On the uptake of cationic liposomes by cells: From changes in elasticity to internalization.

In this study, we assessed the capacity of a previously reported engineered liposomal formulation, which had been tested against model membranes mimicking the lipid composition of the HeLa plasma membrane, to fuse and function as a nanocarrier in cells. We used atomic force microscopy to observe physicochemical changes on the cell surface and confocal microscopy to determine how the liposomes interact with cell membranes and released their load. In addition, we performed viability assays using methotrexate as an active drug to obtain proof of concept of the formulation´s capacity to function as a drug delivery-system. The interaction of engineered liposomes with living cells corroborates the information obtained using model membranes and supports the capacity of the engineered liposomal formulation to serve as a potential nanocarrier.

Studying Lipid Membrane Interactions of a Super-Cationic Peptide in Model Membranes and Living Bacteria.

The super-cationic peptide dendrimers (SCPD) family is a valuable class of antimicrobial peptide candidates for the future development of antibacterial agents against multidrug-resistant gram-negative bacteria. The deep knowledge of their mechanism of action is a major challenge in research, since it may be the basis for future modifications/optimizations. In this work we have explored the interaction between SCPD and membranes through biophysical and microbiological approaches in the case of the G1OLO-L2OL2 peptide. Results support the idea that the peptide is not only adsorbed or close to the surface of the membrane but associated/absorbed to some extent to the hydrophobic-hydrophilic region of the phospholipids. The presence of low concentrations of the peptide at the surface level is concomitant with destabilization of the cell integrity and this may contribute to osmotic stress, although other mechanisms of action cannot be ruled out.

Characterization of monolayers and liposomes that mimic lipid composition of HeLa cells.

In this work, based on several studies, we develop an artificial lipid membrane to mimic the HeLa cell membrane using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) and cholesterol (CHOL). This is then a means to further study the fusion process of specific engineered liposomes. To characterize the mimicked HeLa cell membrane, we determined a series of surface pressure-area (π-A) isotherms and the isothermal compression modulus was calculated together with the dipole moment normal to the plane of the monolayer. The existence of laterally segregated domains was assessed using a fluorescence technique (Laurdan) and two microscopy techniques: Brewster angle microscopy (BAM) and atomic force microscopy (AFM) of Langmuir-Blodgett films (LBs) extracted at 30 mN m-1. To examine the nature and composition of the observed domains, force spectroscopy (FS) based on AFM was applied to the LBs. Finally, two engineered liposome formulations were tested in a fusion assay against mimicked HeLa cell membrane LBs, showing good results and thereby opening the door to further assays and uses.

Effect of cholesterol on monolayer structure of different acyl chained phospholipids.

In this work we have investigated the effect of cholesterol (CHOL) in phospholipid monolayers on a series of phosphatidylcholines differing in acyl chain composition. We have used the CHOL proportion that abolishes the gel (Lß)-to-liquid-crystalline (Lß) transition in bilayers in order to investigate the mixing properties and laterally-segregated domains formed by specific phospholipid-CHOL ratios at the air-water interface. The binary monolayers where formed by mixing CHOL with 1,2-palmitoyl-sn-glycero-3-phos-phatidylcholine (DPPC);1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC); 1-pal-mitoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (PSPC); 1-palmitoyl-2-oleoyl-sn-gly-cero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphatidyl-choline (PLPC), respectively. From surface pressure-area (π-A) isotherms the isothermal compression modulus were calculated, and the mixing properties of the monolayers obtained by performing a basic surface thermodynamic analysis. From the excess Gibbs energy, the interaction parameter and the activity coefficients were also calculated. The study of the monolayers was complemented by determining the molecular dipole moment normal to the plane of the monolayer. The existence of laterally segregated domains was assessed by atomic force microscopy (AFM) of Langmuir-Blodgett films (LBs) extracted at 30 mNm-1. To get insight into the nature and composition of the observed domains force spectroscopy (FS) based on AFM was applied to the LBs.

Mapping phase diagrams of supported lipid bilayers by atomic force microscopy.

In this work, we present the method followed to construct a pseudophase diagram of two phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol). Two different techniques, DSC and AFM, have been used based in the determination of the onset (Tonset ) and completion (Toffset ) temperatures of the gel-to-liquid crystalline phases (Lß â†’Lα ), the first from the endotherms from liposomes and the second from the topographic images of supported lipid bilayers. The features of both phase diagrams are discussed emphasizing the influence of Ca2+ presence and the substrate (mica) on the transition undergone by the phospholipid mixture. Microsc. Res. Tech. 80:4-10, 2017. © 2016 Wiley Periodicals, Inc.

Enhanced topical delivery of hyaluronic acid encapsulated in liposomes: A surface-dependent phenomenon.

In the present study, we investigated the release and permeation of hyaluronic acid (HA) encapsulated in liposomes when deposited onto two surfaces: cellulose, a model widely used for investigating transport of drugs; and human skin, a natural biointerface used for transdermal drug delivery. We prepared and characterised liposomes loaded with HA and liposomes incorporating two penetration enhancers (PEs): the non-ionic surfactant Tween 80, and Transcutol P, a solubilising agent able to mix with polar and non-polar solvents. In vitro and ex vivo permeation assays showed that PEs indeed enhance HA-release from liposomes. Since one of the possible mechanisms postulated for the action of liposomes on skin is related to its adsorption onto the stratum corneum (SC), we used atomic force microscopy (AFM) topography and force volume (FV) analysis to investigate the structures formed after deposition of liposome formulations onto the investigated surfaces. We explored the possible relationship between the formation of planar lipid structures on the surfaces and the permeation of HA.

Unspecific membrane protein-lipid recognition: combination of AFM imaging, force spectroscopy, DSC and FRET measurements.

In this work, we will describe in quantitative terms the unspecific recognition between lactose permease (LacY) of Escherichia coli, a polytopic model membrane protein, and one of the main components of the inner membrane of this bacterium. Supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol) in the presence of Ca(2+) display lateral phase segregation that can be distinguished by atomic force microscopy (AFM) as well as force spectroscopy. LacY shows preference for fluid (Lα) phases when it is reconstituted in POPE : POPG (3:1, mol/mol) proteoliposomes at a lipid-to-protein ratio of 40. When the lipid-to-protein ratio is decreased down to 0.5, two domains can be distinguished by AFM. While the upper domain is formed by self-segregated units of LacY, the lower domain is constituted only by phospholipids in gel (Lß) phase. On the one hand, classical differential scanning calorimetry (DSC) measurements evidenced the segregation of a population of phospholipids and point to the existence of a boundary region at the lipid-protein interface. On the other hand, Förster Resonance Energy Transfer (FRET) measurements in solution evidenced that POPE is selectively recognized by LacY. A binary pseudophase diagram of POPE : POPG built from AFM observations enables to calculate the composition of the fluid phase where LacY is inserted. These results are consistent with a model where POPE constitutes the main component of the lipid-LacY interface segregated from the fluid bulk phase where POPG predominates.

Effect of lactose permease presence on the structure and nanomechanics of two-component supported lipid bilayers.

In this paper we present a comparative study of supported lipid bilayers (SLBs) and proteolipid sheets (PLSs) obtained from deposition of lactose permease (LacY) of Escherichia coli proteoliposomes in plane. Lipid matrices of two components, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), at a 3:1, mol/mol ratio, were selected to mimic the inner membrane of the bacteria. The aim was to investigate how species of different compactness and stiffness affect the integration, distribution and nanomechanical properties of LacY in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or 1,2-palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). Both compositions displayed phase separation and were investigated by atomic force microscopy (AFM) imaging and force-spectroscopy (FS) mode. PLSs displayed two separated, segregated domains with different features that were characterised by FS and force-volume mode. We correlated the nanomechanical characteristics of solid-like gel phase (Lß) and fluid liquid-crystalline phase (Lα) with phases emerging in presence of LacY. We observed that for both compositions, the extended PLSs showed a Lß apparently formed only by lipids, whilst the second domain was enriched in LacY. The influence of the lipid environment on LacY organisation was studied by performing protein unfolding experiments using the AFM tip. Although the pulling experiments were unspecific, positive events were obtained, indicating the influence of the lipid environment when pulling the protein. A possible influence of the lateral surface pressure on this behaviour is suggested by the higher force required to pull LacY from DPPE:POPG than from POPE:POPG matrices. This is related to higher forces governing protein-lipid interaction in presence of DPPE.

Phospholipid-lactose permease interaction as reported by a head-labeled pyrene phosphatidylethanolamine: a FRET study.

Förster resonance energy transfer (FRET) measurements were performed in preceding works to study the selectivity between a single-tryptophan mutant of lactose permease (LacY) of Escherichia coli (used as the donor) and phospholipid probes labeled with pyrene at the acyl chain moiety (used as the acceptor). In the present work, we report the results obtained by using the same LacY mutant (W151/C154G) and binary lipid mixtures of phosphatidylethanolamine (PE) differing in the acyl chain composition and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (3:1 mol/mol) doped with a phospholipid probe labeled with pyrene at the headgroup. The use of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(1-pyrenesulfonyl) ammonium salt (HPyr-PE), which bears two unsaturated acyl chains, enabled the investigation of the specific interaction between LacY and HPyr-PE. The main conclusions raised from our results suggest that (i) for phase-separated systems, LacY would be located in fluid domains nominally enriched in POPG, and if a given proportion of PE is present in this phase, it will be mainly located around LacY; and (ii) in the absence of phase separation, LacY is preferentially surrounded by PE and, in particular, seems to be sensitive to the lipid spontaneous curvature.

Phosphatidylethanolamine-lactose permease interaction: a comparative study based on FRET.

In this work we have investigated the selectivity of lactose permease (LacY) of Escherichia coli (E. coli) for its surrounding phospholipids when reconstituted in binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). Förster resonance energy transfer (FRET) measurements have been performed to investigate the selectivity between a single tryptophan mutant of LacY used as donor (D), and two analogues of POPE and POPG labeled with pyrene in the acyl chains (Pyr-PE and Pyr-PG) used as acceptors. As a difference from previous works, now the donor has been single-W151/C154G/D68C LacY. It has been reported that the replacement of the aspartic acid in position 68 by cysteine inhibits active transport in LacY. The objectives of this work were to elucidate the phospholipid composition of the annular region of this mutant and to determine whether the mutation performed, D68C, induced changes in the protein-lipid selectivity. FRET efficiencies for Pyr-PE were always higher than for Pyr-PG. The values of the probability of each site in the annular ring being occupied by a label (µ) were similar at the studied temperatures (24 °C and 37 °C), suggesting that the lipid environment is not significantly affected when increasing the temperature. By comparing the results with those obtained for single-W151/C154G LacY, we observe that the mutation in the 68 residue indeed changes the selectivity of the protein for the phospholipids. This might be probably due to a change in the conformational dynamics of LacY.

Membrane protein-lipid selectivity: enhancing sensitivity for modeling FRET data.

Förster resonance energy transfer (FRET) is a powerful method for the characterization of membrane proteins lipid selectivity. FRET can be used to quantify distances between a single donor and a single acceptor molecule; however, for FRET donors and acceptors scattered in the bilayer plane, multiple donor-acceptor pairs and distances are present. In addition, when studying protein/lipid selectivity, for a single tryptophan used as a donor; several lipid acceptors may be located at the boundary region (annular lipids) of the protein. Therefore, in these experiments, a theoretical analysis based on binomial distribution of multiple acceptors around the membrane proteins is required. In this work, we performed FRET measurements between single tryptophan lactose permease (W151/C154G LacY) of Escherichia coli and pyrene-labeled phospholipids (Pyr-PE, Pyr-PG, and Pyr-PC) reconstituted in palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline, and 1,2-dioleoyl-sn-glycero-3-phospho-choline at 25 and 37 °C. To increase the sensitivity of the method and to ascertain the lipid selectivity for LacY, we reconstituted the protein in the pure phospholipids doped with 1.5% of labeled phospholipids. From fitting the theoretical model to the experimental FRET efficiencies, two parameters were calculated: the probability of a site in the annular ring being occupied by a labeled pyrene phospholipid and the relative association constant between the labeled and unlabeled phospholipids. The experimental FRET efficiencies have been interpreted taking into account the particular folding of the protein in each phospholipid matrix. Additional information on the annular lipid composition for each system has been obtained by exciting W151/C154G LacY and monitoring the emission intensities for monomer and excimer of the pyrene spectra. The results obtained indicate a higher selectivity of LacY for PE over PG and PC and pointed to a definite role of the acyl chains in the overall phospholipid-protein interaction.

Miscibility behavior and nanostructure of monolayers of the main phospholipids of Escherichia coli inner membrane.

We report a thermodynamic study of the effect of calcium on the mixing properties at the air-water interface of two phospholipids that mimic the inner membrane of Escherichia coli: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. In this study, pure POPE and POPG monolayers and three mixed monolayers, χ(POPE) = 0.25, 0.5, and 0.75, were analyzed. We show that for χ(POPE) = 0.75, the values of the Gibbs energy of mixing were negative, which implies attractive interactions. We used atomic force microscopy to study the structural properties of Langmuir-Blodgett monolayers that were transferred onto mica substrate at lateral surface pressures of 25 and 30 mN m(-1). The topographic images of pure POPE and POPG monolayers exhibited two domains of differing size and morphology, showing a step height difference within the range expected for liquid-condensed and liquid-expanded phases. The images captured for χ(POPE) = 0.25 were featureless, and for χ(POPE) = 0.5 small microdomains were observed. The composition that mimics quantitatively the proportions found in the inner membrane of E. coli , χ(POPE) = 0.75, showed large liquid condensed domains in the liquid expanded phase. The extension of each domain was quantitatively analyzed. Because calcium is used in the formation of supported bilayers of negatively charged phospholipids, the possible influence of the nanostructure of the apical on the distal monolayer is discussed.

Acyl chain differences in phosphatidylethanolamine determine domain formation and LacY distribution in biomimetic model membranes.

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the two main components of the inner membrane of Escherichia coli. It is well-known that inner membrane contains phospholipids with a nearly constant polar headgroup composition. However, bacteria can regulate the degree of unsaturation of the acyl chains in order to adapt to different external stimuli. Studies on model membranes of mixtures of PE and PG, mimicking the proportions found in E. coli, can provide essential information on the phospholipid organization in biological membranes and may help in the understanding of membrane proteins activity, such as lactose permease (LacY) of E. coli. In this work we have studied how different phosphatidylethanolamines differing in acyl chain saturation influence the formation of laterally segregated domains. Three different phospholipid systems were studied: DOPE:POPG, POPE:POPG, and DPPE:POPG at molar ratios of 3:1. Lipid mixtures were analyzed at 24 and 37 °C through three different model membranes: monolayers, liposomes, and supported lipid bilayers (SLBs). Data from three different techniques, Langmuir isotherms, Laurdan generalized polarization, and atomic force microscopy (AFM), evidenced that only the DPPE:POPG system exhibited coexistence between gel (L(ß)) and fluid (L(α)) phases at both 24 and 37 °C . In the POPE:POPG system the L(ß)/L(α) coexistence appears at 27 °C. Therefore, in order to investigate the distribution of LacY among phospholipid phases, we have used AFM to explore the distribution of LacY in SLBs of the three phospholipid systems at 27 °C, where the DOPE:POPG is in L(α) phase and POPE:POPG and DPPE:POPG exhibit L(ß)/L(α) coexistence. The results demonstrate the preferential insertion of LacY in fluid phase.

Lactose permease lipid selectivity using Förster resonance energy transfer.

The phospholipid composition that surrounds a membrane protein is critical to maintain its structural integrity and, consequently, its functional properties. To understand better this in the present work we have performed FRET measurements between the single tryptophan residue of a lactose permease Escherichia coli mutant (single-W151/C154G LacY) and pyrene-labeled phospholipids (Pyr-PE and Pyr-PG) at 37 degrees C. We have reconstituted this LacY mutant in proteoliposomes formed with heteroacid phospholipids, POPE and POPG, and homoacid phospholipids DOPE and DPPE, resembling the same PE/PG proportion found in the E. coli inner membrane (3:1, mol/mol). A theoretical model has been fitted to the experimental data. In the POPE/POPG system, quantitative model calculations show accordance with the experimental values that requires an annular region composed of approximately approximately 90 mol% PE. The experimental FRET efficiencies for the gel/fluid phase-separated DOPE/POPG system indicate a higher presence of PG in the annular region, from which it can be concluded that LacY shows clear preference for the fluid phase. Similar conclusions are obtained from analysis of excimer-to-monomer (E/M) pyrene ratios. To test the effects of this on cardiolipin (CL) on the annular region, myristoyl-CL and oleoyl-CL were incorporated in the biomimetic POPE/POPG matrix. The experimental FRET efficiency values, slightly larger for Pyr-PE than for Pyr-PG, suggest that CL displaces POPE and, more extensively, POPG from the annular region of LacY. Model fitting indicates that CL enrichment in the annular layer is, in fact, solely produced by replacing PG and that myristoyl-CL is not able to displace PE in the same way that oleoyl-CL does. One of the conclusions of this work is the fact that LacY inserts preferentially in fluid phases of membranes.

Preferential insertion of lactose permease in phospholipid domains: AFM observations.

We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli (E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids.

Evidence of phosphatidylethanolamine and phosphatidylglycerol presence at the annular region of lactose permease of Escherichia coli.

Biochemical and structural work has revealed the importance of phospholipids in biogenesis, folding and functional modulation of membrane proteins. Therefore, the nature of protein-phospholipid interaction is critical to understand such processes. Here, we have studied the interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) mixtures with the lactose permease (LacY), the sugar/H(+) symporter from Escherichia coli and a well characterized membrane transport protein. FRET measurements between single-W151/C154G LacY reconstituted in a lipid mixture composed of POPE and POPG at different molar ratios and pyrene-labeled PE or PG revealed a different phospholipid distribution between the annular region of LacY and the bulk lipid phase. Results also showed that both PE and PG can be part of the annular region, being PE the predominant when the PE:PG molar ratio mimics the membrane of E. coli. Furthermore, changes in the thermotropic behavior of phospholipids located in this annular region confirm that the interaction between LacY and PE is stronger than that of LacY and PG. Since PE is a proton donor, the results obtained here are discussed in the context of the transport mechanism of LacY.

Calcium-induced formation of subdomains in phosphatidylethanolamine-phosphatidylglycerol bilayers: a combined DSC, 31P NMR, and AFM study.

We study the effect of Ca(2+) on the lateral segregation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). Supported lipid bilayers (SLBs) were observed by atomic force microscopy (AFM). Since SLBs are formed from liposomes of POPE:POPG, we examined the effect of calcium on these suspensions by differential scanning calorimetry (DSC) and (31)P nuclear magnetic resonance spectroscopy ((31)P NMR). AFM images revealed the existence of two separated phases, the higher showing a region with protruding subdomains. Force spectroscopy (FS) was applied to clarify the nature of each phase. The values of breakthrough force (F(y)), adhesion force (F(adh)), and height extracted from the force curves were assigned to the corresponding gel (L(beta)) and fluid (L(alpha)) phase. The endotherms obtained by DSC suggest that, in the presence of Ca(2+), phase separation already exists in the suspensions of POPE:POPG used to form SLBs. Due to the temperature changes applied during preparation of SLBs a (31)P NMR study was performed to assess the lamellar nature of the samples before spreading them onto mica. With in situ AFM experiments we showed that the binding of Ca(2+) to POPG-enriched domains only induces the formation of subdomains in the L(beta) phase.

Phase changes in supported planar bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine.

We studied the thermal response of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) by comparing the differential scanning calorimetry (DSC) data of liposomes with atomic force microscopy (AFM) observations on supported planar bilayers. Planar bilayers were obtained by using the Langmuir-Blodgett (LB) technique: the first leaflet transferred at 30 mN m(-1) and the second at 25 mN m(-1). The topographic evaluation of supported POPE bilayers above room temperature showed changes between 43.8 and 59.8 degrees C. These observations are discussed in relation to the main roughness (Ra) variations and are interpreted as the result of the lamellar liquid crystalline (Lalpha) to inverted hexagonal (HII) phase transition. High-magnification images obtained at 45 degrees C revealed intermediate structures in the transformation. Force spectroscopy (FS) was subsequently applied to gain further structural and nanomechanical insight into the POPE planar bilayers as a function of temperature. These measurements show that the threshold force (Fy), which is the maximum force, that the sample can withstand before breaking, increases from 1.91+/-0.11 nN at 21 degrees C up to 3.08+/-0.17 nN at 43.8 degrees C. This behavior is interpreted as a consequence of the formation of intermediate structures or stalks in the transition from the L alpha to H II phase.

Atomic force microscopy characterization of supported planar bilayers that mimic the mitochondrial inner membrane.

In this study we examined the properties of supported planar bilayers (SPBs) formed from phospholipid components that comprise the mitochondrial inner membrane. We used 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL). Liposomes of binary POPE:POPC (1:1, mol:mol) and ternary (POPE:POPC:CL (0.5:0.3:0.2, mol:mol:mol) composition were used in the formation of SPBs on mica. The characterization of the SPBs was carried out below (4 degrees C) and above (24 and 37 degrees C) the phase transition temperature (Tm) of the mixtures in solution. We observed: (i) that the thickness of the bilayers, calculated from a cross-sectional analysis, decreased as the visualization temperature increased; (ii) the existence of laterally segregated domains that respond to temperature in SPBs of POPE:POPC:CL; (iii) a decrease in height and an increase in roughness (Ra) of SPBs after cytochrome c (cyt c) injection at room temperature. To obtain further insight into the nature of the interaction between cyt c and the bilayers, the competition between 8-anilino-1-naphthalene sulfonate (ANS) and the protein for the same binding sites in liposomes was monitored by fluorescence. The results confirm the existence of preferential interaction of cyt c with CL containing liposomes. Taking these results and those of previous papers published by the group, we discuss the preferential adsorption of cyt c in CL domains. This provides support for the relevance of these phospholipids as a proton trap in the oxidative phosphorylation process that occurs in the energy transducing membranes.

Unveiling a complex phase transition in monolayers of a phospholipid from the annular region of transmembrane proteins.

The lateral packing properties of phospholipids that surround transmembrane proteins are fundamental in the biological activity of these proteins. In this work, Langmuir monolayers of one such lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), are studied with a combination of pressure-area isotherm analysis, Brewster angle microscopy, and atomic force microscopy of extracted films. The analysis reveals a sequence of phase transitions LE-LC-LC' occurring in a narrow packing range. The lateral pressures and area densities of these phases provided meanings for the packing requirements in the annular lipid region of typical transmembrane proteins.