Melittin, a honeybee venom­derived antimicrobial peptide, may target methicillin­resistant Staphylococcus aureus.

Methicillin­resistant Staphylococcus aureus (MRSA) is difficult to treat using available antibiotic agents. Honeybee venom has been widely used as an oriental treatment for several inflammatory diseases and bacterial infections. The venom contains predominantly biologically active compounds, however, the therapeutic effects of such materials when used to treat MRSA infections have not been investigated extensively. The present study evaluated bee venom and its principal active component, melittin, in terms of their antibacterial activities and in vivo protection against MRSA infections. In vitro, bee venom and melittin exhibited comparable levels of antibacterial activity, which was more marked against MRSA strains, compared with other Gram­positive bacteria. When MRSA­infected mice were treated with bee venom or melittin, only the latter animals were successfully rescued from MRSA­ induced bacteraemia or exhibited recovery from MRSA­infected skin wounds. Together, the data of the present study demonstrated for the first time, to the best of our knowledge, that melittin may be used as a promising antimicrobial agent to enhance the healing of MRSA­induced wounds.

Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 µM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent.

Melittin peptides exhibit different activity on different cells and model membranes.

Melittin (MLT) is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogs were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes and hemolysis. Although lytic activity against model membranes decreased for the MLT dimer, hemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation.

Highly efficient macromolecule-sized poration of lipid bilayers by a synthetically evolved peptide.

Peptides that self-assemble, at low concentration, into bilayer-spanning pores which allow the passage of macromolecules would be beneficial in multiple areas of biotechnology. However, there are few, if any, natural or designed peptides that have this property. Here we show that the 26-residue peptide "MelP5", a synthetically evolved gain-of-function variant of the bee venom lytic peptide melittin identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers under conditions where melittin and other pore-forming peptides do not. In surface-supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at peptide:lipid ratios as low as 1:25000. The increase in bilayer conductance due to MelP5 is dramatically higher, per peptide, than the increase due to the parent sequence of melittin or other peptide pore formers. Here we also develop two novel assays for macromolecule leakage from vesicles, and we use them to characterize MelP5 pores in bilayers. We show that MelP5 allows the passage of macromolecules across vesicle membranes at peptide:lipid ratios as low as 1:500, and under conditions where neither osmotic lysis nor gross vesicle destabilization occur. The macromolecule-sized, equilibrium pores formed by MelP5 are unique as neither melittin nor other pore-forming peptides release macromolecules significantly under the same conditions. MelP5 thus appears to belong to a novel functional class of peptide that could form the foundation of multiple potential biotechnological applications.

Synthesis and in vitro testing of new potent polyacridine-melittin gene delivery peptides.

The combination of a polyacridine peptide modified with a melittin fusogenic peptide results in a potent gene transfer agent. Polyacridine peptides of the general formula (Acr-X)(n)-Cys were prepared by solid-phase peptide synthesis, where Acr is Lys modified on its epsilon-amine with acridine, X is Arg, Leu, or Lys and n is 2, 3, or 4 repeats. The Cys residue was modified by either a maleimide-melittin or a thiolpyridine-Cys-melittin fusogenic peptide resulting in reducible or non-reducible polyacridine-melittin peptides. Hemolysis assays established that polyacridine-melittin peptides retained their membrane lytic potency relative to melittin at pH 7.4 and 5. When combined with plasmid DNA, the membrane lytic potency of polyacridine-melittin peptides was neutralized. Gene transfer experiments in multiple cell lines established that polyacridine-melittin peptides mediate expression as efficiently as PEI. The expression was very dependent upon a disulfide bond linking polyacridine to melittin. The gene transfer was most efficient when X is Arg and n is 3 or 4 repeats. These studies establish polyacridine peptides as a novel DNA binding anchor peptide.

Targeting the cancer stroma with a fibroblast activation protein-activated promelittin protoxin.

Fibroblast-Activation Protein-α (FAP) is a membrane-bound serine protease that is expressed on the surface of reactive stromal fibroblasts present within the majority of human epithelial tumors but is not expressed by normal tissues. FAP is a postprolyl peptidase that differs from other dipeptidyl prolyl peptidases such as diprolylpeptidase 4 in that it also has gelatinase and collagenase endopeptidase activity. Therefore, FAP represents a potential pan-tumor target whose enzymatic activity can be exploited for the intratumoral activation of prodrugs and protoxins. To evaluate FAP as a tumor-specific target, putative FAP-selective peptide protoxins were constructed through modification of the prodomain of melittin, a 26 amino acid amphipathic cytolytic peptide that is the main toxic component in the venom of the common European honeybee Apis milefera. Melittin is synthesized as promelittin, containing a 22 amino acid NH(2)-terminal prodomain rich in the amino acids proline and alanine. In this study, peptides containing truncated melittin prodomain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. Once optimized, modified promelittin peptides were generated in which previously identified FAP substrate sequences were introduced into the prodomain. Peptide protoxins were identified that were efficiently activated by FAP and selectively toxic to FAP-expressing cell lines with an IC(50) value in the low micromolar range that is similar to melittin. Intratumoral injection of an FAP-activated protoxin produced significant lysis and growth inhibition of human breast and prostate cancer xenografts with minimal toxicity to the host animal.

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action.

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.

Inhibition of plant-pathogenic bacteria by short synthetic cecropin A-melittin hybrid peptides.

Short peptides of 11 residues were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Xanthomonas vesicatoria and compared to the previously described peptide Pep3 (WKLFKKILKVL-NH(2)). The antimicrobial activity of Pep3 and 22 analogues was evaluated in terms of the MIC and the 50% effective dose (ED(50)) for growth. Peptide cytotoxicity against human red blood cells and peptide stability toward protease degradation were also determined. Pep3 and several analogues inhibited growth of the three pathogens and had a bactericidal effect at low micromolar concentrations (ED(50) of 1.3 to 7.3 microM). One of the analogues consisting of a replacement of both Trp and Val with Lys and Phe, respectively, resulted in a peptide with improved bactericidal activity and minimized cytotoxicity and susceptibility to protease degradation compared to Pep3. The best analogues can be considered as potential lead compounds for the development of new antimicrobial agents for use in plant protection either as components of pesticides or expressed in transgenic plants.

[In-vitro activity of two hybrid synthetic peptides having antimicrobial activity against mycobacteria]. / Sensibilidad in vitro de micobacterias a dos péptidos sintéticos híbridos con actividad antimicrobiana.

The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis has been evaluated. The minimal inhibitory concentration was determined by using the broth macrodilution technique. The minimal bactericide concentration in Lowenstein Jensen medium was then obtained. The peptides studied were active, in vitro, against M. smegmatis, but they did not show any activity against the other mycobacteria analyzed.

[Synthesis of peptide fragment of melittin and the function of rheumatoid arthritis cure].

To retain the anti-rheumatoid arthritis activity of melittin and to reduce the hemolysis and hypersusceptibility caused by melittin, a deletion peptide of melittin was synthesized. Its ant-inflammation effect was observed . A hydrophile peptide fragment of melittin was synthesized by standard solid-phase method. The product was analyzed by HPLC and MS. The relevant hemolysis and hypersusceptibility were tested. The rabbits' model of immune arthritis were established and treated. The results showed that the hemolysis rate for peptide fragment was less than 5%, the hypersusceptibility rate was less than 8%. The hydrophile peptide fragment of melittin may retain anti-rheumatoid arthritis activity and reduce the melittin-induced hemolysis and hypersusceptibility.

Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide.

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide-LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the 'endotoxic principle' of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides.

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12).

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella tryphimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect but that the alpha-helical property is not connected with the enhanced antibiotic activity.

Conformation of the cecropin-melittin hybrid, cecropin A(1-8)-melittin (1-18) antibacterial Peptide.

Cecropin A (1-8)-Melittin (1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. The primary sequence of the peptide is as follows: KWKLPKKIGIGAVLKVLTTGLPALIS-NH2. 1H and 13C 2D NMR techniques were used to deduce the conformational parameters of chemical shift, 3JNHalpha coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter-residue nOe's. NMR studies were carried out in water (pH 6.0) and hexafluoroacetone (HFA). The peptide was found in a beta-pleated structure in water, and in HFA it adopts a right-handed alpha-helix conformation. Solution structures generated using restrained molecular dynamics simulations were refined by Mardigras to R factors ranging from 0.5 to 0.6.

Cloaking cytolytic peptides for liposome-based detection and potential drug delivery.

Potent cytolytic peptides with specific tethering and cloaking sites have been synthesised and used to release payload from liposomes in a quantitative manner. A functionally located cloaking site has been modified specifically by simple conjugation without adversely affecting the cytolytic properties of the peptide. The cytolytic activity of modified peptides was then efficiently (>98%) cloaked and uncloaked by ligand-protein or hapten-antibody interactions. The principle of a dual response peptide has been demonstrated using an avidin-cloaked pH-sensitive peptide. Biospecific cloaking/uncloaking provided a new sensitive (approximately 12 pmol) homogeneous diagnostic and also appears potentially suited to bioresponsively targeted release of antimicrobial, anticancer and other drugs now delivered using liposomes.

Solid-state NMR structure determination of melittin in a lipid environment.

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.

Synthesis and study of normal, enantio, retro, and retroenantio isomers of cecropin A-melittin hybrids, their end group effects and selective enzyme inactivation.

In our effort to understand the structural requirements for the antimicrobial activity of cecropin A (CA) and melittin (M), we synthesized the normal, enantio, retro and retroenantio hybrid analogs; we related activity to their sequence, chirality, amide bond direction (helix dipole) and end group charges. To compare the effect of the end groups, each of these analogs was synthesized both with an acid and an amide C-terminus and also with and without an N alpha-acetyl N-terminus. The all-L- and all-D-enantiomers of several cecropin-melittin hybrids were previously found to be equally potent against several bacterial species, and no chiral effect was observed. This general rule has now been confirmed and extended. However, two exceptions have been found. All-L-CA(1-13)M(1-13) acid was 5 times and 9 times less potent than the all-D-analog, respectively, toward gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa. All-L-CA(1-7)M(2-9) acid was 5 times and 14 times less active against S. aureus and P. aeruginosa, respectively, than its all-D acid isomer. The corresponding D- and L-retro analogs differed only marginally. A role for proteolytic enzymes has been implicated as a cause for these differences in the activities of L- and D-enantiomers. In all cases, blocking the alpha-amine by acetylation had no significant effect on potency. The retro and retroenantio analogs of CA(1-13)M(1-13) acid were as potent as their normal and enantio analogs against all the test bacteria. The C-terminal amides also showed similar potency against four test bacteria. It should be noted that the negative end of the helix dipole of a normal peptide points toward the C-terminus, whereas it points away in the case of a retro derivative when viewed in the direction of the normal sequence.

Release of lipid vesicle contents by an antibacterial cecropin A-melittin hybrid peptide.

A synthetic cecropin A(1-8)-melittin(1-18) hybrid peptide, with antimalarial and antibacterial properties, promotes leakage of aqueous contents of phospholipid vesicles, as determined by measuring the induced release of vesicle-entrapped fluorescence probes. The release of vesicle contents corresponds to an all-or-none mechanism. High molecular weight entrapped solutes (fluorescence-labeled dextrans, 20 and 4 kDa molecular mass) are also released by the peptide. This fact and the high peptide stoichiometry required for the release of vesicle contents suggest a detergent-like disruption of the bilayer. The leakage process is not related to any membrane event requiring lipid-mixing between bilayers. The peptide destabilizes both negatively and neutrally charged phospholipid vesicles. The thermal variation of the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene-labeled vesicles is modified by the peptide. Circular dichroism and tryptophan fluorescence emission spectra reveal conformational changes in the peptide molecule upon interaction with the lipid vesicles. These changes are consistent with an increased alpha-helical content and a less polar environment for the single tryptophan residue of the peptide. The leakage induced in phosphatidylserine vesicles is a faster process than in phosphatidylcholine vesicles, while the peptide is more effective in releasing the contents of the latter type of vesicles. This suggests that acidic phospholipids may modulate the effect of the peptide on membranes.

Hydrophobic effects on antibacterial and channel-forming properties of cecropin A-melittin hybrids.

The design of cecropin-melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile8 in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH2, [CA(1-7)M(2-9)NH2] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile8 reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile8 by a hydrophobic leucine maintained good activity and Ala8 was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser8 strongly reduced potency against all five organisms. Deletion of Leu15 decreased activity, but addition of Thr16 maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic alpha-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in alpha-helicity with an increase in hexafluoro 2-propanol concentration.

Immunogenicity studies with haptenated melittin peptides: implication for membrane involvement during the recognition step.

Melittin peptides carrying 2,4-dinitro-6-carboxyphenyl (Dncp) haptenic groups regularly evoked anti-hapten IgG responses in mice or guinea pigs when the hapten was C-terminally attached. Single haptens on the N-terminal helix in several positions gave poor or no responses in the early stages but adequate titres after prolonged immunization. Peptides with Dncp at the C-terminus as an invariant feature and a second Dncp in various positions along the peptide chain did not fail to produce adequate responses. The hampering effect is not due to a defect at the T-cell level but involves the recognition step on the B-cell. It is implied that the haptenic interaction with the paratope of the recognizing immunoglobulin on the B-cell involves the cell membrane in an important way. It is also suggested that late antibody responses should not be overlooked during the development of proteinaceous immunogens for vaccination.

Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein.

Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.