAS spectra were observed. Both isoforms exhibited similar behavior towards cholesterol monolayer. However, the interactions of AnxA6-2 with cholesterol ester monolayer were most favorable compared to AnxA6-1. Our experimental data are discussed in relation with the different intracellular localization of the two isoforms and with our constructed model of AnxA6-2 with the known crystal structure of AnxA6-1 showing the persistence of the 516-529 α-helix in AnxA6-2 despite the absence of the 524-VAAEIL-529 sequence.

Interaction of AnxA6 with isolated and artificial lipid microdomains; importance of lipid composition and calcium content.

Niemann-Pick type C (NPC) disease is a lipid storage disorder characterized by accumulation of lipids in the late endosome/lysosome (LE/LY) compartment. In our previous report we isolated membranes of the LE/LY compartment from NPC L1 skin fibroblasts with a mutation in the NPC1 gene and found that they were characterized by low fluidity which likely contributed to the impaired function of membrane proteins involved in storage and turnover of cholesterol. In this report we isolated lipid microdomains (DRMs) from membranes of various cellular compartments and observed an increased amount of DRMs in the LE/LY compartment of NPC L1 cells in comparison to control cells, with no change in the DRM content in the plasma membrane. In addition, in the NPC cells, the majority of the cholesterol-interacting protein, AnxA6, which participates in the transport and distribution of cholesterol, translocated to DRMs upon a rise in Ca(2+) concentration. The mechanism of this translocation was further studied in vitro using Langmuir monolayers. We found that Ca(2+) is the main factor which regulates the interaction of AnxA6 with monolayers composed of neutral lipids, such as DPPC and sphingomyelin, and may also determine AnxA6 localization in cholesterol and sphingomyelin enriched microdomains, thus contributing to the etiology of the NPC disease.

Conformational analyses of bacillomycin D, a natural antimicrobial lipopeptide, alone or in interaction with lipid monolayers at the air-water interface.

Bacillomycin D is a natural antimicrobial lipopeptide belonging to the iturin family. It is produced by Bacillus subtilis strains. Bacillomycin D is characterized by its strong antifungal and hemolytic properties, due to its interaction with the plasma membrane of sensitive cells. Until now, only few limited analyses were conducted to understand the biological activities of bacillomycin D at the molecular level. Our purpose was to analyze the conformation of bacillomycin D using IR spectroscopy and to model its interactions with cytoplasmic membranes using Langmuir interfacial monolayers. Our findings indicate that bacillomycin D contains turns and allow to model its three-dimensional structure. Bacillomycin D formed a monolayer film at the air-water interface and kept its turn conformation, as shown by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). To identify the membrane lipid target of bacillomycin D, its interactions with pure lipid monolayers were analyzed and an original behavior of the lipopeptide toward cholesterol-containing monolayers was shown. This original behavior was lost when bacillomycin D was interacting with pure cholesteryl acetate monolayers, suggesting the involvement of the alcohol group of cholesterol in the lipopeptide-cholesterol interaction.

Interactions of the antifungal mycosubtilin with ergosterol-containing interfacial monolayers.

Mycosubtilin, an antimicrobial lipopeptide produced by Bacillus subtilis, is characterized by strong antifungal activities. The molecular mechanisms of its biological activities on the membranes of the sensitive yeasts or fungi have not yet been clearly elucidated. Our purpose was to mimic the mycosubtilin interactions with these membranes using various Langmuir monolayers. Since the major sterol of yeasts or fungi is ergosterol, the interactions of mycosubtilin with monolayers constituted by ergosterol, DPPC/ergosterol or DPPC/sphingomyelin/ergosterol were examined at different initial surface pressures (Πi). Plotting the mycosubtilin-induced surface pressure increases versus Πi allowed to determine that the exclusion pressures of mycosubtilin from these different monolayers is higher than the surface prevailing within the biological membranes. However, this behavior was lost when mycosubtilin was interacting with ergosteryl acetate-containing monolayers. This suggests the involvement of the sterol alcohol group in the mycosubtilin interactions within membranes. Furthermore, the behavior of mycosubtilin with stigmasterol, similar to that observed with ergosterol, differs from that previously observed with cholesterol, suggesting a role of the alkyl side chain of the sterols. The adsorption of mycosubtilin to ergosterol monolayers induced changes in the lipopeptide orientation at the air-water interface as revealed by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Moreover, imaging the air-water interface by Brewster angle microscopy (BAM) indicates that mycosubtilin induced changes in the organization and morphology of monolayers containing pure ergosterol with the appearance of small condensed dots, suggesting again that the target of mycosubtilin might be the ergosterol present in the membranes of the sensitive yeasts or fungi.

Annexins as organizers of cholesterol- and sphingomyelin-enriched membrane microdomains in Niemann-Pick type C disease.

Growing evidence suggests that membrane microdomains enriched in cholesterol and sphingomyelin are sites for numerous cellular processes, including signaling, vesicular transport, interaction with pathogens, and viral infection, etc. Recently some members of the annexin family of conserved calcium and membrane-binding proteins have been recognized as cholesterol-interacting molecules and suggested to play a role in the formation, stabilization, and dynamics of membrane microdomains to affect membrane lateral organization and to attract other proteins and signaling molecules onto their territory. Furthermore, annexins were implicated in the interactions between cytosolic and membrane molecules, in the turnover and storage of cholesterol and in various signaling pathways. In this review, we focus on the mechanisms of interaction of annexins with lipid microdomains and the role of annexins in membrane microdomains dynamics including possible participation of the domain-associated forms of annexins in the etiology of human lysosomal storage disease called Niemann-Pick type C disease, related to the abnormal storage of cholesterol in the lysosome-like intracellular compartment. The involvement of annexins and cholesterol/sphingomyelin-enriched membrane microdomains in other pathologies including cardiac dysfunctions, neurodegenerative diseases, obesity, diabetes mellitus, and cancer is likely, but is not supported by substantial experimental observations, and therefore awaits further clarification.

Specific interactions of mycosubtilin with cholesterol-containing artificial membranes.

Mycosubtilin is a natural antimicrobial lipopeptide produced by Bacillus subtilis strains. It is characterized by its hemolytic and strong antifungal activities. Mycosubtilin interacts with the plasma membranes of sensitive cells. However, the molecular mechanisms of its biological activities have not been completely elucidated. Our purpose was therefore to analyze the interactions of mycosubtilin with biological membranes by using biomimetic membranes such as Langmuir monolayers and multilayers. Structural changes of mycosubtilin, involving its peptide backbone and the side chain of its tyrosyl residue, were observed when the lipopeptide was interacting with cholesterol-containing multilayers. The interactions of mycosubtilin with monolayers constituted by pure lipids and by phosholipid/cholesterol or phospholipid/sphingomyelin/cholesterol were also examined. An original behavior of mycosubtilin toward cholesterol-containing monolayers was found. However, this original behavior was lost when mycosubtilin was interacting with pure cholesterylacetate monolayers. This suggests the involvement of the alcohol group of cholesterol in mycosubtilin-cholesterol interactions within membranes. Moreover, mycosubtilin induced changes in the organization and morphology of cholesterol-containing monolayers, and large condensed domains with different levels of condensation appeared only in the case of DPPC/sphingomyelin/cholesterol monolayer.

Influence of the lipid composition of biomimetic monolayers on the structure and orientation of the gp41 tryptophan-rich peptide from HIV-1.

The tryptophan-rich peptide of gp41 (so-called gp41W), one of the two envelope glycoproteins of HIV-1, is known to play a crucial role in the fusion between this virus and the host cell membranes. The influence of lipids on this role was investigated using different lipid monolayers at the air-water interface. Gp41W affinity for the lipid monolayer was measured by following the peptide-induced variation in the lateral surface pressure and we demonstrated that gp41W binds to monolayers containing the saturated zwitterionic dipalmitoylphosphatidylcholine (DPPC) as well as to the anionic dipalmitoylphosphatidylglycerol (DPPG) and to mixed monolayers containing DPPC and cholesterol (Chol). The secondary structure of gp41W in the presence of these lipid monolayers was determined by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The data showed that gp41W was an oriented α-helix in the presence of DPPG. However this spectroscopic method was unable to detect the gp41W structure in the presence of DPPC and DPPC/Chol monolayer. The peptide-induced modifications of the DPPC/Chol, DPPC and DPPG monolayer morphology were analyzed by Brewster angle microscopy (BAM). The peptide-induced changes in the DPPG monolayer morphology suggest that gp41W disturbed the lipid intermolecular interactions. Furthermore the peptide delayed the condensed state of DPPC and DPPC/Chol, indicating that, although gp41W was not detected by PM-IRRAS, it was present in these lipid monolayers.

Annexin A6 is recruited into lipid rafts of Niemann-Pick type C disease fibroblasts in a Ca2+-dependent manner.

Niemann-Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.

Interfacial properties and structure stability of the gp41 tryptophan-rich peptide from HIV-1.

The HIV-1 envelope glycoprotein 41 (gp41) undergoes large-scale conformational changes in order to induce the fusion of the virus and cell membranes. Thus, we investigated a possible structure transit at the air-water interface for the tryptophan-rich peptide of gp41 (gp41W). The synthetic peptide (KWASLWNWFNITNWLWYIK), corresponding to gp41W, shows interfacial properties on pure water and Tris buffer at pH 8.5. Isotherm measurements and Brewster angle microscopy (BAM) imaging showed that the behavior of the peptide monolayer was dependent on the subphase composition. A homogenous film was formed on buffer during the peptide monolayer compression, while the appearance of condensed domains on pure water could indicate the oligomerization of gp41W during the surface pressure increase. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) showed that, whatever the subphase, gp41W adopts an α-helix structure at the air-water interface and does not transit for any other structure even at high surface pressures.

Interaction of annexin A6 with cholesterol rich membranes is pH-dependent and mediated by the sterol OH.

Annexin A6 (AnxA6), a calcium- and membrane-binding protein, is well-known to play a role in calcium homeostasis, membrane traffic and membrane organization. It had been suggested that, despite calcium-dependent interaction with anionic phospholipids, AnxA6 displays calcium-independent cholesterol binding properties. In this study, the following questions were addressed: does AnxA6 bind preferentially to cholesterol-containing biomimetic membranes? If so, what is the molecular mechanism of the binding? To answer these questions, human recombinant AnxA6-1 isoform was prepared and used with Langmuir monolayers containing various lipids. The interactions between AnxA6 and the lipid monolayers were examined by kinetic measurements of the interfacial adsorption and Brewster angle microscopy. We focused on the pH effect on the AnxA6 binding to monolayers containing cholesterol. At acidic pH, AnxA6-1 exhibits the highest affinity to monolayers containing the highest amount of cholesterol. Replacing cholesterol by cholesteryl acetate provided evidence that the hydroxyl group of cholesterol plays a role in AnxA6-lipid interactions. In addition, the affinity of recombinant AnxA6-1 tryptophan mutant (W343F) to the air/water interface and to lipid monolayers was tested. Substitution of Trp343 modified the interfacial properties of the protein and its interactions with sterol monolayers. Our results suggest that the linker region containing Trp343 is important for the interactions between AnxA6-1 and cholesterol.

Interactions of the natural antimicrobial mycosubtilin with phospholipid membrane models.

Among the secondary metabolite lipopeptides produced by Bacillus subtilis, mycosubtilin is characterized by its strong antifungal activities. Even though its structure and its cellular target, the cytoplasmic membrane, have been determined, the molecular mechanisms of the biological activity of mycosubtilin have not been completely elucidated. In this work, the interactions between mycosubtilin and cytoplasmic membranes were modelled by using biomimetic systems such as Langmuir monolayers at the air-water interface and lipid multilamellar vesicles. The interactions of mycosubtilin with these biomimetic systems were examined, for the first time, by using specific techniques such as polarization modulation infrared reflection absorption spectroscopy, Brewster angle microscopy and high-resolution magic angle spinning NMR. Our findings indicate that mycosubtilin alone, at the air-water interface, forms a monolayer film and keeps its turn conformation. In the presence of DMPC, mycosubtilin binds to phospholipid monolayers, in a surface pressure-dependent manner. This binding results in the appearance of condensed domains which can be due to the formation of mycosubtilin clusters and/or to the lipopeptide aggregation with some phospholipid molecules and/or the formation of liquid-condensed domains of DMPC. Furthermore, in multilamellar vesicles, the mycosubtilin-DMPC interactions take place at the level of the aliphatic chains of the phospholipid because the phase transition temperature of DMPC decreased in the presence of mycosubtilin.

Hyaluronidase binds differently DPPC, DPPS or GlcNAc-bearing glycolipid biomimetic monolayers.

Bovine testis hyaluronidase (btHyal) had been shown to have direct effects on cancer cells and to be a useful adjuvant in several medicines. Furthermore this enzyme had been found to be membrane-associated. Thus, in this work, the interactions between btHyal and membranes were analyzed by using lipid monolayers at the air-water interface as a biomimetic membrane system. This allowed us to define the btHyal interactions with two residues of hyaluronic acid (a btHyal substrate), GlcNAc and carboxylic group, which are present in cholesteryl-triethoxy-N-acetylglucosamine (Chol-E3-GlcNAc) and in DPPS, respectively. btHyal bound preferentially Chol-E3-GlcNAc monolayers and showed a decreasing affinity for Chol-E3-GlcNAc-DPPC monolayers containing decreasing amount of glycolipid, suggesting a crucial role of the glycolipid GlcNAc. Furthermore the significant btHyal binding to DPPS was not affected by the presence of free GlcNAc in the subphase. These results and the absence of significant binding of btHyal to pure DPPC monolayer suggest that the protein interacts with the lipid monolayer by mimicking the enzyme-substrate interactions or by electrostatic interactions.

Conformational and interfacial analyses of K3A18K3 and alamethicin in model membranes.

The involvement of membrane-bound peptides and the influence of protein conformations in several neurodegenerative diseases lead us to analyze the interactions of model peptides with artificial membranes. Two model peptides were selected. The first one, an alanine-rich peptide, K3A18K3, was shown to be in alpha-helix structures in TFE, a membrane environment-mimicking solvent, while it was mostly beta-sheeted in aqueous buffer as revealed by infrared spectroscopy. The other, alamethicin, a natural peptide, was in a stable alpha-helix structure. To determine the role of the peptide conformation on the nature of its interactions with lipids, we compared the structure and topology of the conformational-labile peptide K3A18K3 and of the alpha-helix rigid alamethicin in both aqueous and phospholipid environments (Langmuir monolayers and multilamellar vesicles). K3A18K3 at the air-water interface showed a pressure-dependent orientation of its beta-sheets, while the alpha-helix axis of alamethicin was always parallel to the interface, as probed by polarization modulation infrared reflection absorption spectroscopy. The beta-sheeted K3A18K3 peptide was uniformly distributed into DPPC condensed domains, while the helical-alamethicin insertion distorted the DPPC condensed domains, as evidenced by Brewster angle microscopy imaging of the air/interface. The beta-sheeted K3A18K3 interacted with DMPC multilamellar vesicles via hydrophilic interactions with polar heads and the helical-alamethicin via hydrophobic interactions with alkyl chains, as shown by infrared spectroscopy and solid state NMR. Our findings are consistent with the prevailing assumption that the conformation of the peptide predetermines the mode of interaction with lipids. More precisely, helical peptides tend to be inserted via hydrophobic interactions within the hydrophobic region of membranes, while beta-sheeted peptides are predisposed to interact with polar groups and stay at the surface of lipid layers.

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content.

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.

Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism.

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri = 200) and is complete for Ri = 50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

Temperature-dependent localization of GPI-anchored intestinal alkaline phosphatase in model rafts.

In plasma membranes, most of glycosylphosphatidylinositol (GPI)-anchored proteins would be associated with rafts, a category of ordered microdomains enriched in sphingolipids and cholesterol (Ch). They would be also concentrated in the detergent resistant membranes (DRMs), a plasma membrane fraction extracted at low temperature. Preferential localization of GPI-anchored proteins in these membrane domains is essentially governed by their high lipid order, as compared to their environment. Changes in the temperature are expected to modify the membrane lipid order, suggesting that they could affect the distribution of GPI-anchored proteins between membrane domains. Validity of this hypothesis was examined by investigating the temperature-dependent localization of the GPI-anchored bovine intestinal alkaline phophatase (BIAP) into model raft made of palmitoyloleoylphosphatidylcholine/sphingomyelin/cholesterol (POPC/SM/Chl) supported membranes. Atomic force microscopy (AFM) shows that the inserted BIAP is localized in the SM/Chl enriched ordered domains at low temperature. Above 30 degrees C, BIAP redistributes and is present in both the 'fluid' POPC enriched and the ordered SM/Chl domains. These data strongly suggest that in cells the composition of plasma membrane domains at low temperature differs from that at physiological temperature.

Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase.

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.