Examination of the peptide sequence requirements for lipid-binding. Alternative pathways for promoting the interaction of amphipathic alpha-helical peptides with phosphatidylcholine

To examine the relationship between peptide sequence and the interaction of amphipathic alpha-helical peptides with phosphatidylcholines, various methods of mixing the peptide and lipid were explored. A series of amphipathic alpha-helical peptides containing from 10 to 18 residues were synthesized by solid-phase techniques. An 18-residue peptide and two relatively hydrophobic 10-residue peptides did not disrupt dimyristoylphosphatidylcholine liposomes when added to the lipid in buffer. However, when the peptides were premixed with lipid in a suitable organic solvent and then reconstituted with aqueous buffer, clear micelles were formed, indicating association of the amphipathic alpha-helical peptide with lipid. In general, the best solvent for this purpose was trifluoroethanol. The circular dichroic and fluorescence spectra of peptides which readily formed clear mixtures when mixed in buffer with dimyristoylphosphatidylcholine liposomes were similar when prepared either by the alternative pathway technique using trifluoroethanol or by a cholate removal technique. For the peptides which did not clear liposomes in buffer, first mixing with dimyristoylphosphatidylcholine in trifluoroethanol resulted in an increase in the alpha-helicity of the peptides as judged by circular dichroic spectra and a blue-shift in the fluorescence emission maxima of the single tryptophan residue in each peptide. These data are consistent with formation of an amphipathic alpha-helix in lipid by peptides which based on mixing experiments with dimyristoylphosphatidylcholine liposomes in buffer at the phase transition temperature of the lipid would be considered ineffective in lipid binding. Thus, simple mixing of peptides with liposomes may give misleading results concerning the intrinsic affinity of a particular peptide sequence for lipid. In addition, the data demonstrate that relatively hydrophobic amphipathic alpha-helical peptides which do not form small micelles with dimyristoylphosphatidylcholine spontaneously in aqueous solution may interact with lipid as typical amphipathic alpha-helices when mixed by an alternative pathway.     

Controlling association of vesicle embedded peptides by alteration of the physical state of the lipid matrix

We report here the reversible association of a designed peptide embedded in a lipid membrane through a stimulus-sensitive trigger that changes the physical state of the bilayer matrix. A peptide designed with the classical 4-3 heptad repeat of coiled coils, equipped with leucine residues at all canonical interface positions, TH1, was rendered membrane soluble by replacement of all exterior residues with randomly selected hydrophobic amino acids. Insertion of TH1 into large unilamellar phosphatidylcholine vesicles was followed by monitoring tryptophan fluorescence. Peptide insertion was observed when the lipids were in the liquid-crystalline state [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] but not when they were in the crystalline phase [1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)]. Formation of a trimeric alpha-helical bundle in lipid bilayers was followed by fluorescence resonance energy transfer. Global fit analysis revealed a monomer--trimer equilibrium with a dissociation constant of around 10(-5) [corrected] MF(2). A lipid mixture composed of DPPC and POPC exhibiting a phase transition at 34 degrees C between a crystalline/liquid-crystalline coexistence region and a completely miscible liquid-crystalline phase was used to control the formation of the trimeric peptide bundle. TH1 is phase excluded in crystalline DPPC domains below 34 degrees C, leading to a larger number of trimers. However, when the DPPC domains are dispersed at temperatures above 34 degrees C, the number of trimers is reduced.     

Association of acylated cationic decapeptides with dipalmitoylphosphatidylserine-dipalmitoylphosphatidylcholine lipid membranes.

The interaction of three acylated and cationic decapeptides with lipid membranes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) has been studied by means of fluorescence spectroscopy and differential scanning calorimetry (DSC). The synthetic model decapeptides that are N-terminally linked with C(2), C(8), and C(14) acyl chains contain four basic histidine residues in their identical amino acid sequence. A binding model, based on changes in the intrinsic fluorescent properties of the peptides upon association with the DPPC-DPPS membranes, is used to estimate the peptide-membrane dissociation constants. The results clearly show that all three peptides have a higher affinity to liposomes containing DPPS lipids due to non-specific electrostatic interactions between the cationic peptides and the anionic DPPS lipids. Furthermore, it is found that the acyl chain length of the peptides plays a crucial role for the binding. A preference for fluid phase membranes as compared to gel phase membranes is generally observed for all three peptides. DSC is used to characterise the influence of the three peptides on the thermodynamic phase behaviour of the binary DPPC-DPPS lipid mixture. The extent of peptide association deduced from the heat capacity measurements suggests a strong binding and membrane insertion of the C(14) acylated peptide in accordance with the fluorescence measurements.     

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.     

Interaction of melittin with model membranes: effect on the size and permeability of liposomes

The influence of melittin, a monomer devoid of the phospholipase activity, on the size and permeability of liposomes from egg lecithin (PC), dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) has been investigated by the methods of fluorescence spectroscopy, quasi-elastic light scattering and freeze-fracture electron microscopy. While studying calcein release from liposomes under the influence of melittin it has been shown that binding of melittin with a bilayer is a fast process which depends on the concentration lipid: protein (Ri) ratio as well as on the phase state of the lipid. The lipids being in the liquid-crystalline forms (PC and DMPC) are characterized by a more rapid release of the dye-stuff from liposomes than DPPC vesicles being in gel state with the same Ri. Under the influence of different melittin concentrations heterogeneity of the system and its medium hydrodynamic size of particles at first increases (100 less than or equal to Ri less than 500) due to their fusion and then these parameters decrease to the initial values.     

Phospholipid miscibility in ternary mixtures

The gel to liquid-crystalline phase transition of aqueous dispersions of phospholipid mixtures was investigated by means of the repartition of the spin label 2,2,6,6-tetramethylpiperidine-I-oxyl between aqueous space and lipid hydrocarbon region. The dimyristoylphosphatidylcholine (DMPC)/dibehenoylphosphatidylcholine (DBPC) and dipalmitoylphosphatidylcholine (DPPC)/DBPC phase diagrams indicate gel phase immiscibility, whereas the distearoylphosphatidylcholine (DSPC)/DBPC phase diagram indicates non-ideal gel phase miscibility at low DBPC molar fractions. Aqueous dispersions of DMPC/DPPC/DBPC ternary mixtures show two distinct phase transitions, the first associated with the melting of a DMPC/DPPC phase and the second with the melting of a DBPC phase. Aqueous dispersions of DMPC/DSPC/DBPC ternary mixtures show to phase transitions at low DSPC molar fractions; the first is probably associated with the melting of a DMPC/DSPC phase, and the second with the melting of a DBPC/DSPC phase. At high DSPC molar fractions, only one phase transition is observed; this suggests that all the lipids are mixed in gel state membranes.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylethanolamine Bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and members of the homologous series of zwitterionic n-saturated diacyl phosphatidylethanolamines (PEs). Analogs of L24, in which the lysine residues were replaced by 2,3-diaminopropionic acid (acetyl-DAP2-Leu24-DAP2-amide (L24DAP)) or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (Ac-K2-W-L22-W-K2-amide (WL22W)), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the PE bilayers is altered by these peptides in a hydrophobic mismatch-independent manner, in contrast to the hydrophobic mismatch-dependent manner observed previously with zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) bilayers. Moreover, all three peptides reduce the phase transition temperature to a greater extent in PE bilayers than in PC and PG bilayers, indicating a greater disruption of PE gel-phase bilayer organization. Moreover, the lysine-anchored L24 reduces the phase transition temperature, enthalpy, and the cooperativity of PE bilayers to a much greater extent than DAP-anchored L24DAP, whereas replacement of the terminal leucines by tryptophan residues (Ac-K2-W-L22-W-K2-amide) only slightly attenuates the effects of this peptide on the chain-melting phase transition of the host PE bilayers. All three peptides form very stable alpha-helices in PE bilayers, but small conformational changes occur in response to mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. These results suggest that the lysine snorkeling plays a significant role in the peptide-PE interactions and that cation-pi-interactions between lysine and tryptophan residues may modulate these interactions. Altogether, these results suggest that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer but also by the electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the polar headgroups of PE bilayers.     

螺旋脂质体的研究——Ⅰ.含有心磷脂的两相体系形成螺旋脂质体的条件

用CL(心磷脂)与DMPC(二肉豆蔻酰磷脂酰胆碱)或DPPC(二棕榈酰磷脂酰胆碱)所组成的两组体系制备脂质体,可形成少量管状脂质体.加Ca~(2+)或其它二价阳离子后可形成单股或双股螺旋.对产生这类螺旋脂质体的各种条件进行了研究.     

Molecular organization in striated domains induced by transmembrane alpha-helical peptides in dipalmitoyl phosphatidylcholine bilayers

Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions.     

Association of a pH-sensitive peptide with membrane vesicles: role of amino acid sequence

The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.     

Spectroscopic studies of the tyrosine residues of human plasma apolipoprotein A-II

Human apolipoprotein A-II (apo A-II) in solution and associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was investigated by a combination of absorbance and fluorescence methods. Each apo A-II polypeptide chain contains four tyrosine residues but no tryptophan residues. Two and three tyrosine residues, respectively, appear to be buried for apo A-II in aqueous solution and in the lipid-associated protein. The spectroscopic properties of the tyrosine residues of lipid-associated apo A-II were also investigated. Plots of fluorescence intensity against temperature revealed a discontinuity in the region of the phase transition; however, over the same temperature range, there was no change in the exposure of tyrosine residues to the aqueous environment or in their mobility as measured by fluorescence polarization. Near-ultraviolet circular dichroic measurements demonstrated that the environments of the tyrosine residues of lipid-associated apo A-II and nitrated apo A-II were different from that of the apo A-II in solution or in a denatured state. Similar measurements also revealed that the microenvironments around tyrosines of apo A-II bound to DMPC in the gel phase are different from those observed in the liquid crystalline phase. Using environmentally sensitive fluorescence lipid probes, we have previously demonstrated that the polarity of the lipid/water interface of DMPC changes through a phase transition. The observations presented here indicate that these environmental changes also occur at the lipid/protein interface.     

Fluidity-dependence of membrane adhesiveness can be explained by thermotropic shifts in surface potential

It is demonstrated that the transition of both dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) from the gel to liquid-crystalline phase is paralleled by a pronounced increase in the negative surface potential of liposomes composed of either lipid. The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory is applied to show that this phenomenon can serve as a simple explanation of diverse adhesive properties of solid and fluid lipid bilayers.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

Role of peptide structure in lipid-peptide interactions: a fluorescence study of the binding of pentagastrin-related pentapeptides to phospholipid vesicles

The binding of pentagastrin and three other structurally related pentapeptides to phospholipid vesicles has been studied by fluorescence spectroscopy. The fluorescence of the tryptophan residues of these peptides exhibits an increased quantum yield upon binding to phospholipid vesicles. This is accompanied by a blue shift of the maximum emission, indicative of the incorporation of the tryptophan residue into a more hydrophobic environment. The affinity of the peptides for a zwitterionic phospholipid, dimyristoylphosphatidylcholine (DMPC), increases in the following order: N-t-Boc-beta-Ala-Trp-Met-Gly-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2. Comparison of the interaction of these various peptides with this phospholipid indicates that although the interaction is largely of hydrophobic nature, the structure of the polar amino acids and their electrostatic charge have significant influence on the nature of the bindings. In addition, the sequence of polar and apolar amino acids appears to be of importance. The higher affinity for DMPC of N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 as compared to its "reversed" analogue N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2 suggests that the ability of the peptides to fold into amphiphatic structures can enhance their lipid binding affinity. For all peptides the interaction with DMPC is greater at 8 degrees C, i.e., below the lipid phase transition temperature, than at 40 degrees C, i.e., above the lipid phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides.

To investigate the influence of proline residues on the activity of alpha-helical peptides, variants were synthesized with insertions of proline residues to create peptides without proline, or with one or two prolines. The influence of the proline-induced bends was assessed by circular dichroism in the presence of liposomes, and the ability of the peptides to kill microorganisms, to permeabilize the outer and cytoplasmic membranes of Escherichia coli, to bind to liposomes, to form channels in planar lipid bilayers, and to synergize with conventional antibiotics. Representative peptides adopted alpha-helical conformations in phosphatidylcholine/phosphatidylglycerol (POPC/POPG, 7:3) liposomes as well as in 60% trifluoroethanol solution, as revealed by circular dichroism (CD) spectroscopy. However, the percent of helicity decreased as the number of proline residues increased. Tryptophan fluorescence spectroscopy showed that all of these peptides inserted into the membranes of liposomes as indicated by a blue shift in the emission maximum and an increase in the fluorescence intensity of the single tryptophan at residue 2. Quenching experiments further prove that the tryptophan residue was no longer accessible to the aqueous quencher KI. The peptide that lacked proline exhibited the highest activity [minimal inhibitory concentrations (MICs) of 0.5-4 microg/mL] against all tested Gram-negative and Gram-positive bacteria, but was hemolytic at 8 microg/mL. The single-proline peptides exhibited intermediate antibacterial activity. Peptides with two proline residues were even less active with moderate MICs only against E. coli. With only one exception from each group, the peptides were nonhemolytic. The ability of the peptides to demonstrate synergy in combination with conventional antibiotics increased as the antibacterial effectiveness decreased. All peptides bound to bacterial lipopolysaccharide and permeabilized the outer membrane of E. coli to similar extents. However, their ability to permeabilize the cytoplasmic membrane of E. coli as assessed by the unmasking of cytoplasmic beta-galactosidase decreased substantially as the number of proline residues increased. Correspondingly, increasing the number of proline residues caused a decreased ability to form channels in planar lipid bilayers, and the hemolytic, proline-free peptide tended to cause rapid breakage of planar membranes. Thus, the number of bends created by insertion of proline residues is an important determinant of antimicrobial, hemolytic, and synergistic activity.     

Membrane fluidity as affected by the insecticide lindane

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of lindane with model and native membranes. Lindane disorders the gel phase of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), since it broadens and shifts the main phase transition, but no apparent effect is detected in the fluid phase. These effects of lindane are more pronounced in bilayers of short-chain lipids, e.g., DMPC. In equimolar mixtures containing DMPC and DSPC, lindane preferentially interacts with the more fluid lipid species inducing lateral phase separations. However, in mixtures of DMPC and DPPC, the insecticide only broadens and shifts the main phase transition, i.e., an effect similar to that observed in bilayers of pure lipids. Lindane has no apparent effect in DMPC bilayers enriched with high cholesterol content (greater than or equal to 30 mol%), whereas disordering effects can still be detected in bilayers with low cholesterol (less than 30 mol%). Apparently, lindane does not perturb the fluid phase of representative native membranes, namely, mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes in agreement with the results obtained in fluid phospholipid bilayers, despite the reasonable incorporation of the insecticide in these membranes, as previously reported (Antunes-Madeira, M.C. and Madeira, V.M.C. (1985) Biochim. Biophys. Acta 820, 165-172).     

Interaction of selected anthocyanins with erythrocytes and liposome membranes

Anthocyanins are one of the main flavonoid groups. They are responsible for, e.g., the color of plants and have antioxidant features and a wide spectrum of medical activity. The subject of the study was the following compounds that belong to the anthocyanins and which can be found, e.g., in strawberries and chokeberries: callistephin chloride (pelargonidin-3-O-glucoside chloride) and ideain chloride (cyanidin-3-O-galactoside chloride). The aim of the study was to determine the compounds’ antioxidant activity towards the erythrocyte membrane and changes incurred by the tested anthocyanins in the lipid phase of the erythrocyte membrane, in liposomes composed of erythrocyte lipids and in DPPC, DPPC/cholesterol and egg lecithin liposomes. In particular, we studied the effect of the two selected anthocyanins on red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC and DPPC/cholesterol liposomes. Fluorimetry with the Laurdan and Prodan probes indicated increased packing density in the hydrophilic phase of the membrane in the presence of anthocyanins. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The compounds slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The study has shown that both anthocyanins are incorporated into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The investigation proved that the compounds penetrate only the outer part of the external lipid layer of liposomes composed of erythrocyte lipids, DPPC, DPPC/cholesterol and egg lecithin lipids, changing its packing order. Fluorimetry studies with DPH-PA proved that the tested anthocyanins are very effective antioxidants. The antioxidant activity of the compounds was comparable with the activity of Trolox®.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.