Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Solid-state NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from magainin 2 and melittin

The mechanism of membrane interaction of two amphipathic antimicrobial peptides, MSI-78 and MSI-594, derived from magainin-2 and melittin, is presented. Both the peptides show excellent antimicrobial activity. The 8-anilinonaphthalene-1-sulfonic acid uptake experiment using Escherichia coli cells suggests that the outer membrane permeabilization is mainly due to electrostatic interactions. The interaction of MSI-78 and MSI-594 with lipid membranes was studied using 31P and 2H solid-state NMR, circular dichroism, and differential scanning calorimetry techniques. The binding of MSI-78 and MSI-594 to the lipid membrane is associated with a random coil to alpha-helix structural transition. MSI-78 and MSI-594 also induce the release of entrapped dye from POPC/POPG (3:1) vesicles. Measurement of the phase-transition temperature of peptide-DiPoPE dispersions shows that both MSI-78 and MSI-594 repress the lamellar-to-inverted hexagonal phase transition by inducing positive curvature strain. 15N NMR data suggest that both the peptides are oriented nearly perpendicular to the bilayer normal, which infers that the peptides most likely do not function via a barrel-stave mechanism of membrane-disruption. Data obtained from 31P NMR measurements using peptide-incorporated POPC and POPG oriented lamellar bilayers show a disorder in the orientation of lipids up to a peptide/lipid ratio of 1:20, and the formation of nonbilayer structures at peptide/lipid ratio>1:8. 2H-NMR experiments with selectively deuterated lipids reveal peptide-induced disorder in the methylene units of the lipid acyl chains. These results are discussed in light of lipid-peptide interactions leading to the disruption of membrane via either a carpet or a toroidal-type mechanism.     

Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: a case study of MSI-367

In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA)(3)-NH(2)] was designed and synthesized with the objective of generating cell-selective nonlytic peptides, which have a significant bearing on cell targeting. The peptide exhibited potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined via membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, β-turn structure in aqueous buffer and extended and turn conformations upon binding to lipid vesicles. Differential scanning calorimetry experiments with 1,2-dipalmitoleoyl-sn-glycero-3-phosphatidylethanolamine bilayers indicated the induction of positive curvature strain and repression of the fluid lamellar to inverted hexagonal phase transition by MSI-367. Results of isothermal titration calorimetry (ITC) experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. (2)H nuclear magnetic resonance (NMR) of deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) multilamellar vesicles confirmed the limited effect of the membrane-embedded peptide at the lipid-water interface. (31)P NMR data indicated changes in the lipid headgroup orientation of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed via sum frequency generation vibrational spectroscopy. Circular dichroism, ITC, and (31)P NMR data for Escherichia coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.     

Membrane composition determines pardaxin's mechanism of lipid bilayer disruption

Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the ³¹P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. ²H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. ³¹P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, ³¹P and ²H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via ²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.     

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.     

Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37

LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.     

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.     

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.     

Kinetics and thermodynamics of association of a fluorescent lysophospholipid derivative with lipid bilayers in liquid-ordered and liquid-disordered phases

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) of the fluorescent lysophospholipid derivative NBD-lyso-1-myristoylphosphatidylethanolamine in l(d) and l(o) phase lipid bilayer membranes. The lipid bilayers, studied as LUV, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine, in the l(d) phase; and from two Chol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and Chol (molar ratio of 1:1) and SpM and Chol (molar ratio of 6:4), both in the l(o) phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were measured between 15 degrees C and 35 degrees C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes were derived from these data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and an estimation was made of hypothetical partition coefficients and the respective energetic parameters for partitioning between the different lipid phases if these were to coexist in the same membrane. We show that, contrary to general belief, the association of NBD-lysoMPE with lipid bilayers is not a diffusion-controlled process, the rate-limiting step in insertion being the formation of a free area in the membrane surface of an adequate size for insertion to occur.     

The oxysterol 3β-hydroxy-5-oxo-5,6-secocholestan-6-al changes the phase behavior and structure of phosphatidylethanolamine–phosphatidylcholine mixtures

An oxidized form of cholesterol, atheronal, is a form found in vivo that has been associated with human pathologies. We have studied mixtures of this oxidized sterol with the phospholipids phosphatidylethanolamine and phosphatidylcholine. We used phospholipids either with palmitoyl and oleoyl acyl chains on the C1 and C2 carbon atoms of glycerol or with both acyl chains being palmitoleoyl. We also compared the effects of atheronal on the curvature properties of these lipids with the action of cholesterol. We studied the bilayer to hexagonal phase transition temperature of mixtures of these lipids using differential scanning calorimetry as well as the dimensions of the hexagonal phase cylinders using X-ray diffraction. Disordering of the lamellar phase was also qualitatively assessed by the loss of sharp diffraction peaks. Our results demonstrate that the modulation of membrane curvature in these systems depends not only on the nature of the sterol, but also on the acyl chain composition of the phospholipids used. In addition, some of the effects of atheronal could be ascribed to reaction of the aldehyde and ketone groups of this oxidized sterol with the amino group of phosphatidylethanolamine.     

Lipid Polymorphism Induced by Surfactant Peptide SP-B1-25

Pulmonary surfactant protein B (SP-B) is an essential protein for lowering surface tension in the alveoli. SP-B_1-25, a peptide comprised of the N-terminal 25 amino-acid residues of SP-B, is known to retain much of the biological activity of SP-B. Circular dichroism has shown that when SP-B_1-25 interacts with negatively charged lipid vesicles, it contains significant helical structure for the lipid compositions and peptide/lipid ratios studied here. The effect of SP-B_1-25 on lipid organization and polymorphisms was investigated via DSC, dynamic light scattering, transmission electron microscopy, and solid-state NMR spectroscopy. At 1-3 mol% peptide and physiologic temperature, SP-B_1-25 partitions at the interface of negatively charged PC/PG lipid bilayers. In lipid mixtures containing 1-5 mol% peptide, the structure of SP-B_1-25 remains constant, but ²H and ³¹P NMR spectra show the presence of an isotropic lipid phase in exchange with the lamellar phase below the T_m of the lipids. This behavior is observed for both DPPC/POPG and POPC/POPG lipid mixtures as well as for both the PC and PG components of the mixtures. For 1-3 mol% SP-B_1-25, a return to a single lamellar phase above the lipid mixture T_m is observed, but for 5 mol% SP-B_1-25 a significant isotropic component is observed at physiologic temperatures for DPPC and exchange broadening is observed in ²H and ³¹P NMR spectra of the other lipid components in the two mixtures. DLS and TEM rule out the formation of micellar structures and suggest that SP-B_1-25 promotes the formation of a fluid isotropic phase. The ability of SP-B_1-25 to fuse lipid lamellae via this mechanism, particularly those enriched in DPPC, suggests a specific role for the highly conserved N-terminus of SP-B in the packing of lipid lamellae into surfactant lamellar bodies or in stabilizing multilayer structures at the air-liquid interface. Importantly, this behavior has not been seen for the other SP-B fragments of SP-B_8-25 and SP-B_59-80, indicating a critical role for the proline rich first seven amino acids in this protein.     

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and ³¹P and ²H solid-state NMR spectroscopy. SP-B_59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B_59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B_59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B_59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL₄, a peptide mimetic of SP-B which was originally designed using SP-B_59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.     

Comparative calorimetric and spectroscopic studies of the effects of lanosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative DSC and Fourier transform infrared spectroscopic studies of the effects of cholesterol and lanosterol on the thermotropic phase behavior and organization of DPPC bilayers. Lanosterol is the biosynthetic precursor of cholesterol and differs in having three rather than two axial methyl groups projecting from the β-face of the planar steroid ring system and one axial methyl group projecting from the α-face, whereas cholesterol has none. Our DSC studies indicate that the incorporation of lanosterol is more effective than cholesterol is in reducing the enthalpy of the pretransition. Lanosterol is also initially more effective than cholesterol in reducing the enthalpies of both the sharp and broad components of the main phase transition. However, at sterol concentrations of 50 mol %, lanosterol does not abolish the cooperative hydrocarbon chain-melting phase transition as does cholesterol. Moreover, at higher lanosterol concentrations (~30–50 mol %), both sharp and broad low-temperature endotherms appear in the DSC heating scans, suggestive of the formation of lanosterol crystallites, and of the lateral phase separation of lanosterol-enriched phospholipid domains, respectively, at low temperatures, whereas such behavior is not observed with cholesterol at comparable concentrations. Our Fourier transform infrared spectroscopic studies demonstrate that lanosterol incorporation produces a less tightly packed bilayer than does cholesterol, which is characterized by increased hydration in the glycerol backbone region of the DPPC bilayer. These and other results indicate that lanosterol is less miscible in DPPC bilayers than is cholesterol, but perturbs their organization to a greater extent, probably due primarily to the rougher faces and larger cross-sectional area of the lanosterol molecule and perhaps secondarily to its decreased ability to form hydrogen bonds with adjacent DPPC molecules. Nevertheless, lanosterol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers, although this phase is not as tightly packed as comparable cholesterol/DPPC mixtures.     

Binding and insertion of alpha-helical anti-microbial peptides in POPC bilayers studied by molecular dynamics simulations

We have performed molecular dynamics simulations of the interactions of two alpha-helical anti-microbial peptides, magainin2 and its synthetic analog of MSI-78, with palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers. We used various initial positions and orientations of the peptide with respect to the lipid bilayer, including a surface-bound state parallel to the interface, a trans-membrane state, and a partially inserted state. Our 20 ns long simulations show that both magainin2 and MSI-78 are most stable in the lipid environment, with the peptide destabilized to different extents in both aqueous and lipid/water interfacial environments. We found that there are strong specific interactions between the lysine residues of the peptides and the lipid head-group regions. MSI-78, owing to its large number of lysines, shows better binding characteristics and overall stability when compared to magainin2. We also find that both peptides destabilize the bilayer environment, as observed by the increase in lipid tail disorder and the induction of local curvature on the lipid head-groups by the peptides. From all the simulations, we conclude that the hydrogen bonding interactions between the lysines of the peptides and the oxygens of the polar lipid head-groups are the strongest and determine the overall peptide binding characteristics to the lipids.     

Study of the interaction of an α-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K₂-L₂₄-K₂-amide (L₂₄), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L₂₄ decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L₂₄ in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L₂₄:PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L₂₄. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and ²H NMR spectroscopy study of the interaction of the L₂₄ and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between ²H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L₂₄ suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

A charge-modified general amber force field for phospholipids: improved structural properties in the tensionless ensemble

Accurately predicting the structural properties of phospholipid with a fully atomistic molecular model is critical for the study of pure phospholipid bilayers, mixed bilayer systems and bilayers containing proteins. The general amber force field (GAFF) has traditionally required the presence of a surface tension parameter to correctly model phospholipid bilayer properties such as area per lipid and order parameters. In this work, the GAFF partial charges for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphochiline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were re-parameterised utilising high-level ab initio calculations and the restrained electrostatic potential method. Simulations of pure POPA, POPC and POPG bilayers using the charge-modified GAFF and no applied surface tension are compared with available experimental data, the original GAFF model and the recent Lipid14 variant. The results indicate a significant improvement in the accuracy of the lipid model for reproducing experimental observables without the need for a surface tension parameter. The successful application of modifying the lipid charge distributions represents an alternative to the use of a surface tension parameter within GAFF, and highlights the importance of the partial charge calculations when modelling lipid bilayers.     

Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers

The interaction of an antimicrobial peptide, MSI-78, with phospholipid bilayers has been investigated using atomic force microscopy, circular dichroism, and nuclear magnetic resonance (NMR). Binding of amphipathic peptide helices with their helical axis parallel to the membrane surface leads to membrane thinning. Atomic force microscopy of supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers in the presence of MSI-78 provides images of the membrane thinning process at a high spatial resolution. This data reveals that the membrane thickness is not reduced uniformly over the entire bilayer area. Instead, peptide binding leads to the formation of distinct domains where the bilayer thickness is reduced by 1.1 +/- 0.2 nm. The data is interpreted using a previously published geometric model for the structure of the peptide-lipid domains. In this model, the peptides reside at the hydrophilic-hydrophobic boundary in the lipid headgroup region, which leads to an increased distance between lipid headgroups. This picture is consistent with concentration-dependent 31P and 2H NMR spectra of MSI-78 in mechanically aligned DMPC bilayers. Furthermore, 2H NMR experiments on DMPC-d54 multilamellar vesicles indicate that the acyl chains of DMPC are highly disordered in the presence of the peptide as is to be expected for the proposed structure of the peptide-lipid assembly.