Peptide-induced Pore Research Articles

Free Energy Analysis of Peptide-Induced Pore Formation in Lipid Membranes by Bridging Atomistic and Coarse-Grained Simulations.

Antimicrobial peptides (AMPs) are attractive materials for combating the antimicrobial resistance crisis because they can kill target microbes by directly disrupting cell membranes. Although thousands of AMPs have been discovered, their molecular mechanisms of action are still poorly understood. One broad mechanism for membrane disruption is the formation of membrane-spanning hydrophilic pores which can be stabilized by AMPs. In this study, we use molecular dynamics simulations to investigate the thermodynamics of pore formation in model single-component lipid membranes in the presence of one of three AMPs: aurein 1.2, melittin and magainin 2. To overcome the general challenge of modeling long time scale membrane-related behaviors, including AMP binding, clustering, and pore formation, we develop a generalizable methodology for sampling AMP-induced pore formation. This approach involves the long equilibration of peptides around a pore created with a nucleation collective variable by performing coarse-grained simulations, then backmapping equilibrated AMP-membrane configurations to all-atom resolution. We then perform all-atom simulations to resolve free energy profiles for pore formation while accurately modeling the interplay of lipid-peptide-solvent interactions that dictate pore formation free energies. Using this approach, we quantify free energy barriers for pore formation without direct biases on peptides or whole lipids, allowing us to investigate mechanisms of pore formation for these 3 AMPs that are a consequence of unbiased peptide diffusion and clustering. Further analysis of simulation trajectories then relates variations in pore lining by AMPs, AMP-induced lipid disruptions, and salt bridges between AMPs to the observed pore formation free energies and corresponding mechanisms. This methodology and mechanistic analysis have the potential to generalize beyond the AMPs in this study to improve our understanding of pore formation by AMPs and related antimicrobial materials.

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 hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic \u03b1-helical peptides

We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.

Location of Peptide-Induced Submicron Discontinuities in the Membranes of Vesicles Using ImageJ.

Cell penetrating peptide transportan 10 and antimicrobial peptide melittin formed submicron pores in the lipid membranes of vesicles which are explained by the leakage of water-soluble fluorescent probes from the inside of vesicles to the outside. It is hypothesized that these submicron pores induce submicron discontinuities in the membranes. Considering this hypothesis, a technique has developed to locate the submicron discontinuities in the membranes of giant unilamellar vesicles (GUVs) using ImageJ. In this technique, at first the edges of membrane of a 'single GUV' are detected and then these edges are used to locate the submicron discontinuities. Two continuous rings are observed after applying the ImageJ in GUVs which indicated the edges of membrane. In contrast, the submicron discontinuations are detected at the edges of transportan 10 and melittin induced pore formed membranes. This investigation might be helpful for the elucidation of mechanism of the peptide-induced pore formation in the membranes of vesicles.

Quantitative accounting of dye leakage and photobleaching in single lipid vesicle measurements: Implications for biomacromolecular interaction analysis

Highly parallel measurements on single, tethered lipid vesicles enable real-time monitoring of dynamic membrane interactions of relevance to medical, pharmaceutical, and biotechnological applications. Monitoring the time-dependent release of entrapped fluorescent dyes is a popular measurement approach, although it is often challenging to accurately extract quantitative biochemical parameters. Key issues include dye leakage and imaging-related photobleaching, and corrective measures are needed. Herein, we present an extended analytical framework to collect and interpret time-lapsed fluorescence microscopy imaging data, and demonstrate its utility for tracking membrane-peptide interactions. Our approach is focused on improving platform design and data analysis. First, we identified suitable membrane compositions to minimize dye leakage while enhancing the biomimetic character of lipid vesicles. Second, a data normalization procedure was developed to correct for experimental artifacts, namely dye leakage and photobleaching, and hence improve measurement accuracy. This analytical procedure was applied to experimentally determine the rate of peptide-induced pore formation in single lipid vesicles, and there was up to a nearly three-fold decrease in the measured rate, as compared to uncorrected data. Taken together, the results present a broadly applicable analytical framework to account for experimental artifacts and improve measurement accuracy in highly parallel, single lipid vesicle arrays.

Effect of lipid composition and amino acid sequence upon transmembrane peptide-accelerated lipid transleaflet diffusion (flip-flop)

We examined how hydrophobic peptide-accelerated transleaflet lipid movement (flip-flop) was affected by peptide sequence and vesicle composition and properties. A peptide with a completely hydrophobic sequence had little if any effect upon flip-flop. While peptides with a somewhat less hydrophobic sequence accelerated flip-flop, the half-time remained slow (hours) with substantial (0.5mol%) peptide in the membranes. It appears that peptide-accelerated lipid flip-flop involves a rare event that may reflect a rare state of the peptide or lipid bilayer. There was no simple relationship between peptide overall hydrophobicity and flip-flop. In addition, flip-flop was not closely linked to whether the peptides were in a transmembrane or non-transmembrane (interfacial) inserted state. Flip-flop was also not associated with peptide-induced pore formation. We found that peptide-accelerated flip-flop is initially faster in small (highly curved) unilamellar vesicles relative to that in large unilamellar vesicles. Peptide-accelerated flip-flop was also affected by lipid composition, being slowed in vesicles with thick bilayers or those containing 30% cholesterol. Interestingly, these factors also slow spontaneous lipid flip-flop in the absence of peptide. Combined with previous studies, the results are most consistent with acceleration of lipid flip-flop by peptide-induced thinning of bilayer width.

Deciphering How Pore Formation Causes Strain-Induced Membrane Lysis of Lipid Vesicles.

Pore formation by membrane-active antimicrobial peptides is a classic strategy of pathogen inactivation through disruption of membrane biochemical gradients. It remains unknown why some membrane-active peptides also inhibit enveloped viruses, which do not depend on biochemical gradients. Here, we employ a label-free biosensing approach based on simultaneous quartz crystal microbalance-dissipation and ellipsometry measurements in order to investigate how a pore-forming, virucidal peptide destabilizes lipid vesicles in a surface-based experimental configuration. A key advantage of the approach is that it enables direct kinetic measurement of the surface-bound peptide-to-lipid (P:L) ratio. Comprehensive experiments involving different bulk peptide concentrations and biologically relevant membrane compositions support a unified model that membrane lysis occurs at or above a critical P:L ratio, which is at least several-fold greater than the value corresponding to the onset of pore formation. That is consistent with peptide-induced pores causing additional membrane strain that leads to lysis of highly curved membranes. Collectively, the work presents a new model that describes how peptide-induced pores may destabilize lipid membranes through a membrane strain-related lytic process, and this knowledge has important implications for the design and application of membrane-active peptides.

Designing α-helical peptides with enhanced synergism and selectivity against Mycobacterium smegmatis: Discerning the role of hydrophobicity and helicity

Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant Mycobacterium tuberculosis. In this study, the effect of key physicochemical parameters including hydrophobicity and helicity of α-helical peptides on anti-mycobacterial activity and synergism with rifampicin was investigated. The most hydrophobic analogue, W(LLKK)2W, displayed low selectivity against mycobacteria while peptides with intermediate hydrophobicity were shown to be equally active, yet significantly less toxic. Furthermore, proline substitution impeded the formation of stable amphipathic structures, rendering P(LLKK)2P as one of the least active analogues. Terminal capping with isoleucine was found to promote α-helical folding and the resultant peptide demonstrated the highest selectivity and minimal cytotoxicity against mammalian macrophages. Flow cytometric analysis revealed that enhancements in hydrophobicity and α-helicity increased the rate and extent of peptide-mediated membrane permeabilization. This finding corroborated the hypothesis that synergism between the peptides and rifampicin was likely mediated via peptide-induced pore formation. The rapid, concentration-dependent membrane depolarization, leakage of intracellular ATP and calcein release from PE/PG LUVs supported the membrane-lytic mechanism of action of the peptides. Together, these findings suggest that hydrophobicity and α-helicity significantly impact anti-mycobacterial activity and optimization of both parameters is necessary to develop synthetic analogues with superior selectivity indices and enhanced synergistic potential with conventional antibiotics. Statement of significanceThere is an urgent clinical need for the discovery of new antimicrobials, effective not just for drug susceptible, but also rapidly emerging drug-resistant TB. Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant M. tuberculosis. In this study, we evaluated a series of synthetic α-helical (LLKK)2 peptides over a range of hydrophobicities for their activity against mycobacteria and provide the first report on the modulating effect of hydrophobicity and α-helicity on the antimicrobial mechanisms of synthetic AMPs and their synergism with first-line antibiotics. These findings demonstrate the applicability of strategies employed here for the rational design of AMPs with the aim of improving cell selectivity and synergistic interactions when co-administered with first-line antibiotics in the fight against drug-resistant tuberculosis.

Interaction of amphipathic [formula omitted]-helical peptides with a lipid membrane: Adsorption and pore formation

Amphipathic α-helical peptides often exhibit antimicrobial or antiviral properties. Adsorption of such peptides at a lipid membrane may result in pore formation. Current phenomenological models of the latter process imply that the peptide–peptide lateral interaction is repulsive and that the conditions for pore formation depend on the difference of the peptide energies at the membrane surface and in a pore. There is, however, experimental evidence that the kinetics of peptide adsorption at small vesicles (about 100 nm diameter) may be cooperative and accordingly the peptide–peptide lateral interaction may be attractive. In addition, the experiments indicate that the peptide-induced pore formation is often observed at the conditions close to those corresponding to pore formation under externally induced tensile stress where the difference of the peptide energies at the membrane surface and in a pore is irrelevant. Here, a model describing both types of peptide–peptide lateral interactions at a membrane is proposed. In addition, a new scenario of peptide-induced pore formation naturally explaining the similarity of this process under different conditions is suggested.

Energetic View on Membrane Pore Formation

Energetic View on Membrane Pore Formation

Nucleation in mesoscopic systems under transient conditions: Peptide-induced pore formation in vesicles

Attachment of lytic peptides to the lipid membrane of virions or bacteria is often accompanied by their aggregation and pore formation, resulting eventually in membrane rupture and pathogen neutralization. The membrane rupture may occur gradually via formation of many pores or abruptly after the formation of the first pore. In academic studies, this process is observed during interaction of peptides with lipid vesicles. We present an analytical model and the corresponding Monte Carlo simulations focused on the pore formation in such situations. Specifically, we calculate the time of the first nucleation-limited pore-formation event and show the distribution of this time in the regime when the fluctuations of the number of peptides attached to a vesicle are appreciable. The results obtained are used to clarify the mechanism of the pore formation and membrane destabilization observed recently during interaction of highly active α-helical peptide with sub-100-nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature. The model proposed and the analysis presented are generic and may be applicable to other meso- and nanosystems.

Modeling peptide binding to anionic membrane pores

Peptide-induced pore formation in membranes can be dissected into two steps: pore formation and peptide binding to the pore. A computational method is proposed to study the second step in anionic membranes. The electrostatic potential is obtained from numerical solutions to the Poisson-Boltzmann equation and is then used in conjunction with IMM1 (implicit membrane model 1). A double charge layer model is used to incorporate the effects of the membrane dipole potential. Inhomogeneity of the charge density in the pore, characterized by explicit membrane simulations of toroidal pores, is included in the model. This approach was applied to two extensively studied peptides, magainin and melittin. In agreement with previous work, binding to toroidal pores is more favorable than binding to the flat membrane. The dependence of binding energy on anionic content exhibits different patterns for the two peptides, in correlation with the different lipid selectivity that has been observed experimentally.

The Lipid Dependence of Antimicrobial Peptide Activity Is an Unreliable Experimental Test for Different Pore Models

Antimicrobial peptides usually kill bacteria by making their membranes permeable. Two main models (barrel-stave and Shai-Matsuzaki-Huang) have been proposed to describe the peptide-induced pores. Although several experimental tests can be exploited to discriminate between these two models, the dependence of peptide activity on lipid properties (intrinsic curvature and membrane thickness) is routinely used for this purpose. Here, we show that, contrary to what is currently accepted, this criterion is unreliable.

Mechanisms of Peptide-Induced Pore Formation in Lipid Bilayers Investigated by Oriented 31P Solid-State NMR Spectroscopy

There is a considerable interest in understanding the function of antimicrobial peptides (AMPs), but the details of their mode of action is not fully understood. This motivates extensive efforts in determining structural and mechanistic parameters for AMP’s interaction with lipid membranes. In this study we show that oriented-sample 31P solid-state NMR spectroscopy can be used to probe the membrane perturbations and -disruption by AMPs. For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring. These lipids display reduced dynamics. Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.

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.

The Single GUV Method Reveals the Antimicrobial Peptide-induced Pore Formation in Lipid Membranes

The Single GUV Method Reveals the Antimicrobial Peptide-induced Pore Formation in Lipid Membranes

Direct Visualization of Antibiotic-induced Pores in Phospholipid Vesicles by Cryo Electron Microscope

Cytolytic peptides, such as Magainin, Melittin, and Alamethicin are ubiquitously present within the animal kingdom as a part of the host-defense system. Magainin-2 lyses a wide range of both gram-negative and gram-positive bacteria and a range of cancer cells. Unlike most commercial antibiotics, which interact with specific protein targets, Magainin 2 and other peptides in this class have been shown to interact directly with the lipid bilayer; therefore, it is believed that bacteria will be largely unable to develop resistance to this class of antibiotics. It is believed to initially interact with acidic lipids in the bacterial membranes through electrostatic interactions, forming an amphiphillic helix, followed by hydrophobic interactions inducing pore formation, but the issue remains controversial. A number of methods have been used to study the structure of possible pores; however, none of those methods could directly observe the pores themselves. We present a new method for studying peptide/lipid interactions, which employs cryo-EM to directly image Magainin-induced pores in phospholipid vesicles. Images of DMPC/DMPG lipid vesicles with Magainin showed both perturbed and unperturbed vesicles, while vesicles without Magainin were unperturbed: perturbed vesicles exhibited power spectra similar to neutron scattering experiments in the presence of Magainin. To estimate pore size, we completed a set of simulations with randomly distributed pores on spherical vesicles. The mean pore size obtained by simulation was ∼83A, which is compatible with prior neutron scattering data. In addition, since the vesicle images are projections, we performed cryo-electron tomography experiments to reconstruct the 3-D structure of the pores. For the first time, we were able to visualize antibiotic peptide-induced pores on phospholipids vesicles, and the pore size is consistent with the simulation result.

Single Molecule Studies of the Interactions between β-Amyloid 1-40 and Supported Planar Lipid Membrane

Recent evidence supports the hypothesis that the early oligomers formed by the β-Amyloid peptide are cytotoxic and may feature in Alzheimer's disease (AD). While the mechanism of this cytotoxicity remains unclear, interactions of these oligomers with neuronal membrane are believed to be involved. Identifying the cytotoxic species is challenging because the β-Amyloid oligomers are extremely heterogeneous, metastable, and form at very low physiological concentrations (nM). In our study, we use single molecule spectroscopy (SMS) to study the interactions between β-Amyloid 1-40 and supported planar lipid membranes. The evolution of β-Amyloid species on lipid membranes were monitored for up to a few days. The results indicate that the interactions between β-Amyloid 1-40 and planar lipid membranes follow three stages. First, a very small fraction of β-Amyloid peptide binds to the membranes with high affinity (Kd < 470pM), covering the membrane surface uniformly and also diffusing within the lipid molecules. In the second stage, β-Amyloid peptides assemble to form oligomers in the membrane. We observed at least two different pathways of oligomer formation, depending on the aqueous β-Amyloid peptide concentration. In the final stage, after prolonged incubation with the lipid membranes, β-Amyloid peptides start forming mesh-like deposits. With the high sensitivity and spatial and temporal resolution of single molecule spectroscopy, we successfully traced the interactions between β-Amyloid 1-40 peptide and planar lipid membranes at the molecular level. Our results are also in agreement with the molecular model of pore-forming peptides suggested by H.W. Huang et al. (1).1. Huang, H.W., F.Y. Chen, and M.T. Lee. 2004. Molecular Mechanism of Peptide-Induced Pores in Membranes. Physical Review Letters. 92:198304.

Structure of transmembrane pore induced by Bax-derived peptide: Evidence for lipidic pores

The structures of transmembrane pores formed by a large family of pore-forming proteins and peptides are unknown. These proteins, whose secondary structures are predominantly alpha-helical segments, and many peptides form pores in membranes without a crystallizable protein assembly, contrary to the family of beta-pore-forming proteins, which form crystallizable beta-barrel pores. Nevertheless, a protein-induced pore in membranes is commonly assumed to be a protein channel. Here, we show a type of peptide-induced pore that is not framed by a peptide structure. Peptide-induced pores in multiple bilayers were long-range correlated into a periodically ordered lattice and analyzed by X-ray diffraction. We found the pores induced by Bax-derived helical peptides were at least partially framed by a lipid monolayer. Evidence suggests that the formation of such lipidic pores is a major mechanism for alpha-pore-forming proteins, including apoptosis-regulator Bax.

Coarse-Grained Simulation Studies of Peptide-Induced Pore Formation

We investigate the interactions between lipid bilayers and amphiphilic peptides using a solvent-free coarse-grained simulation technique. In our model, each lipid is represented by one hydrophilic and three hydrophobic beads. The amphiphilic peptide is modeled as a hydrophobic-hydrophilic cylinder with hydrophilic caps. We find that with increasing peptide-lipid attraction the preferred state of the peptide changes from desorbed, to adsorbed, to inserted. A single peptide with weak attraction binds on the bilayer surface, while one with strong attraction spontaneously inserts into the bilayer. We show how several peptides, which individually bind only to the bilayer surface, cooperatively insert. Furthermore, hydrophilic strips along the peptide cylinder induce the formation of multipeptide pores, whose size and morphology depend on the peptides’ overall hydrophilicity, the distribution of hydrophilic residues, and the peptide-peptide interactions. Strongly hydrophilic peptides insert less readily, but prove to be more destructive to bilayer integrity.