Ionic interactions in nanofiltration of beta casein peptides

Nanofiltration (NF) membrane technology shows interesting potentials for separating organic components on the basis of solute charge and size in the range of 300-1000 g mol-1. Separation properties of two inorganic NF membranes were studied with a set of 10 small peptides (molecular mass range: 300-900 g mol-1; 3 < pI < 10) contained in a well-characterized tryptic beta casein hydrolysate. Peptides transmission strongly depended on ionic interactions in the system. Physicochemical conditions such as ionic strength and especially pH were crucial to the separation, because the membrane and peptides showed amphoteric properties. Thus, the three categories of peptides (acid, basic, neutral) were separated according to their pI because of presumed concentration gradients of charged peptides at the membrane: positive for basic peptides and negative for acid peptides. At optimum pH 8 this led to high transmissions of basic peptides (even over 100%), intermediate transmissions for neutral peptides, and low transmissions for acid peptides. The addition of multicharged cationic and anionic species in the hydrolysate induced a markedly enhanced selectivity when the polyelectrolyte was a membrane coion and a complete reversion of selectivity when it was a membrane counterion. Copyright 1998 John Wiley & Sons, Inc.     

Steric and electrostatic interactions govern nanofiltration of amino acids

Crossflow nanofiltration experiments were performed to investigate the factors influencing the removal of amino acids by a commercially available polymeric thin-film composite membrane. The removals of five monoprotic (Ala, Val, Leu, Gly, and Thr), one diprotic (Asp), and one dibasic (Arg) amino acids in a range of permeate fluxes, feed pH values, and ionic strengths were analyzed using a phenomenological model of membrane transport. At any given pH and ionic strength, reflection coefficients (rejection at asymptotically infinite flux) of monoprotic amino acids increased with molar radius demonstrating the role of steric interactions on their removal. Additionally, consistent with Donnan exclusion, higher reflection coefficients were obtained when the membrane and the amino acids both carried the same nature of charge (positive or negative). In other words, both co-ion repulsion and molecular size determined amino acids removal. Importantly, the removal of effectively neutral amino acids were significantly higher than neutral sugars and alcohols of similar size demonstrating that even near their isoelectric point, zwitterionic characteristics preclude them from being considered as strictly neutral.     

Separation of beta-casein peptides through UF inorganic membranes.

Ultrafiltration of peptide mixtures is studied under various operating conditions (transmembrane pressure, tangential flow-rate) using two ultrafiltration inorganic membranes M5 and M1 with molecular weight cut-offs, MWCO 10 and 70 kD, respectively. It is shown that the separation of peptides is controlled by a dual mechanism: size exclusion and electrostatic repulsion. When the ionic strength is high enough to screen out the electrostatic interactions, experimental data are in good agreement with a sieving model developed to estimate the intrinsic transmission from the molecular weight of a component and from the MWCO of the membranes. Although the transmission so found is altered by concentration polarisation and pore blocking mechanisms, the results explain the apparent low transmission of peptides by ultrafiltration membranes. If the ionic strength of the fluid is low, electrostatic interactions can influence the transport phenomena, provided that the molecules are highly charged (at pHs away from the pI). For attractive interactions, an apparent partition coefficient larger than 1 is observed. Otherwise, the transmission is lower than predicted by the sieving theoretical equation, as if the partition coefficient were smaller than 1.     

Separation albumin-PEG: transmission of PEG through ultrafiltration membranes

Transmission of polyethylene glycol (PEG) through ultrafiltration membranes has been studied under various operating conditions of pressure, crossflow, and concentration, using different membranes cut-offs and two module designs with the aim of understanding the separation of PEG from BSA. The influence of protein adsorption and fouling of the choice of a membrane has also been considered. Retention depends in general on the molecule to average pore size ratio, as expected, but also on concentration polarization. Accordingly, all operating and design parameters favoring concentration polarization lead to higher transmission. At high fluxes, flexible macromolecules can pass through the membrane, even if the random coil is larger than the apparent average pore. From a process selectivity point of view, the best way to separate PEG from BSA would be to use a membrane totally retaining BSA and to enhance concentration polarization of PEG. Unfortunately, such conditions also increase fouling and concentration polarization by BSA, which limits flux and thus PEG concentration polarization and transmission. Consequences of such conditions on separation efficiency are discussed. (c) 1993 Wiley & Sons, Inc.     

Anti-tumoral effect of scorpion peptides: Emerging new cellular targets and signaling pathways

Scorpion toxins have been the subject of many studies exploring their pharmacological potential. The high affinity and the overall selectivity to various types of ionic channels endowed scorpion toxins with a potential therapeutic effect against many channelopathies. These are diseases in which ionic channels play an important role in their development. Cancer is considered as a channelopathy since overexpression of some ionic channels was highlighted in many tumor cells and was linked to the pathology progression.Interestingly, an increasing number of studies have shown that scorpion venoms and toxins can decrease cancer growth in vitro and in vivo. Furthermore through their ability to penetrate the cell plasma membrane, certain scorpion toxins are able to enhance the efficiency of some clinical chemotherapies. These observations back-up the applicability of scorpion toxins as potential cancer therapeutics.In this review, we focused on the anti-cancer activity of scorpion toxins and their effect on the multiple hallmarks of cancer. We also shed light on effectors and receptors involved in signaling pathways in response to scorpion toxins effect. Until now, the anticancer mechanisms described for scorpion peptides consist on targeting ion channels to (i) inhibit cell proliferation and metastasis; and (ii) induce cell cycle arrest and/or apoptosis through membrane depolarization leading to hemostasis deregulation and caspase activation. Putative targets such as metalloproteinases, integrins and/or growth factor receptors, beside ion channels, have been unveiled to be affected by scorpion peptides.     

Comparison of p25 presequence peptide and melittin. Red blood cell haemolysis and band 3 aggregation.

The 25 residue presequence (p25) for subunit IV of yeast cytochrome oxidase had previously been shown to possess structural and behavioural characteristics in common with the bee venom polypeptide, melittin. The present study extends the results of leakage experiments on model-membrane systems to the haemolysis of human erythrocytes, which both peptides are shown to accomplish in a manner sensitive to membrane potential. In addition, the laser flash-induced transient dichroism technique for measuring protein rotational diffusion has been used to show that both peptides aggregate band 3, the major integral membrane protein of the erythrocyte. Aggregation cannot be reversed by high ionic strength; this serves to differentiate these peptides from other positively charged species such as polylysine that aggregate band 3 at low ionic strength. These results suggest that aggregation of membrane proteins may possibly prove to be a feature of the interaction of p25 signal peptide with mitochondrial membranes.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.     

Cellular uptake [correction of utake] of the Tat peptide: an endocytosis mechanism following ionic interactions

The cellular delivery of various biological compounds has recently been improved by conjugating them to short peptides known as protein transduction domains or cell penetrating peptides. These peptides include Tat, Antennapedia and arginine-rich peptides. The common feature of these peptides is their highly cationic nature. Up to now, the cellular uptake of about 50 different peptides and proteins coupled to Tat or Antennapedia peptides has been reported. The ability to deliver molecules into cells is not limited to peptide moieties, since oligonucleotides, peptide nucleic acids or other low molecular weight entities have been successfully internalized. Moreover, most of these examples have been accompanied by the expected biological response. More surprisingly, the uptake of large structures such as liposomes, phages, nanoparticles or adenoviruses has also been documented. Indeed the mechanism by which these very different entities could enter cells following a putative common pathway appeared more and more intriguing after each new reported example of cellular uptake mediated by these peptides. After a long period of uncertainty regarding the mechanism of entry, data from several groups now argue for an energy-dependent process of entry. The entry of most of these molecules is likely to be inhibited by low temperature incubation or in the presence of various drugs applied to inhibit the energy-dependent pathway of cell entry. Moreover, the binding of the highly cationic Tat peptide to various anionic membrane components probably initiates the first step of the cell internalization process.     

The actions of melittin on membranes

The molecular mechanisms underlying the various effects of melittin on membranes have not been completely defined and much of the evidence described indicates that different molecular mechanisms may underlie different actions of the peptide. Ideas about the formation of transbilayer aggregates of melittin under the influence of a transbilayer potential, and for bilayer structural perturbation arising from the location of the peptide helix within the head group region of the membrane have been made based on the crystal structure of the peptide, the kinetics and concentration dependence of melittins membrane actions, together with simple ideas about the conformational properties of amphipathic helical peptides and their interactions with membranes. Physical studies of the interaction of melittin with model membranes have been useful in determining the potential of the peptide to adopt different locations, orientations and association states within membranes under different conditions, but the relationship of the results obtained to the actions of melittin in cell membranes or under the influence of a membrane potential are unclear. Experimental definition of the interaction of melittin with more complex membranes, including the erythrocyte membrane or in bilayers under the influence of a transmembrane potential, will require direct study in these membranes. Experiments employing labeled melittins for ESR, NMR or fluorescence experiments are promising both for their sensitivity (ESR and fluorescence) and the ability to focus on the peptide within the background of endogenous proteins within cell membranes. The study of melittin in model membranes has been useful for the development of methodology for determination of membrane protein structures. Despite the structural complexity of integral membrane proteins, it is interesting that in some respects their study be more straightforward, lacking as they do the elusive properties of melittin (and other structurally labile membrane peptides) which limit the possibility of defining their interaction with membranes in terms of a single conformation, location, orientation and association state within the membrane.     

Aggregation of liposomes induced by the toxic peptides Alzheimer's Abetas, human amylin and prion (106-126): facilitation by membrane-bound GM1 ganglioside

To compare both the peptide molecular self-aggregation and the interaction with membrane lipids of the Alzheimer's amyloid beta (Abeta)40, Abeta42 peptides, and the cytotoxic peptides human amylin and prion (106-126) peptides, we applied a liposome aggregation technology. The kinetics of the changes in the optical density (DeltaOD) of liposome suspensions generated by the aggregation of liposomes induced by these peptides, allowed us to comparatively analyze their phospholipid affinity and self-aggregation. The kinetic curves showed an initial nonlinear region where d(DeltaOD)/dt followed first order kinetics corresponding to the binding of the peptides to the membrane of the liposome, a linear region where d(DeltaOD)/dt was constant, corresponding to the interaction between two membrane-bound peptide molecules, and a final slower increasing nonlinear region that corresponds to nucleation or seeding of aggregation. The analysis of the aggregation curves demonstrated that amylin and prion peptides also showed affinity for the acidic phospholipid phosphatidylserine (PS), as it has previously been shown for the Alzheimer's Abeta40, Abeta42 peptides. Abeta42 showed the highest, and amylin the lowest, affinity for the liposome membrane. When bound to the membrane of the liposomes, all the peptides preserved the self-aggregation characteristics observed in solution. Aging the Abeta40 and Abeta42 peptide solutions that permit molecular self-aggregation reduced their capacity to induce liposome aggregation. The self-aggregation of membrane-bound prion molecules was several orders of magnitude higher than that observed for the other toxic peptides. Incorporation of the ganglioside GM1 into the membrane of liposomes enhanced the peptide-induced liposome aggregation. Kinetic analysis revealed that this enhancement was due to facilitation of the formation of bridges between membrane-bound peptide molecules, demonstrating that the peptide-membrane interaction and the peptide amyloidogenesis are independent functions performed at separate molecular regions.     

Charged lipid vesicles: effects of salts on bending rigidity, stability, and size

The swelling behavior of charged phospholipids in pure water is completely different from that of neutral or isoelectric phospholipids. It was therefore suggested in the past that, instead of multilamellar phases, vesicles represent the stable structures of charged lipids in excess water. In this article, we show that this might indeed be the case for dioleoylphosphatidylglycerol and even for dioleoylphosphatidylcholine in certain salts. The size of the vesicles formed by these lipids depends on the phospholipid concentration in a way that has been predicted in the literature for vesicles of which the curvature energy is compensated for by translational entropy and a renormalization of the bending moduli (entropic stabilization). Self-consistent field calculations on charged bilayers show that the mean bending modulus kc and the Gaussian bending modulus k have opposite sign and /k/>kc, especially at low ionic strength. This has the implication that the energy needed to curve the bilayer into a closed vesicle Eves=4pi(2kc+k) is much less than one would expect based on the value of kc alone. As a result, Eves can relatively easily be entropically compensated. The radii of vesicles that are stabilized by entropy are expected to depend on the membrane persistence length and thus on kc. Experiments in which the vesicle size is studied as a function of the salt and the salt concentration correlate well with self-consistent field predictions of kc as a function of ionic strength.     

Membrane fluidity,composition, and charge affect the activity and selectivity of the AMP ascaphin-8

Ascaphins are cationic antimicrobial peptides that have been shown to have potential in the treatment of infectious diseases caused by multidrug-resistant pathogens (MDR). However, to date, their principal molecular target and mechanism of action are unknown. Results from peptide prediction software and molecular dynamics simulations confirmed that ascaphin-8 is an alpha-helical peptide. For the first time, the peptide was described as membranotrophic using biophysical approaches including calcein liposome leakage, Laurdan general polarization, and dynamic light scattering. Ascaphin-8’s activity and selectivity were modulated by rearranging the spatial distribution of lysine (Var-K5), aspartic acid (Var-D4) residues, or substitution of phenylalanine with tyrosine (Var-Y). The parental peptide and its variants presented high affinity toward the bacterial membrane model (≤2 μM), but lost activity in sterol-enriched membranes (mammal and fungal models, with cholesterol and ergosterol, respectively). The peptide-induced pore size was estimated to be >20 nm in the bacterial model, with no difference among peptides. The same pattern was observed in membrane fluidity (general polarization) assays, where all peptides reduced membrane fluidity of the bacterial model but not in the models containing sterols. The peptides also showed high activity toward MDR bacteria. Moreover, peptide sensitivity of the artificial membrane models compared with pathogenic bacterial isolates were in good agreement.     

Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration

Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers.

In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu5]-enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid- and phenylpropionic acid-based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C-terminal amino acids of opioid peptides (L-Leu, [Leu5]-enkephalin; D-Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N-terminal amino acid (L-Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30-50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes-Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu5]-enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.     

Role of diffusion potential on the flow of angiotensin II,bradykinin and related molecules through cellophane membranes

The diffusion of electrically charged peptides (angiotensin II, bradykinin and [Suc¹]angiotensin II) across tight cellophane membranes, obtained by different degrees of acetylation, shows a kinetic behaviour which was interpreted in the literature as indicative of the existence of different molecular conformations presenting slow interconversion velocities and different permeabilities across the membrane. A diffusion potential (Δψ) was found to be present across the membrane along diffusion experiments performed in low ionic strength. Upon annihilation of δψ by chemical voltage clamping (by equally increasing the ionic strength on both bathing solutions) the diffusion rate was decreased and the flow followed first order kinetics, indicating a major role of Δψ in the process. As the ionic strength increase could also affect molecular conformation, the role of Δψ on the diffusion of those molecules was tested by fitting flux and Δψ experimental results by an integrated form of Nernst-Planck flux equation. It is concluded that the deviation from first order diffusion kinetics, observed in low ionic strength, is solely due to the diffusion potential, and not to the existence of more than one molecular conformation in aqueous solution. This study was extended to amino acids and other related charged molecules.     

Membrane-active host defense peptides--challenges and perspectives for the development of novel anticancer drugs

Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means.Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Toroidal pores formed by antimicrobial peptides show significant disorder

A large variety of antimicrobial peptides have been shown to act, at least in vitro, by poration of the lipid membrane. The nanometre size of these pores, however, complicates their structural characterization by experimental techniques. Here we use molecular dynamics simulations, to study the interaction of a specific class of antimicrobial peptides, melittin, with a dipalmitoylphosphatidylcholine bilayer in atomic detail. We show that transmembrane pores spontaneously form above a critical peptide to lipid ratio. The lipid molecules bend inwards to form a toroidally shaped pore but with only one or two peptides lining the pore. This is in strong contrast to the traditional models of toroidal pores in which the peptides are assumed to adopt a transmembrane orientation. We find that peptide aggregation, either prior or after binding to the membrane surface, is a prerequisite to pore formation. The presence of a stable helical secondary structure of the peptide, however is not. Furthermore, results obtained with modified peptides point to the importance of electrostatic interactions in the poration process. Removing the charges of the basic amino-acid residues of melittin prevents pore formation. It was also found that in the absence of counter ions pores not only form more rapidly but lead to membrane rupture. The rupture process occurs via a novel recursive poration pathway, which we coin the Droste mechanism.