Free energy analysis of membrane pore formation process in the presence of multiple melittin peptides

Understanding the molecular mechanism underlying pore formation in lipid membranes by antimicrobial peptides is of great importance in biological sciences as well as in drug design applications. Melittin has been widely studied as a pore forming peptide, though the molecular mechanism for pore formation is still illusive. We examined the free energy barrier for the creation of a pore in lipid membranes with and without multiple melittin peptides. It was found that six melittin peptides significantly stabilized a pore, though a small barrier (a few k_BT) for the formation still existed. With five melittin peptides or fewer, the pore formation barrier was much higher, though the established pore was in a local energy minimum. Although seven melittins effectively reduced the free energy barrier, a single melittin peptide left the pore after a long time MD simulation probably because of the overcrowded environment around the bilayer pore. Thus, it is highly selective for the number of melittin peptides to stabilize the membrane pore, as was also suggested by the line tension evaluations. The free energy cost required to insert a single melittin into the membrane is too high to explain the one-by-one insertion mechanism for pore formation, which also supports the collective melittin mechanism for pore formation.     

Cooperative antimicrobial action of melittin on lipid membranes: A coarse-grained molecular dynamics study

We conducted a series of coarse-grained molecular dynamics (CG-MD) simulations to investigate the complicated actions of melittin, which is an antimicrobial peptide (AMP) derived from honey bee venom, on a lipid membrane. To accurately simulate the AMP action, we developed and used a protein CG model as an extension of the pSPICA force field (FF), which was designed to reproduce several thermodynamic quantities and structural properties. At a low peptide-to-lipid (P/L) ratio (1/102), no defect was detected. At P/L = 1/51, toroidal pore formation was observed due to collective insertion of multiple melittin peptides from the N-termini. The pore formation was initiated by a local increase in membrane curvature in the vicinity of the peptide aggregate. At a higher P/L ratio (1/26), two more modes were detected, seemingly not controlled by the P/L ratio but by a local arrangement of melittin peptides: 1. Pore formation accompanied by lipid extraction by melittin peptides:a detergent-like mechanism. 2. A rapidly formed large pore in a significantly curved membrane: bursting. Thus, we observed three pore formation modes (toroidal pore formation, lipid extraction, and bursting) depending on the peptide concentration and local arrangement. These observations were consistent with experimental observations and hypothesized melittin modes. Through this study, we found that the local arrangements and population of melittin peptides and the area expansion rate by membrane deformation were key to the initiation of and competition among the multiple pore formation mechanisms.     

Interactions of peptides with liposomes: pore formation and fusion

Leakage from liposomes induced by several peptides is reviewed and a pore model is described. According to this model peptide molecules become incorporated into the vesicle bilayer and aggregate reversibly or irreversibly within the surface. When a peptide aggregate reaches a critical size, peptide translocation can occur and a pore is formed. With the peptide GALA the pores are stable and persist for at least 10 minutes. The model predicts that for a given lipid/peptide ratio, the extent of leakage should decrease as the vesicle diameter decreases, and for a given amount of peptide bound per vesicle less leakage would be observed at higher temperatures due to the increase in reversibility of surface aggregates of the peptide. Effect of membrane composition on pore formation is reviewed. When cholesterol was included in the liposomes the efficiency of inducation of leakage by the peptide GALA was reduced due to reduced binding and increased reversibility of surface aggregation of the peptide. Phospholipids which contain less ordered acyl-chains and have a slightly wedge-like shape, can better accommodate peptide surface aggregates, and consequently insertion and translocation of the peptide may be less favored. Demonstrations of antagonism between pore formation and fusion are presented. The choice of factors which promote vesicle aggregation, e.g., larger peptides, increased vesicle and peptide concentration results in enhanced vesicle fusion at the expense of formation of intravesicular pores. FTIR studies with HIV-1 fusion peptides indicate that in systems where extensive vesicle fusion occurred the beta conformation of the peptides was predominant, whereas the alpha conformation was exhibited in cases where leakage was the main outcome. Antagonism between leakage and fusion was exhibited by 1-palmitoyl-2-oleoylphosphatidylglycerol vesicles, where the order of addition of peptide (HIV(arg)) or Ca(2+)dictated whether pore formation or vesicle fusion would occur. The current study emphasizes that the addition of Ca(2+), which promotes vesicle aggregation can also reduce peptide translocation in isolated vesicles.     

Surface aggregation and membrane penetration by peptides: relation to pore formation and fusion

The peptide GALA undergoes a conformational change to an amphipathic alpha -helix when the pH is reduced, inducing leakage of contents from vesicles. Leakage from neutral or negativelycharged vesicles at pH 5.0 was similar and could be adequately explained by a mathematical model which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of M =10+/-2 peptides. Increasing cholesterol content in the membranes resulted in reduced leakage, and increased reversibility of surface aggregation of the peptide. Employing fluorescently labelled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol containing liposomes. Orientation of the peptide GALA in bilayers was determined by a bodipy-avidin/ biotin binding assay. The peptide was labelled by biotin at the N- or Cterminus and bodipy-avidin molecules were added externally or were preencapsulated in the vesicles. The peptides are arranged in the pore perpendicularly to the membrane, such that 3/4 of the N-termini are on the internal side of the membrane. The pores are stable and persist for at least 10 min. When the peptides form an aggregate of size smaller than M, the orientation of the peptide is mostly parallel to the surface and the biotinylated peptide does not translocate. When a critical size of the aggregate is attained, a rearrangement of the peptide occurs, which amounts to rapid penetration and formation of a pore structure. Induction of fusion by peptides may be antagonistic to pore formation, the outcome being dependent on vesicle aggregation.     

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.     

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

Mechanisms of leakage

Abstract

Two mechanisms of leakage from liposomes are discussed, (i) Cations such as Ca²⁺ induce graded release whose rate depends mainly on vesicle collisions and is associated in the case of several acidic phospholipids with fusion events. A certain degree of leakage also occurs in between collisions. Consequently, the leakage per fusion is reduced at larger lipid and Ca concentrations, (n) Certain peptides induce leakage by pore formation, which shows selectivity to the size of the entrapped molecules and occurs by an all or none mechanism; vesicles either leak or retain all of their contents. A model for final extents and kinetics of leakage due to pore forming peptides is described. This model assumes that pore forming peptides become incorporated into the vesicle bilayer and aggregate to form a pore. Recent developments in the model enable considerations of a reversible or irreversible surface aggregation of peptides. Results of final extents and kinetics of leakage induced by pore forming peptides can be well explained and predicted by this formalism. Studies demonstrate that Ca can play a dual role in affecting leakage. A case is presented where Ca ⁺ inhibits and can even arrest pore formation by a peptide, while promoting vesicle fusion. Conversely, formation of pore structures by a peptide can inhibit vesicle fusion.     

Attempts to define functional domains of gap junction proteins with synthetic peptides.

To map the binding sites involved in channel formation, synthetic peptides representing sequences of connexin 32 were tested for their ability to inhibit cell-cell channel formation. Both large peptides representing most of the two presumed extracellular loops of connexin32 and shorter peptides representing subsets of these larger peptides were found to inhibit cell-cell channel formation. The properties of the peptide inhibition suggested that the binding site is complex, involving several segments of both extracellular loops. One of the peptides (a 12-mer) did not inhibit but instead was found to form channels in membranes. Both in oocyte membranes and in bilayers, the channels formed by the peptide were asymmetrically voltage dependent. Their unit conductances ranged from 20 to 160 pS. These data are discussed in the form of a model in which the connexin sequence represented by the peptide is part of a beta structure providing the lining of the channel pore.     

Thermodynamic profiling of peptide membrane interactions by isothermal titration calorimetry: a search for pores and micelles

Antimicrobial peptides are known to interact strongly with negatively charged lipid membranes, initially by peripheral insertion of the peptide into the bilayer, which for some antimicrobial peptides will be followed by pore formation, and successive solubilization of the membranes resulting in mixed peptide-lipid micelles. We have investigated the mode of action of the antimicrobial peptide mastoparan-X using isothermal titration calorimetry (ITC) and cryo-transmission electron microscopy (cryo-TEM). The results show that mastoparan-X induces a range of structural transitions of POPC/POPG (3:1) lipid membranes at different peptide/lipid ratios. It has been established that ITC can be used as a fast method for localizing membrane transitions and when combined with DLS and cryo-TEM can elucidate structural changes, including the threshold for pore formation and micellation. Cryo-TEM was employed to confirm the structural changes associated with the thermodynamic transitions found by ITC. The pore-formation process has furthermore been investigated in detail and the thermodynamic parameters of pore formation have been characterized using a system-specific temperature where the enthalpy of peptide partitioning becomes zero (T_zero). This allows for an exclusive study of the pore-formation process. The use of ITC to find T_zero allows for characterization of the thermodynamic parameters of secondary processes on lipid membranes.     

Effect of cholesterol and charge on pore formation in bilayer vesicles by a pH-sensitive peptide.

The effect of cholesterol on the bilayer partitioning of the peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) and its assembly into a pore in large unilamellar vesicles composed of neutral and negatively charged phospholipids has been determined. GALA undergoes a conformational change from a random coil to an amphipathic alpha-helix when the pH is reduced from 7.0 to 5.0, inducing at low pH leakage of contents from vesicles. Leakage from neutral or negatively charged vesicles at pH 5.0 was similar and could be adequately explained by the mathematical model (Parente, R. A., S. Nir, and F. C. Szoka, Jr., 1990. Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. Biochemistry. 29:8720-8728) which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of 10 +/- 2 peptides. Increasing cholesterol content in the membranes resulted in a reduced efficiency of the peptide to induce leakage. Part of the cholesterol effect was due to reduced binding of the peptide to cholesterol-containing membranes. An additional effect of cholesterol was to increase reversibility of surface aggregation of the peptide in the membrane. Results could be explained and predicted with a model that retains the same pore size, i.e., 10 +/- 2 peptides, but includes reversible aggregation of the monomers to form the pore. Resonance energy transfer experiments using fluorescently labeled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol-containing liposomes, thus reducing the efficiency of pore formation.     

Fluctuations and the Rate-Limiting Step of Peptide-Induced Membrane Leakage

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.     

Osmotically induced membrane tension modulates membrane permeabilization by class L amphipathic helical peptides: nucleation model of defect formation.

The mechanism of action of lytic peptides on membranes is widely studied and is important in view of potential medical applications. Previously (I. V. Polozov, A. I. Polozova, E. M. Tytler, G. M. Anantharamaiah, J. P. Segrest, G. A. Woolley, and R. M., Biochemistry, 36:9237--9245) we analyzed the mechanism of membrane permeabilization by 18L, the archetype lytic peptide featuring the class L amphipathic alpha-helix, according to the classification of Segrest et al. (J. P. Segrest, G. de Loof, J. G. Dohlman, C. G. Brouillette, and G. M. Anantharamaiah, 1990, Proteins, 8:103--117). We concluded that the 18L peptide destabilizes membranes, leading to a transient formation of large defects that result in contents leakage and, in the presence of bilayer-bilayer contact, could lead to vesicle fusion. Here we report that this defect formation is strongly enhanced by the membrane tension induced by osmotic swelling of vesicles. Even below standard leakage-inducing peptide/lipid ratios, membrane resistance to osmotic tension drops from hundreds to tens of milliosmoles. The actual decrease is dependent on the peptide/lipid ratio and on the type of lipid. We propose that under membrane tension a peptidic pore serves as a nucleation site for the transient formation of a lipidic pore. The tension is released upon pore expansion with inclusion of more peptides and lipids into the pore lining. This tension modulation of leakage was observed for other class L peptides (mastoparan, K18L) and thus may be of general applicability for the action of membrane active lytic peptides.     

Energetics and self-assembly of amphipathic peptide pores in lipid membranes

We present a theoretical study of the energetics, equilibrium size, and size distribution of membrane pores composed of electrically charged amphipathic peptides. The peptides are modeled as cylinders (mimicking alpha-helices) carrying different amounts of charge, with the charge being uniformly distributed over a hydrophilic face, defined by the angle subtended by polar amino acid residues. The free energy of a pore of a given radius, R, and a given number of peptides, s, is expressed as a sum of the peptides' electrostatic charging energy (calculated using Poisson-Boltzmann theory), and the lipid-perturbation energy associated with the formation of a membrane rim (which we model as being semitoroidal) in the gap between neighboring peptides. A simple phenomenological model is used to calculate the membrane perturbation energy. The balance between the opposing forces (namely, the radial free energy derivatives) associated with the electrostatic free energy that favors large R, and the membrane perturbation term that favors small R, dictates the equilibrium properties of the pore. Systematic calculations are reported for circular pores composed of various numbers of peptides, carrying different amounts of charge (1-6 elementary, positive charges) and characterized by different polar angles. We find that the optimal R's, for all (except, possibly, very weakly) charged peptides conform to the "toroidal" pore model, whereby a membrane rim larger than approximately 1 nm intervenes between neighboring peptides. Only weakly charged peptides are likely to form "barrel-stave" pores where the peptides essentially touch one another. Treating pore formation as a two-dimensional self-assembly phenomenon, a simple statistical thermodynamic model is formulated and used to calculate pore size distributions. We find that the average pore size and size polydispersity increase with peptide charge and with the amphipathic polar angle. We also argue that the transition of peptides from the adsorbed to the inserted (membrane pore) state is cooperative and thus occurs rather abruptly upon a change in ambient conditions.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.     

Many-body effect of antimicrobial peptides: on the correlation between lipid's spontaneous curvature and pore formation

Recently we have shown that the free energy for pore formation induced by antimicrobial peptides contains a term representing peptide-peptide interactions mediated by membrane thinning. This many-body effect gives rise to the cooperative concentration dependence of peptide activities. Here we performed oriented circular dichroism and x-ray diffraction experiments to study the lipid dependence of this many-body effect. In particular we studied the correlation between lipid's spontaneous curvature and peptide's threshold concentration for pore formation by adding phosphatidylethanolamine and lysophosphocholine to phosphocholine bilayers. Previously it was argued that this correlation exhibited by magainin and melittin supported the toroidal model for the pores. Here we found similar correlations exhibited by melittin and alamethicin. We found that the main effect of varying the spontaneous curvature of lipid is to change the degree of membrane thinning, which in turn influences the threshold concentration for pore formation. We discuss how to interpret the lipid dependence of membrane thinning.     

A lipocentric view of peptide-induced pores

Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, nonmediated leakage is not completely improbable. A high activation energy normally keeps unassisted bilayer permeation at a very low frequency, but lipids are able to self-organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions such as phase coexistence, external stress or perturbation associated to binding of nonlipidic molecules. Here, we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation of peptides, lipidic pores induced by different types of stress, and spontaneous transient bilayer defects driven by thermal fluctuations. Within such a lipocentric framework, amphipathic peptides are best described as pore-inducing rather than pore-forming elements. Active peptides bound to membranes can be understood as a source of internal surface tension which facilitates pore formation by diminishing the high activation energy barrier. This first or immediate action of the peptide has some resemblance to catalysis. However, the presence of membrane-active peptides has the additional effect of displacing the equilibrium towards the pore-open state, which is then maintained over long times, and reducing the size of initial individual pores. Thus, pore-inducing peptides, regardless of their sequence and oligomeric organization, can be assigned a double role of increasing the probability of pore formation in membranes to high levels as well as stabilizing these pores after they appear.     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

A synthetic peptide forms voltage-gated porin-like ion channels in lipid bilayer membranes

Design of simple protein structures represents the essential first step toward novel macromolecules and understanding the basic principles of protein folding. Our work focuses on the ion channel formation and structure of peptides having a repeated pattern of glycine residues. Investigation of the ion channel properties of a glycine repeat peptide, VSLGLSIGFSVGVSIGWSFGRSRG revealed the formation of porin-like high conductance, multimeric, non-selective voltage-gated channels in phospholipid bilayer membranes. ATR-IR and CD spectroscopic studies showed an anti-parallel beta sheet structure in membranes. The formation of porin-like ion channels by a beta sheet peptide suggests spontaneous assembly into a beta barrel structure through oligomerization as in pore forming bacterial toxins. The present work is the first example of a short synthetic peptide mimicking the pore characteristics of a complex beta barrel protein and demonstrates that smaller peptides are capable of mimicking the complex functional properties of natural ion channels. This will have implications in understanding the folding of beta sheet proteins in membranes, the mechanism of two state voltage gating, and the role of glycine residues in beta barrel proteins.