Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. (31)P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, (2)H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.     

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: Insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. ³¹P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, ²H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.     

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1 × 10^− 4 M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes—a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15–70 °C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.     

Membrane integrity and amyloid cytotoxicity: a model study involving mitochondria and lysozyme fibrillation products

Recent findings implicate that fibrillation products, the protein aggregates formed during the various steps leading to formation of mature fibrils, induce neurotoxicity predominantly in their intermediate oligomeric state. This has been shown to occur by increasing membrane permeability, eventually leading to cell death. Despite accumulating reports describing mechanisms of membrane permeabilization by oligomers in model membranes, studies directly targeted at characterizing the events occurring in biological membranes are rare. In the present report, we describe interaction of the original native structure, prefibrils and fibrils of hen egg white lysozyme (HEWL) with mitochondrial membranes, as an in vitro biological model, with the aim of gaining insight into possible mechanism of cytotoxicity at the membrane level. These structures were first characterized using a range of techniques, including fluorescence, size-exclusion chromatography, dynamic light scattering, transmission electron microscopy, dot blot analysis and circular dichroism. HEWL oligomers were found to be flexible/hydrophobic structures with the capacity to interact with mitochondrial membranes. Possible permeabilization of mitochondria was explored utilizing sensitive fluorometric and luminometric assays. Results presented demonstrate release of mitochondrial enzymes upon exposure to HEWL oligomers, but not native enzyme monomer or mature fibrils, in a concentration-dependent manner. Release of cytochrome c was also observed, as reported earlier, and membrane stabilization promoted by addition of calcium prevented release. Moreover, the oligomer-membrane interaction was influenced by high concentrations of NaCl and spermine. The observed release of proteins from mitochondria is suggested to occur by a nonspecific perturbation mechanism.     

The possible role of enantiodiscrimination in bilirubin toxicity

The possibility that enantiodiscrimination of bilirubin might be involved in neuronal membrane destabilization, and therefore in bilirubin toxicity, was investigated, by circular dichroism, on model membranes composed of phospholipids. The equilibrium between bilirubin enantiomers is displaced at some extent by the interaction with certain phospholipids. The extent of equilibrium displacement depends on the molecular structure of phospholipids and on the state of charge of bilirubin. The results obtained validate the hypothesis of a possible involvement of chirality in bilirubin toxicity and support a previously suggested model for the molecular mechanism of the interaction of bilirubin with the synaptic membrane.     

The sterol carrier protein-2 amino terminus: a membrane interaction domain.

Sterol carrier protein-2 (SCP2) is a small, 123 amino acid, protein postulated to play a role in intracellular transport and metabolism of lipids such as cholesterol, phospholipids, and branched chain fatty acids. While it is thought that interaction of SCP2 with membranes is necessary for lipid transfer, evidence for this possibility and identification of a membrane interaction domain within SCP2 has remained elusive. As shown herein with circular dichroism and a direct binding assay, SCP2 bound to small unilamellar vesicle (SUV) membranes to undergo significant alteration in secondary structure. The SCP2 amphipathic N-terminal 32 amino acids, comprised of two alpha-helical segments, were postulated to represent a putative phospholipid interaction site. This hypothesis was tested with a series of SCP2 N-terminal peptides, circular dichroism, and direct binding studies. The SCP2 N-terminal peptide (1-32)SCP2, primarily random coil in aqueous buffer, adopted alpha-helical structure upon interaction with membranes. The induction of alpha-helical structure in the peptide was maximal when the membranes contained a high mole percent of negatively charged phospholipid and of cholesterol. While deletion of the second alpha-helical segment within this peptide had no effect on formation of the first alpha-helix, it significantly weakened the peptide interaction with membranes. Substitution of Leu(20) with Glu(20) in the N-terminal peptide disrupted the alpha-helix structure and greatly weakened the peptide interaction with membranes. Finally, deletion of the first nine nonhelical amino acids had no effect either on formation of alpha-helix or on peptide binding to membranes. N-Terminal peptide (1-32)SCP2 competed with SCP2 for binding to SUV. These data were consistent with the N-terminus of SCP2 providing a membrane interaction domain that preferentially bound to membranes rich in anionic phospholipid and cholesterol.     

A multitechnique approach in protein/surfactant interaction study: physicochemical aspects of sodium dodecyl sulfate in the presence of trypsin in aqueous medium

Interaction of sodium dodecyl sulfate (SDS) with a globular protein, trypsin, has been physicochemically studied in aqueous medium in detail using tensiometric, conductometric, calorimetric, fluorimetric, viscometric, and circular dichroism techniques. The results indicate that SDS-trypsin aggregates start to form at a surfactant concentration higher than the critical micelle concentration of pure SDS micelle. In contrast, the counterion binding decreases in the presence of trypsin. The free energies and enthalpies of micellization, interfacial adsorption, and entropy of micellization associated with the interaction have also been calculated. The values show that the interaction phenomenon is entropy controlled and endothermic in nature. The increase in viscosity is observed for the system of SDS-trypsin cluster above the critical micelle concentration of SDS micelle only. The aggregation number and interface polarity decrease compared to the values of micelles without protein. Circular dichroism spectra show the high alpha-helical content and unfolded structure of trypsin in the presence of SDS due to strong electrostatic repulsion leading to a probable "necklace and bead" model in the case of biopolymer-surfactant complexes.     

Differential interactions of the antimicrobial peptide,RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes

BackgroundAntimicrobial peptides (AMPs) are molecules with potential application for the treatment of microorganism infections. We, herein, describe the structure, activity, and mechanism of action of RQ18, an α-helical AMP that displays antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeasts from the Candida genus.MethodsA physicochemical-guided design assisted by computer tools was used to obtain our lead peptide candidate, named RQ18. This peptide was assayed against Gram-positive and Gram-negative bacteria, yeasts, and mammalian cells to determine its selectivity index. The secondary structure and the mechanism of action of RQ18 were investigated using circular dichroism, large unilamellar vesicles, and molecular dynamic simulations.ResultsRQ18 was not cytotoxic to human lung fibroblasts, peripheral blood mononuclear cells, red blood cells, or Vero cells at MIC values, exhibiting a high selectivity index. Circular dichroism analysis and molecular dynamic simulations revealed that RQ18 presents varying structural profiles in aqueous solution, TFE/water mixtures, SDS micelles, and lipid bilayers. The peptide was virtually unable to release carboxyfluorescein from large unilamellar vesicles composed of POPC/cholesterol, model that mimics the eukaryotic membrane, indicating that vesicles' net charges and the presence of cholesterol may be related with RQ18 selectivity for bacterial and fungal cell surfaces.ConclusionsRQ18 was characterized as a membrane-active peptide with dual antibacterial and antifungal activities, without compromising mammalian cells viability, thus reinforcing its therapeutic application.General significanceThese results provide further insight into the complex process of AMPs interaction with biological membranes, in special with systems that mimic prokaryotic and eukaryotic cell surfaces.     

Conformation of a synthetic antigenic peptide from HIV-1 p24 protein induced by ionic micelles

We studied the interaction of the peptide AAMQMLKETINEEAAEWDRVHPVHAGPIA from the HIV-1 p24 protein in the presence of SDS (anionic) and CTABr (cationic) micelles at pH 7.0 by circular dichroism, fluorescence, and electron spin resonance (ESR). The micelles induced secondary structure as well as a blue shift in the tryptophan fluorescence emission, indicating an interaction between the peptide and the micelles. However, different contents of secondary structure elements were found when the peptide interacts with SDS or CTABr micelles. Steady-state anisotropy indicates a constraint on the rotational mobility of the tryptophan residue of the peptide upon interaction with micelles. ESR studies pointed to different locations for the peptide in either micelle. Our results suggested that at least part of the peptide might be located at the hydrophobic core of the CTABr micelles, probably at the C-terminal region, while it is more inserted into the SDS micelles.     

Comparative study on the interaction of cell-penetrating polycationic polymers with lipid membranes

Cell-penetrating peptides are arginine- and lysine-rich cationic peptides that can readily enter cells not only by themselves but also carrying other macromolecular cargos. In fact, we have reported that polycationic polymer such as poly-l-lysine (PLL) and poly-l-arginine (PLA) translocate through negatively charged phospholipid liposome membranes. In this work, we made a comparative study of the interaction of PLL or PLA with lipid membranes consisting of negatively charged phospholipids to understand the role of basic amino acid residue (i.e. arginine and lysine) in the membrane-penetrating activity of polypeptides. PLA and PLL translocated into giant unilamellar vesicle composed of soybean phospholipids. ζ-potential and turbidity measurements demonstrated the electrostatic binding of PLL and PLA to large unilamellar vesicle (LUV). Fluorescence studies using membrane probes revealed that the binding of PLA and PLL to LUV affects the hydration and packing of the membrane interface region, in which the membrane insertion of PLA appeared to be greater than PLL. Differential scanning calorimetry showed that the enthalpy of the gel to liquid-crystalline phase transition for dipalmitoyl phosphatidylglycerol vesicle was greatly reduced by binding of PLL and PLA, in which the reduction is much larger in PLA than in PLL. Circular dichroism measurements in 2,2,2-trifluoroethanol/water mixture or in the presence of LUV indicated that the propensity of PLA to form α-helical structure is greater than PLL. Consistently, attenuated total reflection-Fourier transform infrared spectroscopy revealed that there is greater α-helical structure in PLA bound to LUV compared to PLL, which has much less ordered structure. Furthermore, isothermal titration calorimetry measurements demonstrated that the contribution of enthalpy to the energetics of binding to LUV is two-fold larger in PLA than in PLL. These results suggest that the stronger interaction of arginine residue with negatively charged phospholipid membranes compared to lysine residue appears to facilitate the conformational change in cationic polypeptide and its insertion into lipid membrane interior.     

Conformational studies by circular dichroism, 1H NMR, and computer simulations of bombolitins I and III in aqueous solution containing surfactant micelles

The heptadecapeptides bombolitin I and bombolitin III are two members of a series of biologically active peptides postulated to be membrane active. In order to understand the effects of the membrane on the secondary structure of the peptides, we have carried out the conformational characterization of bombolitins I and III in the presence of SDS micelles using circular dichroism, nuclear magnetic resonance, and computer simulations. The characteristic bands in the circular dichroism spectra indicate an alpha-helix content of approximately 60% in bombolitin III and 70% in bombolitin I. The observation of NOE's quite distinctive for such secondary structure strongly supports the CD results. The conformational preferences of the two bombolitins derived from CD and NMR were then energetically refined with molecular dynamics simulations. The results from the spectroscopic examination were utilized as input for the simulations, the CD results for generation of the initial structure, and the NOE's as constraints during the simulations. The results from the different techniques employed are in complete agreement.     

Exploring interaction of beta-amyloid segment (25-35) with membrane models through paramagnetic probes.

The accumulation of beta-amyloid peptides into senile plaques is one of the hallmarks of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the beta-sheet oligomerization process of beta-amyloid. Abeta(25-35), the sequence of which is GSNKGAIIGLM, is a highly toxic segment of amyloid beta (Abeta)-peptides, which forms fibrillary aggregates. In the present work, two spin-labelled Abeta(25-35) analogues containing the nitroxide group of the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe at the N- or the C-terminus of the peptide sequence, respectively, were synthesized in order to investigate the peptide-membrane interaction. The orientation and associated changes of the peptide conformation in the presence of different artificial membrane models (micelles, liposomes) were evaluated by electron paramagnetic resonance and circular dichroism techniques. The results of this study allowed us to propose a model in which the C-terminal portion of the peptide is highly associated to the membrane, while the N-terminal part extends into the aqueous phase with occasional contacts with the lipid head-group region. Interestingly, the interaction of the C-terminal portion of the peptide is particularly enhanced in the presence of sodium dodecyl sulfate (SDS) molecules.     

Secondary and tertiary structure formation of the beta-barrel membrane protein OmpA is synchronized and depends on membrane thickness

The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of beta-sheet secondary structure and closure of the beta-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other beta-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of alpha-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins.     

Protein-induced formation of cholesterol-rich domains

A major protein of neuronal rafts, NAP-22, binds specifically to cholesterol. We demonstrate by circular dichroism that NAP-22 contains a significant amount of beta-structure that is not sensitive to binding of the protein to membranes, suggesting that a major portion of the protein does not insert deeply into the membrane. The free energy of binding of NAP-22 to liposomes of dioleoylphosphatidylcholine with 40% cholesterol is -7.3 +/- 0.5 kcal/mol. NAP-22 mixed with dipalmitoylphosphatidylcholine and 40% cholesterol partitions into the detergent insoluble fraction in the presence of 1% Triton X-100. NAP-22 also causes this insoluble fraction to become enriched in cholesterol relative to phospholipid, again demonstrating the ability of this protein to segregate cholesterol and phospholipids into domains. Differential scanning calorimetry results demonstrate that NAP-22 promotes domain formation in liposomes composed of cholesterol and phosphatidylcholine. This is shown by NAP-22-promoted changes in the shape and enthalpy of the phase transition of phosphatidylcholine as well as by the appearance of cholesterol crystallite transitions in membranes composed of phosphatidylcholine with either saturated or unsaturated acyl chains. In situ atomic force microscopy revealed a marked change in the surface morphology of a supported bilayer of dioleoylphosphatidylcholine with 0.4 mole fraction of cholesterol upon addition of NAP-22. Prior to the addition of the protein, the bilayer appears to be a molecularly smooth structure with uniform thickness. Addition of NAP-22 resulted in the rapid formation of localized raised bilayer domains. Remarkably, there was no gross disruption or erosion of the bilayer but rather simply an apparent rearrangement of the lipid bilayer structure due to the interaction of NAP-22 with the lipid. Our results demonstrate that NAP-22 can induce the formation of cholesterol-rich domains in membranes. This is likely to be relevant in neuronal membrane domains that are rich in NAP-22.     

Kinetic approach to the interaction of sodium n-dodecyl sulphate with heme enzymes

The kinetics of interaction of sodium n-dodecyl sulphate (SDS) with catalase has been studied by absorbance and fluorescence changes. The results have been compared with circular dichroism spectra and activity measurements. The tertiary structure of catalase is modified by SDS in the monomeric and micellar form. The secondary structure of catalase is altered only in the presence of SDS micelles. On the other hand, neither spectroscopic properties nor activity of horseradish peroxidase change in the presence of SDS below micellar concentration. In the presence of SDS micelles, however, changes of secondary and tertiary structure of this protein are detected. The reason for relatively high stability of horseradish peroxidase in the presence of SDS is discussed.     

Isolation of human platelet plasma membranes with polylysine beads

Human platelet plasma membranes were isolated with polylysine beads according to the technique developed by Jacobson and Branton (1977, Science [Wash. D. C.] 195:302--304). Lactoperoxidase-catalyzed surface iodination revealed that ninefold greater 125I specific activity was associated with the membranes isolated on beads than with whole platelets. Enrichment in the bead membrane preparation of the activities of membrane marker enzymes, bis(p-nitrophenyl)phosphate phosphodiesterase and Na,K-ATPase, was 8.0 and 4.4, respectively. Contamination with enzymes of other organelles, cytochrome oxidase and beta-glucuronidase, was relatively low as compared with membranes isolated by sucrose gradient centrifugation. Analysis by SDS polyacrylamide gel electrophoresis showed that a full complement of surface glycoproteins was present on the membranes isolated with polylysine beads. The polylysine bead technique is a rapid, reproducible and efficient method for the preparation of relatively pure platelet plasma membranes.     

The non-native conformations of cytochrome c in sodium dodecyl sulfate and their modulation by ATP

To understand the interaction of cytochrome c (cyt c) with membranes, a systematic investigation of sodium dodecyl sulfate (SDS)-induced conformational alterations in native horse heart ferricytochrome c (pH 7.0) was carried out using heme absorbance, tryptophan fluorescence and circular dichroism (CD) spectroscopy. ATP interaction with membrane-bound cyt c is known to regulate the process of apoptosis. To understand the effect of nucleotide phosphates on membrane-bound cyt c, we also carried out studies of the interaction of ATP with cyt c in the presence of SDS. Fluorescence and UV-Vis data suggest that SDS induces two different transitions (F to C1, C1 to C2) in cyt c, one in the pre-micellar region and the other in the post-micellar region. The fluorescence data further indicated the increase in distance between Trp 59 and heme in the intermediates in both the regions, suggesting loosening up of cyt c on titration with SDS. The far-UV and near-UV CD data suggest partial loss of secondary and tertiary structure in C1, but complete loss of tertiary structure and no further loss of secondary structure in C2. On titration of C1 and C2 with ATP, the secondary structure is restored. However, the heme ligation pattern and heme exposure change only for C2, but not for C1 on the addition of ATP.     

Caveolin scaffolding region and cholesterol-rich domains in membranes

A protein that constitutes a good marker for a type of cholesterol-rich domain in biological membranes is caveolin. A segment of this protein has a sequence that corresponds to a cholesterol recognition/interaction amino acid consensus (CRAC) motif; this motif has been suggested to cause the incorporation of proteins into cholesterol-rich domains. We have studied the interaction of two peptides containing the CRAC motif of caveolin-1 by differential scanning calorimetry, fluorescence, circular dichroism and magic angle spinning NMR. These peptides promote the segregation of cholesterol into domains from mixtures of the sterol with phosphatidylcholine, as shown by depletion of cholesterol from a portion of the membrane and enrichment of cholesterol in another domain. Cholesterol passes its solubility limit in the cholesterol-rich domain, resulting in the formation of cholesterol crystallites, suggesting that not all of the cholesterol recruited to this domain is bound to the peptide. NMR studies show that the peptides insert somewhat more deeply into membranes when cholesterol is present, but their strongest interaction takes place with the interfacial region of the membrane. We conclude that the peptides we studied containing CRAC sequences are more effective in promoting the formation of cholesterol-rich domains than are shorter peptides of this region of caveolin, which although they contain several aromatic amino acids, they have no CRAC motif. The presence or absence of a CRAC motif, however, is not a sufficient criterion to determine the extent to which a protein will promote the segregation of cholesterol in membranes.     

New short cationic antibacterial peptides. Synthesis,biological activity and mechanism of action

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.     

Conformation of magainin-2 and related peptides in aqueous solution and membrane environments probed by Fourier transform infrared spectroscopy.

The conformational properties of the magainin family of antimicrobial peptides in aqueous solution and in model membranes have been probed by Fourier transform infrared spectroscopy. The magainins were found to be structureless in aqueous solution at neutral pD, confirming other studies by Raman and circular dichroism spectroscopy. Increasing the pD to 10 induced the formation of predominantly alpha-helical secondary structures, with some beta-sheet. In the presence of negatively charged liposomes (dimyristoylphosphatidylglycerol), the peptides folded into alpha-helical secondary structures with some beta-sheet structure evident. On the other hand, in the presence of zwitterionic phospholipids (dimyristoylphosphatidylcholine), the spectra were identical to those in aqueous solution. For some magainins, the interaction with charged liposomes was modulated by the presence of cholesterol; cholesterol was found to promote the formation of beta-sheet structures, as evidenced by the appearance of amide I bands at 1614 and 1637 cm-1. Differences in structure were observed between the amidated and nonamidated forms of some peptides. From the data, a mechanism of antimicrobial action of the magainin family of peptides is proposed.