Pore-forming Peptide Research Articles

Hybrid bilayer membranes on metallurgical polished aluminum

In this work we describe the functionalization of metallurgically polished aluminum surfaces yielding biomimetic electrodes suitable for probing protein/phospholipid interactions. The functionalization involves two simple steps: silanization of the aluminum and subsequent fusion of multilamellar vesicles which leads to the formation of a hybrid bilayer lipid membrane (hBLM). The vesicle fusion was followed in real-time by fast Fourier transform electrochemical impedance spectroscopy (FFT EIS). The impedance-derived complex capacitance of the hBLMs was approximately 0.61 µF cm−2, a value typical for intact phospholipid bilayers. We found that the hBLMs can be readily disrupted if exposed to > 400 nM solutions of the pore-forming peptide melittin. However, the presence of cholesterol at 40% (mol) in hBLMs exhibited an inhibitory effect on the membrane-damaging capacity of the peptide. The melittin-membrane interaction was concentration dependent decreasing with concentration. The hBLMs on Al surface can be regenerated multiple times, retaining their dielectric and functional properties essentially intact.

Membrane perturbation of fullerene and graphene oxide distinguished by pore-forming peptide melittin

Carbon nanomaterials such as fullerenes (C60) and graphene oxide (GO) are considered as promising candidates for diverse applications in biotechnology and biomedicine. However, their potential toxic effects are still under debate. Herein, by using melittin (Mel), a representative pore-forming peptide, as a testing molecule we demonstrated that even the low-concentrated (usually assumed non-toxic) C60 and GO could still mechanically perturb a cell membrane by adsorption and insertion, and consequently influence the function realization of membrane active proteins/peptides. Such perturbations, however, are particle-property and membrane-environment dependent. GO would sensitize both model bilayers and bacterial membranes to Mel, demonstrated as significantly enhanced membrane permeabilization ability or improved antibacterial performance of Mel. In contrast, C60 activates the permeabilization effect of Mel on model membranes, while produces exactly the reverse effect on living bacteria and mammalian cells. Simulations further provide molecular details of the structural disturbance and probe the residue-specific formation of C60-Mel complex in membrane. This work emphasizes the dependence of biological toxicity of nanomaterials on their physico-chemical properties, provides a facile method to detect the subtle structural perturbation of cell membranes at nanoscale, and suggests a necessity for a careful evaluation of the potential influences of nanomaterials on biological processes.

Tuning of a Membrane-Perforating Antimicrobial Peptide to Selectively Target Membranes of Different Lipid Composition.

The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure-function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore-forming peptide into a selective pore-former for different lipid types. A library of 2916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization.

Interplay between Leakage and Fusion of Phosphatidylcholine Liposomes Induced by the Macrolittins, a Synthetically Evolved Family of Pore-Forming Peptides

Peptides that form pores in lipid bilayer membranes can be used in a variety of biotechnological and clinical applications including drug delivery and targeted cancer therapy. According to the results from our previous peptide library screening, novel peptides called “macrolittins” were discovered that release macromolecules from phosphatidylcholine vesicles at low concentration by forming large pores. Here we explore how the uniquely potent functions of an example macrolittin, M159, depend on bilayer properties.

Cytosolic Delivery of Proteins

In medicine, current methods to target intracellular proteins have largely been limited to small molecule drugs as they can freely diffuse across the plasma membrane. However, their lack of specificity and inability to target large protein-protein interfaces are limitations. Proteins, such as antibodies, overcome these limitations but they are too large to diffuse into the cell. Intracellular targets such as RAS and MYC are therefore deemed “ungruggable”. A method to deliver proteins into live cells in their intact form could therefore prove very useful in research and medicine. We present work done on pHD108, a pH-sensitive pore-forming peptide. Through libraries and screens and synthetic molecular evolution, we imparted the parent peptide melittin with the ability to be potent (activity at 1:1000 peptide to lipid ratio), triggered by low-pH only (pH < 6), and form macromolecule-sized pores (∼8.2 nm radii in POPC bilayers). We sought to harness these unique properties for protein delivery into cells. Since endosomes become acidic (pH ∼6), we hypothesized that cells endocytosing protein drug as well as pHD108 peptide will allow the peptide to become active and form pores in the endosome, thus releasing the protein drug into the cytosol. We show evidence supporting this through a novel apoptosis assay based on P. aueruginosa exotoxin a domain III (PE-III, 37 kDA). PE-III is only cytotoxic when delivered into the cytoplasm else it gets degraded in the lysosome. With D-pHD108 treatment, we see apoptosis, indicating endosomolytic activity. We also see that pores in cells are large enough for IgGs to pass through. Modifications to the peptide has revealed an importance in membrane binding as anchoring to membranes through an acyl tail can increase activity. Together, this shows pHD108 is functional for this application but a roadmap for further optimizations should be followed as well.

PH-triggered pore-forming peptides with strong composition-dependent membrane selectivity

Peptides that self-assemble into nanometer-sized pores in lipid bilayers could have utility in a variety of biotechnological and clinical applications if we can understand their physical chemical properties and learn to control their membrane selectivity. To empower such control, we have used synthetic molecular evolution to identify the pH-dependent delivery peptides, a family of peptides that assemble into macromolecule-sized pores in membranes at low peptide concentration but only at pH < ∼6. Further advancements will also require better selectivity for specific membranes. Here, we determine the effect of anionic headgroups and bilayer thickness on the mechanism of action of the pH-dependent delivery peptides by measuring binding, secondary structure, and macromolecular poration. The peptide pHD15 partitions and folds equally well into zwitterionic and anionic membranes but is less potent at pore formation in phosphatidylserine-containing membranes. The peptide also binds and folds similarly in membranes of various thicknesses, but its ability to release macromolecules changes dramatically. It causes potent macromolecular poration in vesicles made from phosphatidylcholine with 14 carbon acyl chains, but macromolecular poration decreases sharply with increasing bilayer thickness and does not occur at any peptide concentration in fluid bilayers made from phosphatidylcholine lipids with 20-carbon acyl chains. The effects of headgroup and bilayer thickness on macromolecular poration cannot be accounted for by the amount of peptide bound but instead reflect an inherent selectivity of the peptide for inserting into the membrane-spanning pore state. Molecular dynamics simulations suggest that the effect of thickness is due to hydrophobic match/mismatch between the membrane-spanning peptide and the bilayer hydrocarbon. This remarkable degree of selectivity based on headgroup and especially bilayer thickness is unusual and suggests ways that pore-forming peptides with exquisite selectivity for specific membranes can be designed or evolved.

Time-resolved SANS reveals pore-forming peptides cause rapid lipid reorganization

Time-resolved SANS showed alamethicin and melittin promote DMPC lipid vesicle mixing and perturb DMPC kinetics in similar ways.

Calcium-Dependent ESCRT Recruitment and Lysosome Exocytosis Maintain Epithelial Integrity During &lt;i&gt;Candida albicans&lt;/i&gt; Invasion

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identified two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induced oscillatory increases in cytosolic [Ca2+], which caused hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process, and repair plasmalemmal tears induced mechanically by invading hyphae by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Molecular Tetrahedrons as Selective and Efficient Ion Transporters via a Two-Station Swing-Relay Mechanism

The traditional approach to utilizing an ion-relay mechanism for ion transport requires three or more ion-relay stations. Herein, we describe a novel strategy, incorporating a swing action to reali...

Macromolecular Crowding Affects Voltage-Dependent Alamethicin Pore Formation in Lipid Bilayer Membranes.

Macromolecular crowding is known to modulate chemical equilibria, reaction rates, and molecular binding events, both in aqueous solutions and at lipid bilayer membranes, natural barriers that enclose the crowded environments of cells and their subcellular compartments. Previous studies on the effects of macromolecular crowding in aqueous compartments on conduction through membranes have focused on single-channel ionic conduction through previously formed pores at thermodynamic equilibrium. Here, the effects of macromolecular crowding on the mechanism of pore formation itself were studied using the droplet interface bilayer (DIB) technique with the voltage-dependent pore-forming peptide alamethicin (alm). Macromolecular crowding was varied using 8 kDa molecular weight polyethylene glycol (PEG8k) or 500 kDa dextran (DEX500k) in two aqueous droplets on both sides of the bilayer membrane. In general, voltage thresholds for pore formation in the presence of crowders in the droplets decreased compared to their values in the absence of crowders, due to excluded volume effects, water binding by PEG, and changes in the ordering of water molecules and hydrogen-bonding interactions involving the polar lipid headgroups. In addition, asymmetric crowder loading (e.g., PEG8k-DEX500k on either side of the membrane) resulted in transmembrane osmotic pressure gradients that either enhanced or degraded the ionic conduction through the pores.

Rational Substitution of ε-Lysine for α-Lysine Enhances the Cell and Membrane Selectivity of Pore-Forming Melittin.

Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, ε-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with ε-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three ε-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept in vivo experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by ε-lysylation.

Effect of helical kink in antimicrobial peptides on membrane pore formation.

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.

What Makes a Good Pore Former: A Study of Synthetic Melittin Derivatives

Pore formation by membrane-active peptides, naturally encountered in innate immunity and infection, could have important medical and technological applications. Recently, the well-studied lytic peptide melittin has formed the basis for the development of combinatorial libraries from which potent pore-forming peptides have been derived, optimized to work under different conditions. We investigate three such peptides, macrolittin70, which is most active at neutral pH; pHD15, which is active only at low pH; and MelP5_Δ6, which was rationally designed to be active at low pH but formed only small pores. There are three, six, and six acidic residues in macrolittin70, pHD15, and MelP5_Δ6, respectively. We perform multi-microsecond simulations in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of hexamers of these peptides starting from transmembrane orientations at neutral pH (all residues at standard protonation), low pH (acidic residues and His protonated), and highly acidic environments in which C-termini are also protonated. Previous simulations of the parent peptides melittin and MelP5 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are repeated in POPC. We find that the most potent pore-forming peptides exhibit strong interpeptide interactions, including salt bridges, H-bonds, and polar interactions. Protonation of the C-terminus promotes helicity and pore size. The proximity of the peptides allows fewer lipid headgroups to line the pores than in previous simulations, making the pores intermediate between barrel stave and toroidal. Based on these structures and geometrical arguments, we attempt to rationalize the factors that under different conditions can increase or decrease pore stability and propose mutations that could be tested experimentally.

Emergence by Design in Self-Assembling Protein Shells.

The use of proteins and peptides as nanoscale components to generate new-to-nature physical entities holds great promise in biocatalysis, therapeutic or diagnostic delivery, and materials templating. The majority of functionalized particles have been based on existing structures found in nature. Developing biomimetic particles in this way takes advantage of highly evolved platforms for organization or encapsulation of functional moieties, offering significant advantages in stoichiometry, multivalency, and sequestration. However, novel assembly paradigms for the modular construction of macromolecular structures are now greatly expanding the functional diversity of protein-based nanoparticles in health and manufacturing. In the February issue of ACS Nano, Kepiro et al. demonstrate the refinement of this concept, engineering the capacity for self-assembly such that it is integral to pore-forming peptide motifs, resulting in superior antibiotic activity of the self-assembled particle. Nature encodes multiple functions in proteins with exquisite efficiency, and emulating this multiplicity may be the ultimate goal of biomimetic nanotechnologies.

Construction of Programmable Nanopore using de novo Designed β-Sheet Peptide

Nanopore structures have been employed in biosensing technologies such as single molecule detection and DNA sequencing. However, it is difficult to adjust the size and shape of nanopores for each target molecule using natural proteins. To construct programmable nanopore, de novo design method, which is able to construct artificial proteins/peptides structure accurately, will be promising. Recently, the de novo design technics have been developed, but there are few clues to design pore-forming proteins in the bilayer lipid membrane (BLM). Therefore, we attempted to design pore-forming peptides, which assemble to construct nanopore, because the sequence space of amino acids is less than proteins. Previously, we designed pore-forming α-helical peptides, but the stability of these nanopores was not enough to use nanopore sensing. This paper describes de novo design of β-sheet peptides to construct a nanopore in BLM. The β-sheet peptides are possible to construct more stable nanopore because of the hydrogen bonds between the β-sheet structures. We designed the peptide using four strategies. First, we adopted a β-hairpin structure as a minimum motif of β-sheet structure. Second, the amphiphilic regions coincided with the transmembrane regions. Third, π-π stacking would stabilize the nanopore structure. Last, charged residues would decide the peptide orientations by applying voltage. To prevent from aggregating, a precursor was synthesized using isoacyl dipeptide. As results, the circular dichroism spectrum and solid-state NMR confirmed the peptide has the β-hairpin structure in BLM. The channel current measurements revealed the pore formations of the peptide using the analysis of the shape of channel current signals. Furthermore, the peptide mostly displayed the pentamer pore structures, and we confirmed the pore stability using molecular dynamics simulation. For the next, we will control the pore size by optimization of amino acid sequences.

Secretion of Pore-Forming Peptide Toxin, Tolaasin, by Ptα Type Strains of Pseudomonas Tolaasii, but not by Ptβ Type Strains

Secretion of Pore-Forming Peptide Toxin, Tolaasin, by Ptα Type Strains of Pseudomonas Tolaasii, but not by Ptβ Type Strains

A Novel Strategy for Enhance Potentiation of Meglumine antimo-niate against Leishmania major In Vitro

We aimed to design a different method of drug delivery for increased transfer of the choice drug (meglumine antimoniate) within the host cells. Therefore, listeriolysin O (LLO), a bacterial product which is a member of pore-forming peptides was used as an enhancer factor with meglumine antimoniate in order to facilitate the transition of the drug across macrophage membrane. LLO was produced in Isfahan University of Medical Sciences in 2016, by expressing the hlyA gene in Escherichia coli and purified using affinity chromatography. Cytotoxicity of the purified protein was investigated in an in vitro model of macrophage Leishmania infection. LLO was cytotoxic against murine macrophage cells (J774-A1) and amastigote forms of L. major (MRHO/IR/75/ER). It was less toxic to macrophages (CC50=2.56 μg ml-1 ±0.09) than to the parasites (IC50=1.72 μg ml-1 ±0.07). Moreover, noncytotoxic concentration of LLO (0.006 ug ml-1) potentiated the cytotoxicity induced by low dose concentration of meglumine antimoniate. Very little dose of meglumine antimoniate was needed when combined with the LLO (IC50=12.63 μg ml-1 ±0.13) in comparison with the cytotoxicity induced when the drug is used alone (IC50=46.17 μg ml-1 ±0.28). The combination of pore-forming proteins with anti-leishmanial agents could increase the advantage of anti-leishmanial drugs. Since lower concentrations of anti-leishmanial drugs can reduce undesirable side effects of chemotherapy trials carried out in animal models and then in humans with this system.

How Do the Properties of Amphiphilic Polymer Membranes Influence the Functional Insertion of Peptide Pores?

Pore-forming peptides are of high biological relevance particularly as cytotoxic agents, but their properties are also applicable for the permeabilization of lipid membranes for biotechnological applications, which can then be translated to the more stable and versatile polymeric membranes. However, their interactions with synthetic membranes leading to pore formation are still poorly understood, hampering the development of peptide-based nanotechnological applications, such as biosensors or catalytic compartments. To elucidate these interactions, we chose the model peptide melittin, the main component of bee venom. Here, we present our systematic investigation on how melittin interacts with and inserts into synthetic membranes, based on amphiphilic block copolymers, to induce pore formation in three different setups (planar membranes and micrometric and nanometric vesicles). By varying selected molecular properties of block copolymers and resulting membranes (e.g., hydrophilic to hydrophobic block ratio, membrane thickness, surface roughness, and membrane curvature) and the stage of melittin addition to the synthetic membranes, we gained a deeper understanding of melittin insertion requirements. In the case of solid-supported planar membranes, melittin interaction was favored by membrane roughness and thickness, but its insertion and pore formation were hindered when the membrane was excessively thick. The additional property provided by micrometric vesicles, curvature, increased the functional insertion of melittin, which was evidenced by the even more curved nanometric vesicles. Using nanometric vesicles allowed us to estimate the pore size and density, and by changing the stage of melittin addition, we overcame the limitations of peptide-polymer membrane interaction. Mirroring the functionality assay of planar membranes, we produced glucose-sensing vesicles. The design of synthetic membranes permeabilized with melittin opens a new path toward the development of biosensors and catalytic compartments based on pore-forming peptides functionally inserted in synthetic planar or three-dimensional membranes.

Cultured bacteria hypermodify peptides

Polytheonamides are extensively modified peptides produced by bacteria in the microbiomes of marine sponges. These pore-forming peptides are toxic to cells, which makes them intriguing as possible cancer drugs. A major problem is that the bacteria that make the peptides can’t be cultured. Now, Jorn Piel of the Swiss Federal Institute of Technology (ETH), Zurich, and coworkers have identified a cluster of enzymes in another bacterium that produces similarly hypermodified peptides (Nat. Chem. 2019, DOI: 10.1038/s41557-019-0323-9). And unlike the bacteria from the marine sponge, Microvirgula aerodenitrificans can be grown in cell culture. M. aerodenitrificans produces its own class of modified peptides called aeronamides. The peptides are ribosomally synthesized with leader and core portions. The enzyme cluster recognizes the leader portion and modifies the core. The modifications include iterative epimerization to introduce 21 d-amino acids, methylation of various carbon and nitrogen atoms in the peptide, a...

Targeting specific membranes with an azide derivative of the pore-forming peptide ceratotoxin A

Pore-forming antimicrobial peptides (AMPs) are attracting interest as cytolytic antibiotics and drug delivery agents with potential use for targeting cancer cells or multidrug-resistant pathogens. Ceratotoxin A (CtxA) is an insect-derived cytolytic AMP with 36 amino acids that is thought to protect the eggs of the medfly Ceratitis capitata against pathogens. Single channel recordings using planar lipid bilayers have shown that CtxA forms pores with well-defined conductance states resembling those of alamethicin; it also forms one of the largest pores among the group of ceratotoxins. In this work, we modified CtxA at its N-terminus with an azide group and investigated its pore-forming characteristics in planar lipid bilayer experiments. We demonstrate the possibility to target specific lipids by carrying out click reactions in-situ on lipid membranes that display a dibenzocyclooctyne (DBCO) moiety on their head group. As a result of covalent linkage of the peptides to the bilayer, pore-formation occurs at 10-fold reduced peptide concentration and with a reduced dependence on the transmembrane voltage compared to unlinked CtxA-azide peptides or native CtxA peptides.