Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Insertion of Dengue E into lipid bilayers studied by neutron reflectivity and molecular dynamics simulations

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that the tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.     

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L_α or the gel phase L_β lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (≥ 44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R_P = 0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.     

Structure and Dynamics of the Myristoyl Lipid Modification of Src Peptides Determined by H Solid-State NMR Spectroscopy

Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used ²H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2-19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears “stiffer” than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: Lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 °C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 °C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.     

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

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. 7.8 nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

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.     

Phospholipid Composition of Membranes Directs Prions Down Alternative Aggregation Pathways

Prion diseases are neurodegenerative disorders of the central nervous system that are associated with the misfolding of the prion protein (PrP). PrP is glycosylphosphatidylinositol-anchored, and therefore the hydrophobic membrane environment may influence the process of prion conversion. This study investigates how the morphology and mechanism of growth of prion aggregates on membranes are influenced by lipid composition. Atomic force microscopy is used to image the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS). Circular dichroism shows that PrP interactions with POPS membranes result in an increase in β-sheet structure, whereas interactions with POPC do not influence PrP structure. Prion aggregation is observed on both zwitterionic and anionic membranes, and the morphology of the aggregates formed is dependent on the anionic phospholipid content of the membrane. The aggregates that form on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger aggregates with a distinctive sponge-like morphology that are disruptive to membranes. These data provide detailed information on the aggregation mechanism of PrP on membranes, which can be described by classic models of growth.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Peptide-lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. ³¹P and ²H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

A 2H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

A H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

Oxidized phospholipids as potential molecular targets for antimicrobial peptides

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.     

General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides

Model compounds of modified hydrophobicity (H), hydrophobic moment (μ) and angle subtended by charged residues (Φ) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. H and μ influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters’ influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Φ, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of μ and Φ on both lipid bilayers and cell membranes.     

A coarse-grained approach to studying the interactions of the antimicrobial peptides aurein 1.2 and maculatin 1.1 with POPG/POPE lipid mixtures

In the present work we investigated the differential interactions of the antimicrobial peptides (AMPs) aurein 1.2 and maculatin 1.1 with a bilayer composed of a mixture of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). We carried out molecular dynamics (MD) simulations using a coarse-grained approach within the MARTINI force field. The POPE/POPG mixture was used as a simple model of a bacterial (prokaryotic cell) membrane. The results were compared with our previous findings for structures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a representative lipid of mammalian cells. We started the simulations of the peptide–lipid system from two different initial conditions: peptides in water and peptides inside the hydrophobic core of the membrane, employing a pre-assembled lipid bilayer in both cases. Our results show similarities and differences regarding the molecular behavior of the peptides in POPE/POPG in comparison to their behavior in a POPC membrane. For instance, aurein 1.2 molecules can adopt similar pore-like structures on both POPG/POPE and POPC membranes, but the peptides are found deeper in the hydrophobic core in the former. Maculatin 1.1 molecules, in turn, achieve very similar structures in both kinds of bilayers: they have a strong tendency to form clusters and induce curvature. Therefore, the results of this study provide insight into the mechanisms of action of these two peptides in membrane leakage, which allows organisms to protect themselves against potentially harmful bacteria.

Open image in new window

Graphical Abstract Aurein pore structure (green) in a lipid bilayer composed by POPE (blue) and POPG (red) mixture. It is possible to see water beads (light blue) inside the pore.

     

Atomically detailed lipid bilayer models for the interpretation of small angle neutron and X-ray scattering data

We present a new atom density profile (ADP) model and a statistical approach for extracting structural characteristics of lipid bilayers from X-ray and neutron scattering data. Models for five lipids with varying head and tail chemical composition in the fluid phase, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), are optimized using a simplex based method to simultaneously reproduce both neutron and X-ray scattering data. Structural properties are determined using statistical analysis of multiple optimal model structures. The method and models presented make minimal assumptions regarding the atomic configuration, while taking into account the underlying physical properties of the system. The more general model and statistical approach yield data with well defined uncertainties, indicating the precision in determining density profiles, atomic locations, and bilayer structural characteristics. Resulting bilayer structures include regions exhibiting large conformational variation. Due to the increased detail in the model, the results demonstrate the possibility of a distinct hydration layer within the interfacial (backbone) region.