Palmitoyloleoyl Phosphatidylglycerol Research Articles

Phospholipid chlorohydrins as chlorine exposure biomarkers in a large animal model

Chlorine is a toxic industrial chemical that has been used as a chemical weapon in recent armed conflicts. Confirming human exposure to chlorine has proven challenging, and there is currently no established method for analyzing human biomedical samples to unambiguously verify chlorine exposure. In this study, two chlorine-specific biomarkers: palmitoyl-oleoyl phosphatidylglycerol chlorohydrin (POPG-HOCl) and the lipid derivative oleoyl ethanolamide chlorohydrin (OEA-HOCl) are shown in bronchoalveolar lavage fluid (BALF) samples from spontaneously breathing pigs after chlorine exposure. These biomarkers are formed by the chemical reaction of chlorine with unsaturated phospholipids found in the pulmonary surfactant, which is present at the gas-liquid interface within the lung alveoli. Our results strongly suggest that lipid chlorohydrins are promising candidate biomarkers in the development of a verification method for chlorine exposure. The establishment of verified methods capable of confirming the illicit use of toxic industrial chemicals is crucial for upholding the principles of the Chemical Weapons Convention (CWC) and enforcing the ban on chemical weapons. This study represents the first published dataset in BALF revealing chlorine biomarkers detected in a large animal. Furthermore, these biomarkers are distinct in that they originate from molecular chlorine rather than hypochlorous acid.

Protein Corona of Anionic Fluid-Phase Liposomes Compromises Their Integrity Rather than Uptake by Cells.

Despite the undisputable role of the protein corona in the biointeractions of liposome drug carriers, the field suffers from a lack of knowledge regarding the patterns of protein deposition on lipid surfaces with different compositions. Here, we investigated the protein coronas formed on liposomes of basic compositions containing combinations of egg phosphatidylcholine (PC), palmitoyloleoyl phosphatidylglycerol (POPG), and cholesterol. Liposome-protein complexes isolated by size-exclusion chromatography were delipidated and analyzed using label-free LC-MS/MS. The addition of the anionic lipid and cholesterol both affected the relative protein abundances (and not the total bound proteins) in the coronas. Highly anionic liposomes, namely those containing 40% POPG, carried corona enriched with cationic proteins (apolipoprotein C1, beta-2-glycoprotein 1, and cathelicidins) and were the least stable in the calcein release assay. Cholesterol improved the liposome stability in the plasma. However, the differences in the corona compositions had little effect on the liposome uptake by endothelial (EA.hy926) and phagocytic cells in the culture (U937) or ex vivo (blood-derived monocytes and neutrophils). The findings emphasize that the effect of protein corona on the performance of the liposomes as drug carriers occurs through compromising particle stability rather than interfering with cellular uptake.

Lipid mediators in the regulation of innate and adaptive immunity.

Lipid mediators in the regulation of innate and adaptive immunity.

Interaction of detergent with complex mimics of bacterial membranes

Detergents are valuable tools to extract membrane proteins for biophysical, biochemical, and structural scrutiny. The detergent-driven solubilization of bilayers made from a single lipid species is commonly described in terms of pseudo-phase diagrams and a three-stage model accounting for three ranges comprising (i) intact vesicles, (ii) vesicle/micelle co-existence, or (iii) mixed micelles. Moreover, the pseudo-phase boundaries thus determined can often be quantitatively rationalized in terms of the molecular shapes of the lipid and the detergent used. Yet, it has remained unclear to what extent this approach can be applied to multi-component lipid membranes that more closely mimic the compositional complexity of cellular membranes. Here, we studied how lipid mixtures composed of palmitoyl oleoyl phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylglycerol (POPG), and tetraoleoyl cardiolipin (TOCL) are solubilized by the commonly used zwitterionic detergent lauryldimethylamine N-oxide using isothermal titration calorimetry. While phase diagrams of the diverse lipid mixtures showed the typical ranges of the three-stage model, we found that POPG-rich POPE/POPG bilayers are more difficult to solubilize than POPG-poor POPE/POPG bilayers. In turn, POPE/POPG/TOCL bilayers became increasingly resistant to detergent with increasing TOCL content. Since POPG is nearly cylindrically shaped and TOCL adopts inverted cone-like shapes under current buffer conditions, our solubilization data do not align with shape-based arguments. Instead, additional electrostatic interactions between lipids and detergents lead to non-additive mixing behavior affecting the resilience of complex lipid bilayers against solubilization.

Periodic bilayer organization in the complexes of Beta-2 Glycoprotein I with anionic lipid membranes

β2 glycoprotein I (β2GPI) is a soluble protein that participates in blood coagulation, clearance of apoptotic bodies and generation of antigens in antiphospholipid syndrome among many other functions. We studied the aggregates formed by β2GPI with the anionic phospholipids palmitoyloleoylphosphatidyl glycerol, dimyristoylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol and cardiolipin using small angle X-ray scattering. The complexes obtained in a medium containing 0.01 M NaCl showed Bragg peaks up to the sixth order in a well-defined integer sequence indicating the presence of a lamellar stacking with a periodicity of 17.8 nm and with largely reduced membrane fluctuations. Modeling the complex signal allowed us to conclude that the coherence length was only two bilayers and that about 15% of the total surface was actually stacked. The space between bilayers allows accommodating an extended β2GPI molecule making a bridge between the interacting bilayers. The interactions between membranes mediated by β2GPI was favored when the membranes were in the liquid crystalline state.

Antimicrobial peptide activity in asymmetric bacterial membrane mimics.

We report on the response of asymmetric lipid membranes composed of palmitoyl oleoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylglycerol, to interactions with the frog peptides L18W-PGLa and magainin 2 (MG2a), as well as the lactoferricin derivative LF11-215. In particular we determined the peptide-induced lipid flip-flop, as well as membrane partitioning of L18W-PGLa and LF11-215, and vesicle dye-leakage induced by L18W-PGLa. The ability of L18W-PGLa and MG2a to translocate through the membrane appears to correlate with the observed lipid flip-flop, which occurred at the fastest rate for L18W-PGLa. The higher structural flexibility of LF11-215 in turn allows this peptide to insert into the bilayers without detectable changes of membrane asymmetry. The increased vulnerability of asymmetric membranes to L18W-PGLa in terms of permeability, appears to be a consequence of tension differences between the compositionally distinct leaflets, but not due to increased peptide partitioning.

In Silico Study of Membrane Lipid Composition Regulating Conformation and Hydration of Influenza Virus B M2 Channel.

The proton conduction of transmembrane influenza virus B M2 (BM2) proton channel is possibly mediated by the membrane environment, but the detailed molecular mechanism is challenging to determine. In this work, how membrane lipid composition regulates the conformation and hydration of BM2 channel is elucidated in silico. The appearance of several important hydrogen-bond networks has been discovered, as the addition of negatively charged lipid palmitoyloleoyl phosphatidylglycerol (POPG) and cholesterol reduces membrane fluidity and augments membrane rigidity. A more rigid membrane environment is beneficial to expand the channel, allow more water to enter the channel, promote channel hydration, and then even affect the proton conduction facilitated by the hydrated channel. Thus, membrane environment could be identified as an important influence factor of conformation and hydration of BM2. These findings can provide a unique perspective for understanding the mechanism of membrane lipid composition regulating conformation and hydration of BM2 and have important significance to the further study of anti-influenza virus B drugs.

Revealing the Mode of Action of Halictine Antimicrobial Peptides: A Comprehensive Study with Model Membranes.

Antimicrobial peptides are innate host defense molecules with the ability to kill pathogens. They have been widely studied for their membrane lytic activity and their potential to overcome the ever-increasing threat of antimicrobial resistance against conventional antibiotics. Here, we focus on two halictines, antimicrobial peptides first obtained from the venom of the eusocial bee Halictus sexcinctus. The peptides, HAL-1 and HAL-2, are cationic (with +3 and +4 charges, respectively) and amphipathic, have 12 amino acid residues, and exhibit high biological activity. For this study, the mechanism of action of HAL-1 and HAL-2 was studied in detail using large and giant unilamellar vesicles composed of pure palmitoyl oleoyl phosphatidyl choline (POPC) and a mixture of POPC and the anionic lipid palmitoyl oleoyl phosphatidyl glycerol (POPG) as biomimetic models of the membranes of eukaryotes and microorganisms, respectively. A set of complementary techniques was put forward: carboxyfluorescein leakage assay, phase contrast optical microscopy, ζ-potential, static and dynamic light scattering, fluorescence and circular dichroism spectroscopies, and isothermal titration calorimetry. The results show that both halictines are able to interact strongly with anionic membranes: The interaction is exothermic and accompanied by structuring of the peptides as an α-helix and deep insertion into the membrane causing substantial membrane permeabilization at very low peptide/lipid molar ratios. Extensive vesicle aggregation was detected only at a high peptide concentration. On the other hand, the interaction of the halictines with POPC is significantly milder. Yet, the peptides were able to permeabilize the POPC membranes to some extent. Comparing both peptides, HAL-1 showed a somewhat stronger effect on model membranes. Fits to the data revealed apparent binding constants on the order of 103-104 M-1 for anionic membranes and 1 order of magnitude lower for zwitterionic bilayers. When lytic activity results were compared at the same bound peptide/lipid ratio, the halictines exhibited a higher activity toward zwitterionic membranes. As novel peptides, small and with powerful activity, these halictines are potential candidates for becoming antimicrobial agents.

In Vitro Functional and Structural Characterization of A Synthetic Clinical Pulmonary Surfactant with Enhanced Resistance to Inhibition

CHF5633 is a novel synthetic clinical pulmonary surfactant preparation composed by two phospholipid species, dipalmitoyl phosphatidylcholine (DPPC) and palmitoyloleoyl phosphatidylglycerol (POPG), and synthetic analogues of the hydrophobic surfactant proteins SP-B and SP-C. In this study, the interfacial properties of CHF5633 in the absence and in the presence of inhibitory serum proteins have been assessed in comparison with a native surfactant purified from porcine lungs and with poractant alpha, a widely used clinical surfactant preparation. The study of the spreading properties of CHF5633 in a Wilhelmy balance, its ability to adsorb and accumulate at air-liquid interfaces as revealed by a multiwell fluorescence assay, and its dynamic behavior under breathing-like compression-expansion cycling in a Captive Bubble Surfactometer (CBS), all revealed that CHF5633 exhibits a good behavior to reduce and sustain surface tensions to values below 5 mN/m. CHF5633 shows somehow slower initial interfacial adsorption than native surfactant or poractant alpha, but a better resistance to inhibition by serum proteins than the animal-derived clinical surfactant, comparable to that of the full native surfactant complex. Interfacial CHF5633 films formed in a Langmuir-Blodgett balance coupled with epifluorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than films made by native porcine surfactant or poractant alpha. This ability of CHF5633 to segregate condensed lipid phases can be related with a marked thermotropic transition from ordered to disordered membrane phases as exhibited by differential scanning calorimetry (DSC) of CHF5633 suspensions, occurring at similar temperatures but with higher associated enthalpy than that shown by poractant alpha. The good interfacial behavior of CHF5633 tested under physiologically meaningful conditions in vitro and its higher resistance to inactivation by serum proteins, together with its standardized and well-defined composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung inflammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.

The small heat shock proteins, HSPB1 and HSPB5, interact differently with lipid membranes.

Increasing evidence shows that heat shock proteins (hsp) escape the cytosol gaining access to the extracellular environment, acting as signaling agents. Since the majority of these proteins lack the information necessary for their export via the classical secretory pathway, attention has been focused on alternative releasing mechanisms. Crossing the plasma membrane is a major obstacle to the secretion of a cytosolic protein into the extracellular milieu. Several mechanisms have been proposed, including direct interaction with the plasma membrane or their release within extracellular vesicles (ECV). HSPB1 (Hsp27), which belongs to the small hsp family, was detected within the membrane of ECV released from stressed HepG2 cells. To further investigate this finding, we studied the interaction of HSPB1 with lipid membranes using liposomes. We found that HSPB1 interacted with liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl oleoyl phosphatidylglycerol (POPG), with different characteristics. Another member of the small hsp family, HSPB5 (αB-crystallin), has also been detected within ECV released from HeLa cells transfected with this gene. This protein was found to interact with liposomes as well, but differently than HSPB1. To address the regions interacting with the membrane, proteoliposomes were digested with proteinase K and the protected domains within the liposomes were identified by mass spectroscopy. We observed that large parts of HSPB1 and HSPB5 were embedded within the liposomes, particularly the alpha-crystallin domain. These observations suggest that the interaction with lipid membranes may be part of the mechanisms of export of these proteins.

Orphan designation: Sinapultide, dipalmitoylphosphatidylcholine palmitoyl-oleoyl phosphatidylglycerol, sodium salt and palmitic acid, Treatment of cystic fibrosis

Orphan designation: Sinapultide, dipalmitoylphosphatidylcholine palmitoyl-oleoyl phosphatidylglycerol, sodium salt and palmitic acid, Treatment of cystic fibrosis

Dynamic Water Hydrogen-Bond Networks at the Interface of a Lipid Membrane Containing Palmitoyl-Oleoyl Phosphatidylglycerol.

Lipid membrane interfaces are complex environments that host essential cellular processes such as binding of proteins or drug molecules. A key open question is how water molecules at the interface of membranes with anionic lipids participate in protein binding. To address this question, we studied the dynamics of water hydrogen bonding at the interface of membranes composed of phosphatidylcholine and phosphatidylglycerol, and implemented an algorithm to identify hydrogen-bonded networks at the interface of a lipid membrane, and to characterize their dynamics and linear connections. We find that the membrane interface is characterized by a rich network of hydrogen-bonded water chains that bridge lipid headgroups, some of which form transient lipid clusters. Water-mediated bridges between with lipid phosphate groups are dynamic, with residence lifetimes on the order of picoseconds. These clusters of water/lipid headgroup hydrogen bonds could provide a platform for the binding of proteins or of drug molecules with cationic groups.

Layer-by-layer films containing emodin or emodin encapsulated in liposomes for transdermal applications

Dermal drug release systems are an important area of research because they can be applied to the skin in a non-invasive procedure using a lower concentration of drugs. In this study, we have developed two types of Layer-by-Layer (LbL) films for releasing emodin (EM). In one system, EM was intercalated with poly(ethylenimine) PEI and poly(vinyl sufonate) (PVS) polyelectrolytes, forming (PEI/PVS)2(PEI/EM)7; in another, EM was incorporated in liposomes obtained by mixing dipalmitoyl phosphatidyl glycerol (DPPG) and palmitoyl oleoyl phosphatidyl glycerol (POPG) lipids, forming (PEI/PVS)2(PEI/DPPG-POPG-EM)7. UV–vis and FTIR spectroscopies were used to characterize the LbL films. These showed that the depositions of material by LbL were efficient, with increases in the absorbance of each bilayer evidencing the presence of EM in the film. The (PEI/PVS)2(PEI/EM)7 and (PEI/PVS)2(PEI/DPPG-POPG-EM)7 films released EM in three and five days, respectively. The cyclic voltammetry (CV) assay of the (PEI/PVS)2(PEI/EM)7 results are in agreement with UV–vis measurements, which suggest that EM was protonated in acid environments, while the CV of (PEI/PVS)2(PEI/DPPG-POPG-EM)7 demonstrated distinct protonation behaviour for EM within the inner liposome structure, even in acid solutions. Therefore, this study presents two systems based on LbL films and provides additional details about the release of EM from these films to create a viable alternative for transdermal applications.

Non-linear van't Hoff behavior in pulmonary surfactant model membranes

Pulmonary surfactant exhibits phase coexistence over a wide range of surface pressure and temperature. Less is known about the effect of temperature on pulmonary surfactant models. Given the lack of studies on this issue, we used electron paramagnetic resonance (EPR) and nonlinear least–squares (NLLS) simulations to investigate the thermotropic phase behavior of the matrix that mimics the pulmonary surfactant lipid complex, i.e., the lipid mixture composed of dipalmitoyl phosphatidylcholine (DPPC), palmitoyl–oleoyl phosphatidylcholine (POPC) and palmitoyl–oleoyl phosphatidylglycerol (POPG). Irrespective of pH, the EPR spectra recorded from 5°C to 25°C in the DPPC/POPC/POPG (4:3:1) model membrane contain two spectral components corresponding to lipids in gel-like and fluid-like phases, indicating a coexistence of two domains in that range. The temperature dependence of the distribution of spin labels between the domains yielded nonlinear van't Hoff plots. The thermodynamic parameters evaluated were markedly different for DPPC and for the ternary DPPC/POPC/POPG (4:3:1) membranes and exhibited a dependence on chemical environment. While enthalpy and entropy changes for DPPC were always positive and presented a quadratic behavior with temperature, those of the ternary mixture were linearly dependent on temperature and changed from negative to positive values. Despite that, enthalpy-entropy compensation takes place in the two systems. The thermotropic process associated with the coexistence of the two domains is entropically–driven in DPPC and either entropically– or enthalpically–driven in the pulmonary surfactant membrane depending on the pH, ionic strength and temperature. The significance of these results to the structure and function of the pulmonary surfactant lipid matrix is discussed.

Synthetic lung surfactants containing SP-B and SP-C peptides plus novel phospholipase-resistant lipids or glycerophospholipids.

BackgroundThis study examines the biophysical and preclinical pulmonary activity of synthetic lung surfactants containing novel phospholipase-resistant phosphonolipids or synthetic glycerophospholipids combined with Super Mini-B (S-MB) DATK and/or SP-Css ion-lock 1 peptides that replicate the functional biophysics of surfactant proteins (SP)-B and SP-C. Phospholipase-resistant phosphonolipids used in synthetic surfactants are DEPN-8 and PG-1, molecular analogs of dipalmitoyl phosphatidylcholine (DPPC) and palmitoyl-oleoyl phosphatidylglycerol (POPG), while glycerophospholipids used are active lipid components of native surfactant (DPPC:POPC:POPG 5:3:2 by weight). The objective of the work is to test whether these novel lipid/peptide synthetic surfactants have favorable preclinical activity (biophysical, pulmonary) for therapeutic use in reversing surfactant deficiency or dysfunction in lung disease or injury.MethodsSurface activity of synthetic lipid/peptide surfactants was assessed in vitro at 37 °C by measuring adsorption in a stirred subphase apparatus and dynamic surface tension lowering in pulsating and captive bubble surfactometers. Shear viscosity was measured as a function of shear rate on a Wells-Brookfield micro-viscometer. In vivo pulmonary activity was determined by measuring lung function (arterial oxygenation, dynamic lung compliance) in ventilated rats and rabbits with surfactant deficiency/dysfunction induced by saline lavage to lower arterial PO2 to <100 mmHg, consistent with clinical acute respiratory distress syndrome (ARDS).ResultsSynthetic surfactants containing 5:3:2 DPPC:POPC:POPG or 9:1 DEPN-8:PG-1 combined with 3% (by wt) of S-MB DATK, 3% SP-Css ion-lock 1, or 1.5% each of both peptides all adsorbed rapidly to low equilibrium surface tensions and also reduced surface tension to ≤1 mN/m under dynamic compression at 37 °C. However, dual-peptide surfactants containing 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 combined with 9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG had the greatest in vivo activity in improving arterial oxygenation and dynamic lung compliance in ventilated animals with ARDS. Saline dispersions of these dual-peptide synthetic surfactants were also found to have shear viscosities comparable to or below those of current animal-derived surfactant drugs, supporting their potential ease of deliverability by instillation in future clinical applications.DiscussionOur findings support the potential of dual-peptide synthetic lipid/peptide surfactants containing S-MB DATK + SP-Css ion-lock 1 for treating diseases of surfactant deficiency or dysfunction. Moreover, phospholipase-resistant dual-peptide surfactants containing DEPN-8/PG-1 may have particular applications in treating direct forms of ARDS where endogenous phospholipases are present in the lungs.

Comparative study of the mechanism of action of the antimicrobial peptide gomesin and its linear analogue: The role of the β-hairpin structure

Gomesin (Gm) is an antimicrobial peptide first isolated from the hemolymph of a Brazilian spider. Its powerful antimicrobial activity is, however, accompanied by hemolysis. As an alternative to this issue, a linear analogue (named GmL) lacking the disulfide bonds was designed. Here, CD spectroscopy, a fluorescence-based leakage assay, isothermal titration calorimetry (ITC) and light scattering are used to study the interaction of both Gm and GmL with large unilamellar vesicles (LUVs) composed of POPC (palmitoyl oleoyl phosphatidylcholine) with 25 and 50mol% POPG (palmitoyl oleoyl phosphatidylglycerol). The activities of Gm and GmL in respect to their binding affinity/enthalpy, ability to permeabilize membranes and to induce vesicle aggregation are correlated with peptide secondary structure. Whereas Gm displays a quite stable β-hairpin motif irrespective of the environment, GmL assumes a random conformation in aqueous solution and in the presence of 25mol% POPG but adopts a β-like structure in the presence of 50mol% POPG. Gm exhibited high lytic activity against both surface charge densities. Instead, the activity of GmL was found to be negligible in the presence of 25mol% POPG LUVs, but comparable to that of the native peptide against 50mol% POPG as a consequence of peptide structuring. We conclude that the activity of Gm and its linear analogue is intimately related to the formation of a β-turn motif, in which the hydrophobic residues form a hydrophobic face able to insert into the membrane and disrupt it.

Free energy of PAMAM dendrimer adsorption onto model biological membranes.

We investigated the thermodynamic, structural, and dynamics changes in dendrimer-membrane systems during dendrimer adsorption to biological membrane systems by combining atomistic molecular dynamics simulations with umbrella sampling techniques to understand the atomistic interactions between the dendrimer and biological membranes. An ethylenediamine core polyamidoamine dendrimer (generation 3) with amine terminal groups and both zwitterionic dipalmitoyl-phosphatidyl-choline (DPPC) and anionic palmitoyl-oleoyl-phosphatidyl glycerol (POPG) lipid bilayer membranes were used as the model dendrimer and biological membranes, respectively, in this study. The free energy of the dendrimer adsorption onto two model membranes with different charge states was quantitatively determined. For the zwitterionic DPPC membrane, the dendrimer has a minimum free energy of approximately 50 kcal/mol, which is 15 kcal/mol higher than that observed in previous studies. The dominant contribution to the adsorption potential energy is the van der Waals attraction between the dendrimer and the DPPC membrane. However, the anionic POPG membrane pulls the positively charged dendrimer with an attractive mean force of about 200 pN, finally positioning the dendrimer in the membrane headgroup region. As a result of these strong attractive dendrimer and membrane interactions, the dendrimer structurally undergoes the transition from spherical to a pancake conformation, which slows its lateral mobility, especially in the presence of the POPG membrane. The bilayer lipid membranes are also perturbed by the dendrimer adsorption.

Study of Orientation and Penetration of LAH4 into Lipid Bilayer Membranes: pH and Composition Dependence

LAH4 is an antimicrobial peptide that is believed to possess both antibiotic and DNA delivery capabilities. It is one of a number of membrane-active peptides that show increased affinity toward anionic lipids. Herein, we have performed molecular dynamics simulations to compare LAH4 effects on anionic palmitoyl-oleoyl-phosphatidylglycerol bilayer, which approximate a prokaryotic membrane environment and zwitterionic palmitoyl-oleoyl-phosphatidylcholine bilayer, which approximate a eukaryotic membrane environment. One particular interest in this work is to study how different kinds of lipid bilayers respond to the attraction of LAH4. Remarkably, our data have shown that the depth of peptide penetration strongly depends on membrane composition and pH. At acidic pH, LAH4 has exhibited a high tendency to interact strongly with and be adsorbed on anionic membrane. We have also shown that electrostatic interactions between His11 and the phosphor atoms of bilayers should have a significant impact on the penetration of LAH4. These results provide insights into the interactions of LAH4 and lipid bilayers at the atomic level, which is useful to understand cell selectivity and mechanism of the peptide action.

Structure–Activity Relationship of the Antimicrobial Peptide Gomesin: The Role of Peptide Hydrophobicity in Its Interaction with Model Membranes

Antimicrobial peptides are part of the innate immune system of animals and plants. Their lytic activity against microorganisms generally depends on their ability to disrupt and permeabilize membranes. Here we study the structure-activity relationship of the antimicrobial peptide gomesin (Gm), from the spider Acanthoscurria gomesiana, with large unilamellar vesicles (LUVs) composed of 3:7 palmitoyloleoyl phosphatidylglycerol: palmitoyloleoyl phosphatidylcholine. Several synthetic analogues of Gm were designed to alter the hydrophobicity/charge of the molecule, whereby selected amino acid residues were replaced by alanine. Isothermal titration calorimetry (ITC) was used to assess the thermodynamic parameters of peptide binding to LUVs and light scattering measurements were made to evaluated peptide-induced vesicle aggregation. The ability of the peptides to permeabilize vesicles was quantified through the leakage of an entrapped fluorescent probe. The activity of peptides could be quantified in terms of the leakage extent induced and their affinity to the membrane, which was largely dictated by the exothermic enthalpy change. The results show that analogues more hydrophobic than Gm display higher activity, whereas peptides more hydrophilic than Gm have their activity almost abolished. Vesicle aggregation, on the other hand, largely increases with peptide charge. We conclude that interaction of Gm with membranes depends on an interplay between surface electrostatic interactions, which drive anchoring to the membrane surface and vesicle aggregation, and insertion of the hydrophobic portion into the membrane core, responsible for causing membrane rupture/permeabilization.

The Interaction Between the Antimicrobial Peptide K-Hya1 and Model Membranes: Distinct Action in Neutral or Negatively Charged Bilayers

Antimicrobial peptides (AMPs) are important molecules for the innate immune defense system of plants and animals. The antimicrobial peptide Hylina1 (Hya1) was isolated from arboreal South African frog Hypsiboas albopunctatus. In this work, we investigate the interaction of an analogue peptide, K-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with model membranes composed of DPPC (dipalmitoyl phosphatidylcholine), DPPG dipalmitoyl phosphatidylglycerol) and DPPC:DPPG 1:1 or of POPC (palmitoyl oleoyl phosphatidylcholine) and POPC:POPG (palmitoyl oleoyl phosphatidylglycerol) with different techniques. Differential Scanning Calorimetry (DSC) profiles show distinct environments in the bilayer with anionic lipids, suggesting a disturbed region due to peptide adsorption, and an unaltered region. On the other hand, in neutral membranes an average perturbation is observed, which suggests a superficial interaction. Steady-state fluorescence spectroscopy of the intrinsic Trp residue shows a deeper insertion of this residue into charged bilayers as compared with neutral membranes. Dye-leakage experiments show that membrane charge also modulates the kinetics of membrane permeabilization, which is much faster for charged bilayers. Optical microscopy of giant unilamellar vesicles (GUVs) in the fluid phase revealed a different mechanism of action of the peptide in the presence or absence of negatively charged lipids. K-Hya1 induces small perturbations in POPC vesicles, causing membrane permeabilization without morphological changes. On the other hand, the peptide induces permeabilization accompanied by a large increase in surface area of POPC:POPG vesicles, suggesting the opening of several pores in the bilayer. Taken together, the results clearly show a peptide-bilayer interaction modulated by the presence of negatively charged lipids. Data can be interpreted as a different orientation of the peptide in the bilayer: parallel to the surface in neutral membranes and stable and crossed in anionic membranes.