Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Interaction of arginine oligomer with model membrane

Short oligomers of arginine (R8) have been shown to cross readily a variety of biological barriers. A hypothesis was put forward that inverted micelles form in biological membranes in the presence of arginine oligomer peptides, facilitating their transfer through the membranes. In order to define the role of peptide-lipid interaction in this mechanism, we prepared liposomes as the model membrane to study the ability of R8 inducing calcein release from liposomes, the fusion of liposomes, R8 binding to liposomes and membrane disturbing activity of the bound R8. The results show that R8 binding to liposome membrane depends on lipid compositions, negative surface charge density and interior water phase pH values of liposomes. R8 has no activity to induce the leakage of calcein from liposomes or improve liposome fusion. R8 does not permeabilize through the membrane spontaneously. These peptides delivering drugs through membranes may depend on receptors and energy.     

Nitrogen dioxide solubility and permeation in lipid membranes

Nitrogen dioxide (NO₂) is an important oxidant molecule in biology that is produced by several biological processes, and it is also an important air pollutant. It can oxidize proteins and lipids with important consequences on their biological functions. Despite its relevance, the interaction of NO₂ with the cell barrier, the lipid membrane, is poorly understood. For instance, can lipid membranes limit NO₂ diffusion? To estimate the permeability of lipid membranes to NO₂ it is necessary to learn more about its solubility in the lipid phase. However, experimental data on NO₂ solubility is very limited. To improve our knowledge on this matter, we used a mixed approach consisting in calculating the solubility of NO₂ and related diatomic and triatomic gases (NO, O₂, CO₂, etc.) in different solvents using quantum calculations and Tomasi’s Polarizable Continuum Model and validating and correcting these results using experimental data available for the related gases. This approach led to an estimated partition coefficient for NO₂ of 2.7 between n-octanol and water, and 1.5 between lipid membranes and water, meaning that NO₂ is a moderately hydrophobic molecule (less than NO, more than CO₂). Based on the solubility-diffusion permeability theory, the permeability coefficient was estimated to be 5 cm s⁻¹, up to 4000 times higher than that of peroxynitrous acid. It is concluded that lipid membranes are not significant barriers to NO₂ transport.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.     

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmune diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles.     

Correlation of general anesthetic potency with solubility in membranes

Recently (Franks, N.P. and Lieb, W.R. (1978) Nature 274, 339–342) it has been claimed that the traditional correlation between anesthetic potency and vegetable oil solubility breaks down when the alkanols are compared to other volatile anesthetics. Lately, however, new information on the partitioning of anesthetics into lipid bilayers has become available. In this report the potency of twenty-one structurally diverse anesthetic agents is shown to correlate well with their ability to partition into phosphatidylcholine bilayers. Thus the original Meyer-Overton oil solubility hypothesis accomodates a wider range of anesthetics, including alkanols, volatile and gaseous agents, and barbiturates, when lipid bilayer solubility is substituted for oil solubility.     

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (π_e) were clearly lower than the biological membrane pressure (24–30 mN m^− 1) and the HIV-1 FP (35 mN m^− 1). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G^E). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive G^E values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP–lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.     

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation "entrance," contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta "hairpin," and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptide 1) binds also to the anionic vesicles, although with a lower affinity than does the ShB peptide, 2) adopts only occasionally the characteristic beta-structure, and 3) has completely lost the ability to traverse the anionic interphase at the vesicle surface and to insert into the hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate those conformed at the inactivation "entrance" of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i.e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta-structure, and its insertion into the channel's hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mutant ShB-L7E peptide to the lack of ability shown by this peptide to cross through the anionic interphase and insert into the hydrophobic domains of the model vesicle target.     

Interaction of benzocaine with model membranes

We measured the absorption properties, water solubility and partition coefficients (P) between n-octanol, egg phosphatidylcholine (EPC) liposomes and erythrocyte ghosts/water for benzocaine (BZC), an ester-type always uncharged local anesthetic. The interaction of BZC with EPC liposomes was followed using Electron Paramagnetic Resonance, with spin labels at different positions in the acyl chain (5, 7, 12, 16-doxylstearic acid methyl ester). Changes in lipid organization upon BZC addition allowed the determination of P values, without phase separation. The effect of BZC in decreasing membrane organization (maximum of 11.6% at approx. 0.8:1 BZC:EPC) was compared to those caused by the local anesthetics tetracaine and lidocaine. Hemolytic tests revealed a biphasic (protective/inductive) concentration-dependent hemolytic effect for BZC upon rat erythrocytes, with an effective BZC:lipid molar ratio in the membrane for protection (RePROT), onset of hemolysis (ReSAT) and 100% membrane solubilization (ReSOL) of 1.0:1, 1.1:1 and 1.3:1, respectively. The results presented here reinforce the importance of considering hydrophobic interactions in the interpretation of the effects of anesthetics on membranes.     

Interaction of synthetic peptides corresponding to the scaffolding domain of Caveolin-3 with model membranes

Caveolin-1 and -3 are among the few proteins in which the functional domains are contiguous and modular. The interaction of synthetic peptides spanning the scaffolding domain of caveolin-3 with model membranes has been investigated. The peptides include the scaffolding domain, the aromatic and positively charged residues at the C-terminal end of this domain as well as deletion of three amino acids TFT, observed in certain patients with limb girdle muscular dystrophy. All of the peptides appear to be peripherally bound to the bilayer surface. However, no preferential binding to sphingomyelin and cholesterol-containing lipid vesicles was observed. Deletion of TFT appears to affect the association with lipid vesicles compared with the native sequence. Association with lipids decreases considerably when TFT as well as the aromatic-rich segment YWFYR, which occurs at the extreme C-terminus of the scaffolding domain, are deleted.     

The cooperative behaviour of antimicrobial peptides in model membranes

A systematic analysis of the hypothesis of the antimicrobial peptides' (AMPs) cooperative action is performed by means of full atomistic molecular dynamics simulations accompanied by circular dichroism experiments. Several AMPs from the aurein family (2.5,2.6, 3.1), have a similar sequence in the first ten amino acids, are investigated in different environments including aqueous solution, trifluoroethanol (TFE), palmitoyloleoylphosphatidylethanolamine (POPE), and palmitoyloleoylphosphatidylglycerol (POPG) lipid bilayers. It is found that the cooperative effect is stronger in aqueous solution and weaker in TFE. Moreover, in the presence of membranes, the cooperative effect plays an important role in the peptide/lipid bilayer interaction. The action of AMPs is a competition of the hydrophobic interactions between the side chains of the peptides and the hydrophobic region of lipid molecules, as well as the intra peptide interaction. The aureins 2.5-COOH and 2.6-COOH form a hydrophobic aggregate to minimize the interaction between the hydrophobic group and the water. Once that the peptides reach the water/lipid interface the hydrophobic aggregate becomes smaller and the peptides start to penetrate into the membrane. In contrast, aurein 3.1-COOH forms only a transient aggregate which disintegrates once the peptides reached the membrane, and it shows no cooperativity in membrane penetration.     

Fusion peptides and the mechanism of viral fusion

Segments of viral fusion proteins play an important role in viral fusion. They are defined by a number of criteria, including the sensitivity of this region of the viral fusion protein to loss of function as a consequence of mutation. In addition, small model peptides designed to mimic this segment of viral fusion proteins often have some membrane perturbing activity. The properties of viral fusion peptides are quite varied. Many are found at the amino terminus of viral fusion proteins. As isolated peptides, they have been found to form both α-helical as well as β-structure. In addition, some viruses have internal fusion peptides. Just as there are several structural motifs for viral fusion peptides, there are also several mechanisms by which they accelerate the process of membrane fusion. These include the promotion of negative curvature, lowering the rupture tension of the lipid monolayer, acting as an anchor to join the fusion membranes, transmitting a force to the membrane or imparting energy to the system by other means. It is not likely that the fusion peptide can fulfill all of these diverse roles and future studies will elucidate which of these mechanisms is most important for the action of individual viral fusion peptides.     

Interaction of the pitavastatin with model membranes

Pitavastatin is a statin drug that, by competitively inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase, can lower serum cholesterol levels of low-density lipoprotein (LDL) accompanied by side effects due to pleiotropic effects leading to statin intolerance. These effects can be explained by the lipophilicity of statins, which creates membrane affinity and causes statin localization in cellular membranes. In the current report, the interaction of pitavastatin with POPC model membranes and its influence on the membrane structure were investigated using ¹H, ²H and ³¹P solid-state NMR spectroscopy. Our experiments show the average localization of pitavastatin at the lipid/water interface of the membrane, which is biased towards the hydrocarbon core in comparison to other statin molecules. The membrane binding of pitavastatin also introduced an isotropic component into the ³¹P NMR powder spectra, suggesting that some of the lamellar POPC molecules are converted into highly curved structures.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of peptides corresponding to mitochondrial presequences with membranes.

The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus. Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane. To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry. These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure. Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior. Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer. High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors. Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site. These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.     

Interaction of bundled Ser-rich amphiphilic peptides with phospholipid membranes.

To investigate properties of hydrophilic bundled peptides and their interactions with phospholipid membranes, bundled peptides named [Trp2]- and [Trp12]-4alpha-46S9, which are composed of four fragments of amphiphilic 24-mer peptide, were designed and synthesized. Tryptophan (Trp) was introduced at the 2nd position from the N-terminal or at the centre (12th) of the helix to monitor the peptide-lipid interaction. Circular dichroism measurements indicated that the peptides had low alpha-helicities in a buffer solution (pH 7.4) and also in the presence of dipalmitoyl-DL-3-phosphatidylcholine (DPPC) vesicles. In the presence of DPPC/dipalmitoyl-DL-3-phosphatidylglycerol (DPPG) (3:1) vesicles, the measurement could not be taken because of turbidity induced by vesicle aggregation. Both peptides had moderate perturbation activity for both the neutral and acidic vesicles at 25 degrees C. The perturbation patterns at 50 degrees C were much different from those at 25 degrees C and the maximum activity reached 100% at a low peptide concentration. The results of the measurement of membrane fusion activity of peptides showed a similar tendency to that found in the perturbation experiment. A quenching experiment indicated that the Trp2 and Trp12 residues in [Trp2]- and [Trp12]-4alpha-46S9 were scarcely embedded in neutral lipid membranes.     

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.     

Interaction of dystrophin fragments with model membranes

The interaction with membrane lipids of recombinant fragments of human dystrophin, corresponding to a single structural repeating unit of the rod domain, was examined. Surface plasmon resonance, constant-pressure isotherms in a Langmuir surface film balance, and interfacial rheology were used to observe binding of the polypeptides and its effects on the properties of the lipid film. Modification of the monolayer properties was found to depend on the presence of phosphatidylserine in the lipid mixture and on the native tertiary fold of the polypeptide; thus a fragment with the minimum chain length required for folding (117 residues) or longer caused a contraction of the surface area at constant pressure, whereas fragments of 116 residues or less had no effect. The full extent of contraction was reached at a surface concentration of lipid corresponding to an average area of about 42 A2 per lipid molecule. A dystrophin fragment with the native, folded conformation induced a large increase in surface shear viscosity of the lipid film, whereas an unfolded fragment had no effect. Within a wide range of applied shear, the shear viscosity remained Newtonian. Binding of liposomes to immobilized dystrophin fragments could be observed by surface plasmon resonance and was again related to the conformational state of the polypeptide and the presence of phosphatidylserine in the liposomes. Our results render it likely that intact dystrophin interacts directly and strongly with the sarcolemmal lipid bilayer and grossly modifies its material properties.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.