Anionic Bilayers Research Articles

Biophysical Basis for Specificity of Action of Human Isoforms of Secretory Phospholipase A 2 During Cell Death

The hydrolytic activity of secretory phospholipase A2 (sPLA2) toward mammalian cells depends on the health of the cell. During the process of programmed cell death, changes occur in the cell membrane that render it susceptible to hydrolytic attack. Various isozymes of sPLA2 respond differently to cell death depending on the timing and origin of the process. This study used flow cytometry to classify subpopulations of S49 lymphoma cells during several modes of programmed death depending on their vulnerability to sPLA2 and identify the basis for isozyme specificity. Most death stimuli (thapsigargin, dexamethasone, actinomycin D, paclitaxel, and methotrexate) caused a reduction in membrane lipid-neighbor interactions detected as increased binding of the fluorescent dye merocyanine 540 and modest permeability to a vital stain, propidium iodide. In each of these cases, all sPLA2 isozymes tested (snake venom and human groups IIa, V, and X) displayed enhanced ability to hydrolyze the cell membrane. In contrast, cells exposed to a calcium ionophore showed the increase in merocyanine 540 binding without accompanying permeability to propidium iodide. Under these conditions, only the snake venom and human group X enzymes hydrolyzed cells that were dying. Lastly, the human group IIa enzyme, although most active of the isoforms tested toward anionic artificial bilayers, hydrolyzed dying cells at a rate that was only 1/100 that of the other isozymes. These results suggested that each of these human isozymes fills different physiological roles in responding to cell death and provides probable molecular explanations for the distinctions.

Membrane surface charge dictates the structure and function of the epithelial Na+/H+exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.

Fusion-Relevant Changes in Lipid Shape of Hydrated Cholesterol Hemisuccinate Induced by pH and Counterion Species

Cholesterol hemisuccinate (CHEMS) is a protonable lipid that is frequently used for the construction of pH-responsive delivery systems. Such systems have a stable, lamellar phase at pH 7, but can form a fusogenic, hexagonal phase at pH 5. This behavior can be explained by binding or release of counterions from the solvent and the related variations of the effective size of the polar lipid part. Here, we use MD simulations to study the ion recruitment to neutral or anionic bilayers of CHEMS in water. For deprotonated (anionic) CHEMS, we observed an almost complete decoration of the bilayer with sodium, potassium, or argininium cations, which challenges the previous hypothesis that the stability of bilayers from anionic CHEMS results from the electrostatic repulsion between the charged head groups. Protonated (neutral) bilayers of CHEMS did not bind sodium or potassium, but did adsorb argininium cations. Whereas the headgroup of protonated CHEMS is bent and strongly tilted away from the bilayer normal, the headgroup of the deprotonated CHEMS is found to become outstretched and significantly less tilted arising from the adsorption of the counterions. The tilt reduction is most pronounced upon adsorption of arginine which also leads to an increase in the otherwise constant area per lipid. In general, the cation binding to the deprotonated CHEMS acts to increase the effective headgroup volume. This change in the lipid shape may be one possible fact explaining the hexagonal-lamellar phase transitions for CHEMS known from experiments.

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: A biophysical approach to transfection efficiency

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C14H29(CH3)2N+(CH2)2N+(CH3)2C14H29]2Br− and herein designated 14–2–14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14–2–14, 14–2–14:Chol (1:1 molar ratio) and 14–2–14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/− charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14–2–14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/− charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.

Orientation, dynamics, and lipid interaction of an antimicrobial arylamide investigated by 19F and 31P solid-state NMR spectroscopy.

A number of arylamides have been synthesized and found to exhibit potent antimicrobial activities against a broad spectrum of Gram-positive and Gram-negative bacteria while exhibiting low toxicity toward eukaryotic cells. These facially amphiphilic foldamers have a relatively rigid intramolecular hydrogen-bonded arylamide as a framework, which places trifluormethyl versus positively charged amino and guanidino groups along opposite faces of the elongated molecule, facilitating interactions with lipid membranes. To better understand the mechanism of action of these antimicrobial foldamers, we have investigated the lipid interaction, depth of insertion, orientation, and dynamics of an arylamide, PMX30016, using (31)P and (19)F solid-state NMR spectroscopy. Static (31)P NMR line shapes of lipid membranes with a range of compositions indicate that PMX30016 does not disrupt the lamellar order of the lipid bilayer but perturbs the lipid headgroup conformation. This headgroup perturbation, manifested as systematic (31)P chemical shift anisotropy increases, is consistent with the well documented "electrometer" effect of lipid membranes in response to the addition of positive charges to membrane surfaces. Paramagnetic relaxation enhancement experiments indicate that the arylamide inserts into the membrane-water interface, just below the headgroup region. Measurement of (19)F-(19)F dipolar couplings within each CF(3) moiety revealed that PMX30016 is oriented with the molecular plane 20 degrees and 30 degrees from the membrane normal of neutral and anionic bilayers, respectively, and the long molecular axis lies parallel to the membrane plane. Thus, this arylamide inserts into the bilayer in a knife-like fashion, consistent with previous vibrational spectroscopy results. Moreover, (19)F NMR line shapes indicate that this molecular knife undergoes fast uniaxial rotation around the bilayer normal. These results suggest that antimicrobial arylamides destabilize anionic lipid membranes primarily by altering the membrane electric potential profile, and the spinning molecular knife may additionally create transient defects in the lipid membrane. Compared to typical antimicrobial peptides, this arylamide has more subtle effects on and is less disruptive of the structure of lipid bilayers.

Atomic force microscopy of supported lipid membranes and their complexes with polycations

The method of atomic force microscopy has been used to investigate the morphology of mica-supported bilayer lipid membranes and stability of their complexes with a cationic polymer, poly-(N-ethyl-4-vinylpyridinium bromide). Lipid bilayers with a minimum of defects were obtained by the fusion of monolamellar neutral or mixed anionic bilayer vesicles (liposomes) on the mica surface, followed by excessive solvent removal by means of rapid rotation of a plate in horizontal plane (spin-coating). It has been shown that the cationic polymer does not interact with the bilayers, where the outer leaflet (i.e., the monolayer exposed to the surrounding aqueous solution) is made of an electroneutral phosphatidylcholine (PC). At the same time, the polymer irreversibly binds to the bilayer containing an anionic lipid.

Structure of the Amyloid-β (1−42) Monomer Absorbed To Model Phospholipid Bilayers: A Molecular Dynamics Study

The amyloid-beta (Abeta) peptide, the 39 to 43 amino acid peptide that plays a substantial role in Alzheimer's disease, has been shown to interact strongly with lipids both in vitro and in vivo. Abeta-lipid interactions have been proposed as a considerable factor in accelerating Abeta aggregation through the templating role of membranes in aggregation disorders. Previous work has shown that anionic lipids are able to significantly increase Abeta aggregation rate and induce a structural conversion in Abeta from a random coil to a beta-structure that is similar to the monomer structure observed in mature fibrils. However, it is unclear if this structural change occurs with the Abeta monomer because of direct interactions with the lipids or if the structural change results from protein-protein interactions during oligomerization. We use extensive replica exchange molecular dynamics simulations of an Abeta monomer bound to a homogeneous model zwitterionic or anionic lipid bilayer. From these simulations, we do not observe any significant beta-structure formation except for a small, unstable beta-hairpin formed on the anionic dioleylphosphatidylserine bilayer. Further, we see that the Asp23-Lys28 salt bridge that plays a role in beta-hairpin formation is not substantially formed on the bilayer surface and that Lys28 preferentially interacts with lipids when bound to the bilayer. These results suggest that the structural conversion seen in experiments are not due to the ordering of monomeric Abeta on the bilayer surface but are a result of protein-protein interactions enhanced by Abeta binding to the cell membrane.

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers

Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction.

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

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

Deamidation Weakens Membrane Binding Properties of Antimicrobial Peptide Anoplin

Anoplin, GLLKRIKTLL-NH2, isolated from the venom sac of solitary spider wasp, Anoplius samariensis, is the smallest linear α-helical antimicrobial peptide found naturally up to date. Previously Cabrera et al. (J. Pept. Sci. 2008) reported that deamidation dramatically decreased antimicrobial activity of the peptide and showed that amidated Anoplin forms pores in toroidal manner in anionic bilayer. In the present work, interactions of two forms of Anoplin (Anoplin-NH2 and Anoplin-COOH) with model cell membrane (zwitterionic DPPC, anionic DPPG or E. coli extract) were further investigated in order to gain a better understanding of the effect of amidations on the kinetics and thermodynamics of the peptide- membrane interactions. Langmuir Blodgett, Atomic Force Microscopy, UV resonance Raman spectroscopy and Calcein leakage assay were used. Results of the study indicate that amidated form of Anoplin has higher membrane binding activity.

Adaptation of a Membrane-active Peptide to Heterogeneous Environment. I. Structural Plasticity of the Peptide

Some membrane-active peptides undergo drastic changes of conformation and/or orientation on water-lipid interfaces. Among the most notable examples is penetratin (pAntp), a short cell-penetrating peptide. To delineate the driving forces behind pAntp-membrane interactions, we used, in this series of two papers, a combined modeling approach that includes: (1) molecular dynamics simulations of pAntp in zwitterionic and anionic lipid bilayers, (2) free energy perturbation calculations of model residue-residue contacts, and (3) detailed analysis of spatial hydrophobic/hydrophilic properties of the peptide/membrane systems. In this first article, we consider the role of conformational plasticity of the peptide in different membrane surroundings, as well as the ability of pAntp to form stable specific residue-residue interactions and make contacts with particular lipids. It was shown that pAntp displays a complicated conformational behavior. Basic and aromatic residues of the peptide form energetically favorable pairs in water and apolar environments, which facilitate membrane insertion of the peptide and stabilization of the membrane-bound state. These residues are also capable of "trapping" lipid heads, thereby affecting their dynamics and microscopic organization of the water-lipid interface. The latter effect is much more pronounced in anionic bilayers and might be related to the initial stage of peptide-induced destabilization of lipid bilayers.

Uranyl–organic bilayer assemblies with flexible aromatic di-, tri- and tetracarboxylic acids

The reaction of uranyl nitrate hexahydrate with three flexible aromatic polycarboxylic acids under hydrothermal conditions has been found to give 2-D assemblies in all three cases. The complex formed with phenylsuccinic acid (H2L1), [UO2(L1)(H2O)] (1), comprises a planar grid-like assembly with all the phenyl substituents directed outwards on the same side. These layers are organized in a face-to-face fashion by inter-layer π-stacking interactions. 1,3,5-Benzenetriacetic acid (H3L2) gives the compound [UO2(H2O)5][UO2(L2)]2·5H2O (2), in which a novel bilayer arrangement results from both the uranyl ions and the ligand behaving as trigonal nodes. Interdigitation of the anionic bilayers leaves spaces occupied by the [UO2(H2O)5]2+ counter ions. Complex 2 is a novel example of a molecular bilayer involving other building blocks than the usual T-shaped nodes and linear spacers. Finally, [(UO2)3(HL3)2(H2O)]·3H2O (3) was obtained with benzophenone-3,3′,4,4′-tetracarboxylic acid (H4L3) generated in situ from the corresponding dianhydride. The high overall denticity of the ligand (bound to as much as six cations) and the twisting of the two aromatic rings around the central bonds result in the formation of a pillared bilayer of the “double floor” type, with channels containing water molecules.

Solution Structure and Model Membrane Interactions of Temporins-SH, Antimicrobial Peptides from Amphibian Skin. A NMR Spectroscopy and Differential Scanning Calorimetry Study

Temporin-SHa and temporin-SHc are 13 residue long antimicrobial peptides from frog skin that have similar sequences but differ markedly in their membrane-damaging properties. Temporin-SHa contains a single basic lysine residue and has a unique antimicrobial spectrum of action among temporins, being very potent against Gram-positive and Gram-negative bacteria, yeasts, fungi, and protozoa. Temporin-SHc, which contains a single basic histidine residue, is inactive against Gram-negative bacteria, has a reduced efficacy against Gram-positive bacteria, but is still active against yeasts and fungi. Temporin-SHb, with no basic residue, has no antimicrobial activity. The three-dimensional structures of the peptides bound to SDS micelles were analyzed by CD and NMR spectroscopy combined with restrained molecular dynamics calculations. The peptides adopt well-defined amphipathic alpha-helical structures extending from residue 3 to residue 12, when bound to SDS micelles. The structures are stabilized by extensive interactions between aliphatic and aromatic side chains on the nonpolar face. Relaxation enhancements caused by paramagnetic probes showed that the peptides adopt nearly parallel orientations to the micelle surface and do not deeply penetrate into the micelle. The interaction of the peptides with model membranes was investigated by differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles and membrane-permeabilization assays on calcein-loaded large unilamellar vesicles. Calorimetric data indicated that both temporin-SHa and -SHc reside at the hydrocarbon core-water interface of the anionic lipid bilayer but interact with anionic bilayers in a very different manner. This suggests that the charge-induced activity of temporins-SH for bacterial cells is due to changes in the membrane-disturbing mechanism of the bound peptides.

Functional and Structural Characterization of 2B Viroporin Membranolytic Domains

Nonstructural 2B viroporin is an intracellularly produced pore-forming protein required for effective enteroviral and rhinoviral replication. The sequence of 2B displays two putative interconnected transmembrane domains, which are predicted to insert into the negatively charged membranes of target organelles forming an integral hairpin. The use of an overlapping peptide library that spanned the complete 2B sequence has recently allowed the mapping of the cell plasma membrane porating activity to the partially amphipathic, amino-terminal transmembrane domain (TM1, residues 35-55). We describe here that although the TM1 peptide was effective in permeabilizing uncharged membranes, it induced marginal lysis of anionic bilayers. In fact, only the peptide representing the highly conserved carboxy-terminal transmembrane domain (TM2, residues 59-82) reproduced the capacity of the full 2B protein to efficiently permeabilize bilayers made of anionic phospholipids. Insertion into lipid monolayers and circular dichroism determinations were, however, consistent with penetration of the TM1 helix into both anionic and zwitterionic membranes, while TM2 interacting with membranes assumed a mixture of conformations. Moreover, addition of TM1 strongly stimulated TM2-induced permeabilization of the anionic membranes. In combination, TM1 and TM2 formed a complex that had structural properties, including a high proportion of extended nonhelical secondary structure, that were distinct from those of the individual peptides. Finally, a comparison of antimicrobial and hemolytic activities further underscored the TM1 domain's cytolytic character. Overall, our data support the idea that the cytolytic activity of TM1 in the negatively charged cell endomembranes targeted by 2B viroporin requires the cooperation of both transmembrane domains.

Detection of Supported Lipid Bilayers Using Their Electric Charge

We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) + cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho- l-serine] (POPS) + cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[ N-( N′, N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel‐like activity, and the role of the amidated C‐terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide's primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside.

Small angle X-ray scattering study on effect of replacement of hydrogen oxide (H2O) by deuterium oxide (D2O) on anionic phospholipid bilayers

In order to clarify the effect of replacement of hydrogen oxide (H2O) by deuterium oxide (D2O) on the structure of phospholipid bilayers, we carried out small angle X-ray scattering (SAXS) measurements of dimyristoylphosphatidylglycerol (DMPG) bilayers in H2O and D2O. DMPG is an anionic phospholipid and spontaneously forms unilamellar vesicles in water. The obtained SAXS intensity curves were analyzed by using a structural model. The analysis showed that the replacement of H2O by D2O induces structural change at interfacial regions of the DMPG bilayers.

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid–crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H–D exchange when incorporated into D 2O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1 > aurein 1.2 > citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1 > aurein 1.2 ≅ citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.

Temperature effect on the transport dynamics of a small molecule through a liposome bilayer

An ion having hydrophobic parts can directly transport through the liposome bilayer without an ion channel and its transport mechanism can be explained by the free-volume theory. This was confirmed by investigating the temperature effect on the transport dynamics of organic cations through anionic liposome bilayers made of unsaturated and saturated lipids by using optical second-harmonic generation (SHG) technique. This study provides useful information to design practical temperature-controlled drug delivery systems.