Domain formation induced by the adsorption of charged proteins on mixed lipid membranes

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation.     

The electrostatics of lipid surfaces

Charged lipids constitute a substantial fraction of all membrane lipids. Their charges vary in quantity and distribution within their headgroup regions. In long range interactions, their charges' value and electrostatic potential in the vicinity of the membrane surface can be approximated by the Guy-Chapman theory. This theory treats the interface as a charged structureless plain surrounded by uniform environments. However, if one considers intermolecular interactions, such assumptions need to be revised. The interface is in reality a thick region containing the residual charges of lipid headgroups. Their arrangement depends on the type of lipid present in the membrane. The variety of lipids and their biological functions suggests that charge distribution determines the extent and type of interaction with surface associated molecules. Numerous examples show that protein behavior at the lipid bilayer surface is determined by the type of lipid present, indicating protein specificity towards certain surface locations and local properties (determined by lipid composition) of a particular type. Such specificity is achieved by a combination of electrostatic, hydrophobic and enthropic effects. Comparing lipid biological activity, it can be stated that residual charge distribution is one of the factors of intermolecular recognition leading to the specific interaction of lipid molecules and selected proteins in various processes, particularly those involved with signal transduction pathways. Such specificity enables a variety of processes occurring simultaneously on the same membrane surface to function without cross-reaction interference.     

Electrostatic free energy and spontaneous curvature of spherical charged layered membrane

A biophysical model for the equilibrium curvature of a composite membrane element is derived taking into account the mechanical bilayer properties and the adjacent charged protein layers. The minimum of the total free energy density with respect to the curvature of such a membrane curved was estimated from the sum of the electrostatic free energy density of the charges of the membrane and the elastic surface energy density due to bending the lipid bilayer membrane. It was shown that the equilibrium curvature, i.e. the spontaneous curvature, of such a charged composite sandwich-like membrane depends inversely on the bending stiffness of the lipid membrane itself and directly on the charge amount inside and outside the membrane to the second power. Furthermore the geometric and electrostatic structure of the protein layers and the physico-chemical environment conditions are involved. Corresponding to the model developed a "standard RBC" membrane element has a negative spontaneous curvature, accounting for a discocyte RBC shape. The shape change from a discocyte to a more stomatocytic shape (increase in the negative spontaneous curvature) after reducing the charges in the glycocalyx is also explained within this model.     

Electrostatic free energy and shift of the phase transition for charged lipid membranes

For a charged membrane in an electrolyte solution the electrostatic free energy is derived treating the system as a diffuse double layer. The dependence of the free energy on external parameters like surface charge density and temperature is obtained and the physical basis discussed. As an application the charges are shown to exert an electrostatic surface pressure on the lipid chain packing which leads to a shift in the phase transition of lipid membranes. The results confirm the interpretation of experimental data as given by Träuble et al. in the accompanying paper.     

Protein adsorption onto auto-assembled polyelectrolyte films

Surface modification by deposition of ordered protein systems constitutes one of the major objectives of bio-related chemistry and biotechnology. In this respect a concept has recently been reported aimed at fabricating multilayers by the consecutive adsorption of positively and negatively charged polyelectrolytes. We investigate the adsorption processes between polyelectrolyte multilayers and a series of positively and negatively charged proteins. The film buildup and adsorption experiments were followed by Scanning Angle Reflectometry (SAR). We find that proteins strongly interact with the polyelectrolyte film whatever the sign of the charge of both the multilayer and the protein. When charges of the multilayer and the protein are similar, one usually observes the formation of protein monolayers, which can become dense. We also show that when the protein and the multilayer become oppositely charged, the adsorbed amounts are usually larger and the formation of thick protein layers extending up to several times the largest dimension of the protein can be observed. Our results confirm that electrostatic interactions dominate protein/polyelectrolyte multilayer interactions.     

Interaction of IAPP and Insulin with Model Interfaces Studied Using Neutron Reflectometry

The islet amyloid polypeptide (IAPP) and insulin are coproduced by the β-cells of the pancreatic islets of Langerhans. Both peptides can interact with negatively charged lipid membranes. The positively charged islet amyloid polypeptide partially inserts into these membranes and subsequently forms amyloid fibrils. The amyloid fibril formation of insulin is also accelerated by the presence of negatively charged lipids, although insulin has a negative net charge at neutral pH-values. We used water-polymer model interfaces to differentiate between the hydrophobic and electrostatic interactions that can drive these peptides to adsorb at an interface. By applying neutron reflectometry, the scattering-length density profiles of IAPP and insulin, as adsorbed at three different water-polymer interfaces, were determined. The islet amyloid polypeptide most strongly adsorbed at a hydrophobic poly-(styrene) surface, whereas at a hydrophilic, negatively charged poly-(styrene sulfonate) interface, the degree of adsorption was reduced by 50%. Almost no IAPP adsorption was evident at this negatively charged interface when we added 100 mM NaCl. On the other hand, negatively charged insulin was most strongly attracted to a hydrophilic, negatively charged interface. Our results suggest that IAPP is strongly attracted to a hydrophobic surface, whereas the few positive charges of IAPP cannot warrant a permanent immobilization of IAPP at a hydrophilic, negatively charged surface at an ionic strength of 100 mM. Furthermore, the interfacial accumulation of insulin at a hydrophilic, negatively charged surface may represent a favorable precondition for nucleus formation and fibril formation.     

Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.     

Lateral dynamics of proteins with polybasic domain on anionic membranes: a dynamic Monte-Carlo study

Positively charged polybasic domains are essential for recruiting multiple signaling proteins, such as Ras GTPases and Src kinase, to the negatively charged cellular membranes. Much less, however, is known about the influence of electrostatic interactions on the lateral dynamics of these proteins. We developed a dynamic Monte-Carlo automaton that faithfully simulates lateral diffusion of the adsorbed positively charged oligopeptides as well as the dynamics of mono- (phosphatidylserine) and polyvalent (PIP₂) anionic lipids within the bilayer. In agreement with earlier results, our simulations reveal lipid demixing that leads to the formation of a lipid shell associated with the peptide. The computed association times and average numbers of bound lipids demonstrate that tetravalent PIP₂ interacts with the peptide much more strongly than monovalent lipid. On the spatially homogeneous membrane, the lipid shell affects the behavior of the peptide only by weakly reducing its lateral mobility. However, spatially heterogeneous distributions of monovalent lipids are found to produce peptide drift, the velocity of which is determined by the total charge of the peptide-lipid complex. We hypothesize that this predicted phenomenon may affect the spatial distribution of proteins with polybasic domains in the context of cell-signaling events that alter the local density of monovalent anionic lipids.     

Increased concentration of polyvalent phospholipids in the adsorption domain of a charged protein

We studied the adsorption of a charged protein onto an oppositely charged membrane, composed of mobile phospholipids of differing valence, using a statistical-thermodynamical approach. A two-block model was employed, one block corresponding to the protein-affected region on the membrane, referred to as the adsorption domain, and the other to the unaffected remainder of the membrane. We calculated the protein-induced lipid rearrangement in the adsorption domain as arising from the interplay between the electrostatic interactions in the system and the mixing entropy of the lipids. Equating the electrochemical potentials of the lipids in the two blocks yields an expression for the relations among the various lipid fractions in the adsorption domain, indicating a sensitive dependence of lipid fraction on valence. This expression is a result of the two-block picture but does not depend on further details of the protein-membrane interaction. We subsequently calculated the lipid fractions themselves using the Poisson-Boltzmann theory. We examined the dependence of lipid enrichment, i.e., the ratio between the lipid fractions inside and outside the adsorption domain, on various parameters such as ionic strength and lipid valence. Maximum enrichment was found for lipid valence in the range between -3 and -4 in physiological conditions. Our results are in qualitative agreement with recent experimental studies on the interactions between peptides having a domain of basic residues and membranes containing a small fraction of the polyvalent phosphatidylinositol 4,5-bisphosphate (PIP2). This study provides theoretical support for the suggestion that proteins adsorbed onto membranes through a cluster of basic residues may sequester PIP2 and other polyvalent lipids.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

Effect of surface ionization of dimyristoylphosphatidic acid vesicle membranes on the main phase-transition enthalpy and temperature

The main phase transition of phospholipid bilayers is a property expressed by the order-disorder conformational change of the lipid tails. Nevertheless, with ionizable phospholipids, changes in the surface charge have large effects on the membrane properties. The free energy of a charged phospholipid membrane depends on the degree of ionization, area per phospholipid molecule, and the temperature. Here, the effect of surface electrostatic charges on the temperature and the enthalpy of the main phase transition of dimyristoylphosphatidic acid vesicle membranes is analyzed. A simple equation is presented that describes the relationship among the surface charge density, the phase-transition temperature, the surface area ratio between solid and liquid membranes, and the excess enthalpy. The theory indicated that the pH-induced shift in the excess enthalpy is attributable to the change in the surface area ratio between the solid and liquid membranes.     

Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues.     

Lamellar cationic lipid‐DNA complexes from lipids with a strong preference for planar geometry: A Minimal Electrostatic Model

We formulate and analyze a minimal model, based on condensation theory, of the lamellar cationic lipid (CL)‐DNA complex of alternately charged lipid bilayers and DNA monolayers in a salt solution. Each lipid bilayer, composed by a random mixture of cationic and neutral lipids, is assumed to be a rigid uniformly charged plane. Each DNA monolayer, located between two lipid bilayers, is formed by the same number of parallel DNAs with a uniform separation distance. For the electrostatic calculation, the model lipoplex is collapsed to a single plane with charge density equal to the net lipid and DNA charge. The free energy difference between the lamellar lipoplex and a reference state of the same number of free lipid bilayers and free DNAs, is calculated as a function of the fraction of CLs, of the ratio of the number of CL charges to the number of negative charges of the DNA phosphates, and of the total number of planes. At the isoelectric point the free energy difference is minimal. The complex formation, already favoured by the decrease of the electrostatic charging free energy, is driven further by the free energy gain due to the release of counterions from the DNAs and from the lipid bilayers, if strongly charged. This minimal model compares well with experiment for lipids having a strong preference for planar geometry and with major features of more detailed models of the lipoplex. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1114–1128, 2014.     

Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis

Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K(p) approximately 1-10 M(-1). Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.     

Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.     

The effect of lipid demixing on the electrostatic interaction of planar membranes across a salt solution

We study the effect of lipid demixing on the electrostatic interaction of two oppositely-charged membranes in solution, modeled here as an incompressible two-dimensional fluid mixture of neutral and charged mobile lipids. We calculate, within linear and nonlinear Poisson-Boltzmann theory, the membrane separation at which the net electrostatic force between the membranes vanishes, for a variety of different system parameters. According to Parsegian and Gingell, contact between oppositely-charged surfaces in an electrolyte is possible only if the two surfaces have exactly the same charge density (sigma(1) = -sigma(2)). If this condition is not fulfilled, the surfaces can repel each other, even though they are oppositely charged. In our model of a membrane, the lipidic charge distribution on the membrane surface is not homogeneous and frozen, but the lipids are allowed to freely move within the plane of the membrane. We show that lipid demixing allows contact between membranes even if there is a certain charge mismatch, /sigma(1)/ not equal /sigma(2)/, and that in certain limiting cases, contact is always possible, regardless of the value of sigma(1)/sigma(2) (if sigma(1)/sigma(2) < 0). We furthermore find that of the two interacting membranes, only one membrane shows a major rearrangement of lipids, whereas the other remains in exactly the same state it has in isolation and that, at zero-disjoining pressure, the electrostatic mean-field potential between the membranes follows a Gouy-Chapman potential from the more strongly charged membrane up to the point of the other, more weakly charged membrane.     

Electrostatically driven lipid–protein interaction: Answers from FRET

Electrostatics govern the association of a large number of proteins with cellular membranes. In some cases, these proteins present specialized lipid-binding modules or membrane targeting domains while in other cases association is achieved through nonspecific interaction of unstructured clusters of basic residues with negatively charged lipids. Given its spatial resolution in the nanometer range, Förster resonance energy transfer (FRET) is a powerful tool to give insight into protein–lipid interactions and provide molecular level information which is difficult to retrieve with other spectroscopic techniques.In this review we present and discuss the basic formalisms of both hetero- and homo-FRET pertinent to the most commonly encountered problems in lipid–protein interaction studies and highlight some examples of implementations of different FRET methodologies to characterize lipid/protein systems in which electrostatic interactions play a crucial role. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.