Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C-terminally truncated gaegurin 4.

Gaegurin 4 (GGN4), a 37-residue antimicrobial peptide, consists of two amphipathic alpha helices (residues 2-10 and 16-32) connected by a flexible loop region (residues 11-15). As part of an effort to develop new peptide antibiotics with low molecular mass, the activities of C-terminally truncated GGN4 analogues were tested. Delta24-37 GGN4, a peptide analogue with 14 residues truncated from the C-terminus of GGN4, showed a complete loss of antimicrobial activity. However, the single substitution of aspartic acid 16 by tryptophan (D16W) in the Delta24-37 GGN4 completely restored the antimicrobial activity, without any significant hemolytic activity. In contrast, neither the D16F nor K15W substitution of the Delta24-37 GGN4 allowed such a dramatic recovery of activity. In addition, the D16W substitution of the native GGN4 significantly enhanced the hemolytic activity as well as the antimicrobial activity. The structural effect of the D16W substitution in the Delta24-37 GGN4 was investigated by CD, NMR, and fluorescence spectroscopy. The results showed that the single tryptophanyl substitution at position 16 of the Delta24-37 GGN4 induced an alpha helical conformation in the previously flexible loop region in intact GGN4, thereby forming an entirely amphipathic alpha helix. In addition, the substituted tryptophan itself plays an important role in the membrane-interaction of the peptide.     

De novo generation of short antimicrobial peptides with simple amino acid composition

As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L₅K₂W₂), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L₄K₅W₂) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides.     

Activity optimization of an undecapeptide analogue derived from a frog-skin antimicrobial peptide

While natural antimicrobial peptides are potential therapeutic agents, their physicochemical properties and bioactivity generally need to be enhanced for clinical and commercial development. We have previously developed a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH₂) with potent antimicrobial and hemolytic activity, which was derived from a 24-residue, natural antimicrobial peptide isolated from frog skin. Here, we attempted to optimize peptide bioactivity by a rational approach to sequence modification. Seven analogues were generated from GA-W2, and their activities were compared with that of a 12-residue peptide, omiganan, which is being developed for clinical and commercial applications. Most of the modifications reported here improved antimicrobial activity. Among them, the GA-K4AL (FAKWAFKWLKK-NH₂) peptide displayed the most potent antimicrobial activity with negligible hemolytic activity, superior to that of omiganan. The therapeutic index of GA-K4AL was improved more than 53- and more than 31-fold against Gram-negative and Gram-positive bacteria, respectively, compared to that of the starting peptide, GA-W2. Given its relatively shorter length and simpler amino acid composition, our sequence-optimized GA-K4AL peptide may thus be a potentially useful antimicrobial peptide agent.     

Mechanism of action and relationship between structure and biological activity of Ctx-Ha: a new ceratotoxin-like peptide from Hypsiboas albopunctatus

The increase in bacterial resistance to current antibiotics has led to the development of new active molecules. We have isolated the antimicrobial peptide Ctx-Ha from the skin secretion of the frog Hypsiboas albopunctatus. The aim of the present work was to elucidate the mechanism of action of this new antimicrobial peptide. The sequence similarity with Ceratotoxin, the pore size, and the pore-like release of carboxyfluorescein from vesicles indicated that Ctx(Ile21)-Ha has a mechanism of action based on the barrel- stave model. In a second part of this work, we synthesized three analogues to provide information about the relationship between the peptide's structure and its biological activity. Ctx(Ile21)-Ha-VD 16, Ctx(Ile21)- Ha-VD 5,16 and Ctx(Ile21)-Ha-I9K were designed to disrupt the peptide's helical structure and change the hydrophobicity/ hydrophilicity and amphipathicity of the apolar face in order to uncouple the antimicrobial activity of Ctx(Ile21)-Ha from its hemolytic activity. To evaluate the effects of the amino acid substitutions on peptide conformation, secondary structure was accessed using CD measurements. The peptides presented a high amount of α-helical structure in the presence of TFE and LPC. The CD data showed that destruction of the amphipathic α-helix by the replacing isoleucine by lysine is less harmful to the structure than D-amino acid substitutions. Biological tests demonstrated that all peptides have activity. Nevertheless, the peptide Ctx(Ile21)-Ha-I9K showed the highest value of therapeutic index. Our findings suggest that these peptides are potential templates for the development of new antimicrobial drugs. These studies highlight the importance of single amino acid modification as a tool to modulate the biological activity of antimicrobial peptides.     

Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.     

Solution structure of the antimicrobial peptide gaegurin 4 by H and 15N nuclear magnetic resonance spectroscopy.

Gaegurin 4 (GGN4) is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an alpha-helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic alpha-helices extending from residues 2-10 and from residues 16-32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix-assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C-terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.     

Design of a novel membrane-destabilizing peptide selectively acting on acidic liposomes

The design of amphipathic peptides resulted in a novel peptide with a selective ability to destabilize lipid bilayers of acidic liposomes. The newly synthesized peptide, termed mast 21, is a 21-residue long amino acid chain and can only act effectively on acidic liposomes lacking cholesterol. Moreover, mast 21 killed gram-positive and gram-negative bacteria, and it had no hemolytic activity. The antimicrobial and hemolytic activities paralleled the results of membrane destabilizing activity using liposomes. Circular dichroism and Trp-fluorescence emission spectra showed changes in the peptide conformation and circumstances around the peptide during interaction with liposomes. These changes were consistent with an increased alpha-helical content and a less polar environment for the tryptophan residue of the peptide. Mast 21 was observed under dark-field microscopy in real time attacking liposomes. Acidic liposomes were attacked, which resulted in peeling of the lipid bilayer with its subsequent destruction.     

The effect of cyclization of magainin 2 and melittin analogues on structure, function, and model membrane interactions: implication to their mode of action

The amphipathic alpha-helical structure is a common motif found in membrane binding polypeptides including cell lytic peptides, antimicrobial peptides, hormones, and signal sequences. Numerous studies have been undertaken to understand the driving forces for partitioning of amphipathic alpha-helical peptides into membranes, many of them based on the antimicrobial peptide magainin 2 and the non-cell-selective cytolytic peptide melittin, as paradigms. These studies emphasized the role of linearity in their mode of action. Here we synthesized and compared the structure, biological function, and interaction with model membranes of linear and cyclic analogues of these peptides. Cyclization altered the binding of melittin and magainin analogues to phospholipid membranes. However, at similar bound peptide:lipid molar ratios, both linear and cyclic analogues preserved their high potency to permeate membranes. Furthermore, the cyclic analogues preserved approximately 75% of the helical structure of the linear peptides when bound to membranes. Biological activity studies revealed that the cyclic melittin analogue had increased antibacterial activity but decreased hemolytic activity, whereas the cyclic magainin 2 analogue had a marked decrease in both antibacterial and hemolytic activities. The results indicate that the linearity of the peptides is not essential for the disruption of the target phospholipid membrane, but rather provides the means to reach it. In addition, interfering with the coil-helix transition by cyclization, while maintaining the same sequence of hydrophobic and positively charged amino acids, allows a separated evaluation of the hydrophobic and electrostatic contributions to binding of peptides to membranes.     

New indolicidin analogues with potent antibacterial activity.

Indolicidin is a 13-residue antimicrobial peptide amide, ILPWKWPWWPWRR-NH2, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two-fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR-NH2 in which Trp-residues 4,6,8,9,11 were replaced in all positions with X = a single non-natural building block; N-substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non-natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N-terminus with a hydrophobic non-natural building block. We prepared 22 analogues of indolicidin and [Phe(4,6,8,9,11)] indolicidin, 11 of each, carrying a hydrophobic non-natural building block attached to the N-terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non-natural building blocks, are promising lead structures for developing future therapeutics.     

Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity.

This review focuses on important structural features affecting the antimicrobial activity of 15-residue derivatives of lactoferricins. Our investigations are based on an alanine-scan of a 15-residue bovine lactoferricin derivative that revealed the absolute necessity of two tryptophan residues for antimicrobial activity. This "tryptophan-effect" was further explored in homologous derivatives of human, caprine, and porcine lactoferricins by the incorporation of one additional tryptophan residue, and by increasing the content of tryptophan in the bovine derivative to five residues. Most of the resulting peptides display a substantial increase in antimicrobial activity. To identify which molecular properties make tryptophan so effective, a series of bovine lactoferricin derivatives were prepared containing non-encoded unnatural aromatic amino acids, which represented various aspects of the physicochemical nature of tryptophan. The results clearly demonstrate that tryptophan is not unique since most of the modified peptides were of higher antimicrobial potency than the native peptide. The size and three-dimensional shape of the inserted "super-tryptophans" are the most important determinants for the high antimicrobial activity of the modified peptides. This review also describes the use of a "soft-modeling" approach in order to identify important structural parameters affecting the antimicrobial activity of modified 15-residue murine lactoferricin derivatives. This QSAR-study revealed that the net charge, charge asymmetry, and micelle affinity of the peptides were the most important structural parameters affecting their antimicrobial activity.     

Rational design of antimicrobial C3a analogues with enhanced effects against Staphylococci using an integrated structure and function-based approach

The anaphylatoxin C3a and its inactivated derivative C3adesArg, generated during complement activation, exert direct antimicrobial effects, mediated via its C-terminal region [Nordahl et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 16879-16884]. During evolution, this region of C3a displays subtle changes in net charge, while preserving a moderate but variable amphipathicity [Pasupuleti et al. (2007) J. Biol. Chem. 282, 2520-2528]. In this study, we mimic these evolutionary changes, employing a design approach utilizing selected amino acid substitutions at strategic and structurally relevant positions in the original human C3a peptide CNYITELRRQHARASHLGLA, followed by structure-activity studies incorporating sequence-dependent QSAR models as tools for generation of C3a peptide variants with enhanced effects. While the native peptide and related amphipathic analogues of moderate positive net charge were active against the Gram-negative Escherichia coli, activity against the Gram-positive Staphylococcus aureus was primarily observed for peptides characterized by a combination of a relatively high net charge (+6-7) and a propensity to adopt an alpha-helical conformation with amphipathic character. Such increased helicity and charge also conferred activity against the fungus Candida albicans. A central histidine residue (H11), evolutionarily conserved among vertebrates, conferred high selectivity toward microbes, while substitutions with leucine rendered the peptides hemolytic. Selected C3a analogues retained their specificity against staphylococci in the presence of human plasma, while showing low cytotoxicity. The work illustrates structure-activity relationships underlying the function and specificity of antimicrobial C3a and related analogues and provides insights into the forces that drive evolution of antimicrobial peptides.     

Properties and structure-activity studies of cyclic beta-hairpin peptidomimetics based on the cationic antimicrobial peptide protegrin I

The properties and structure-activity relationships (SAR) of a macrocyclic analogue of porcine protegrin I (PG-I) have been investigated. The lead compound, having the sequence cyclo-(-Leu-Arg-Leu-Lys-Lys-Arg-Arg-Trp-Lys-Tyr-Arg-Val-d-Pro-Pro-), shows antimicrobial activity against Gram-positive and -negative bacteria, but a much lower haemolytic activity and a much reduced ability to induce dye release from phosphatidylcholine/phosphatidylglycerol liposomes, when compared to PG-I. The enantiomeric form of the lead peptide shows comparable antimicrobial activity, a property shared with other cationic antimicrobial peptides acting on cell membranes. SAR studies involving the synthesis and biological profiling of over 100 single site substituted analogues, showed that the antimicrobial activity was tolerant to a large number of the substitutions tested. Some analogues showed slightly improved antimicrobial activities (2-4-fold lowering of MICs), whereas other substitutions caused large increases in haemolytic activity on human red blood cells.     

Synthesis and antibacterial activity of analogues of the N-terminal fragment of the sarcotoxin IA antimicrobial peptide

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1-18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.     

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18–26 amino acids long. We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male). We have named this peptide “piscidin 4” since it has considerable (to > 65%) N-terminal sequence homology to piscidins 1–3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.     

A helix-induced oligomeric transition of gaegurin 4, an antimicrobial peptide isolated from a Korean frog

Gaegurin 4 (GGN4), a novel peptide isolated from the skin of a Korean frog, Rana rugosa, has broad spectrum antimicrobial activity. A number of amphipathic peptides closely related to GGN4 undergo a coil to helix transition with concomitant oligomerization in lipid membranes or membrane-mimicking environments. Despite intensive study of their secondary structures, the oligomeric states of the peptides before and after the transition are not well understood. To clarify the structural basis of its antibiotic action, we used analytical ultracentrifugation to define the aggregation state of GGN4 in water, ethyl alcohol, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The maximum size of GGN4 in 15% HFIP corresponded to a decamer, whereas it was monomeric in buffer. The oligomeric transition is accompanied by a cooperative 9 nm blue-shift of maximum fluorescence emission and a large secondary structure change from an almost random coil to an alpha-helical structure. GGN4 induces pores in lipid membranes and, using electrophysiological methods, we estimated the diameter of the pores to be exceed 7.3 A, which suggests that the minimal oligomer structure responsible is a pentamer.     

NMR spectroscopic assessment of the structure and dynamic properties of an amphibian antimicrobial peptide (Gaegurin 4) bound to SDS micelles

The structure and dynamics of a 37-residue antimicrobial peptide gaegurin 4 (GGN4) isolated from the skin of the native Korean frog, Rana rugosa, was determined in SDS micelles by NMR spectroscopy. The solution structure of the peptide in SDS micelles was determined from 352 NOE-derived distance constraints and 22 backbone torsion angle constraints. Dynamic properties for the amide backbone were characterized by (1)H-(15)N heteronuclear NOE experiments. The structural study revealed two amphipathic helices spanning residues 2-10 and 16-32 and that the helices were connected by a flexible loop. An intraresidue disulfide bridge was formed between residues Cys31 and Cys37 near the C-terminus. The loop region (11-15) connecting the two helices are were slightly more flexible than these helices themselves. From the fact that since there is no contact NOEs between two helices, it is implied that the GGN4 peptide shows an independent motion of both helices which has an angle of about 60 degrees -120 degrees from each other.     

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Structure-activity relationships of antimicrobial peptides from the skin of Rana esculenta inhabiting in Korea

The anuran (frogs and toads) skin is a rich source of antimicrobial peptides that can be developed therapeutically. We searched the skin secretions of Korean Rana esculenta for antimicrobial peptides, and isolated two cationic peptides with antimicrobial activity and little hemolytic activity: a 46-residue peptide of the esculentin-1 family and a 24-residue peptide of the brevinin-1 family. Their sequences showed some differences from the esculentins-1 and brevinins-1 of European Rana esculenta, indicating that sequence diversification of anuran skin antimicrobial peptides can arise from differences in habitat as well as from species differences. The 46-residue peptide named esculentin-1c had broad antimicrobial activity, while the 24-residue peptide named brevinin-1Ed exhibited limited activity. The solution structure of brevinin-1Ed was in good agreement with that of other brevinin-1-like peptides, with an amphipathic alpha-helix spanning residues 3-20, stabilized in membrane-mimetic environments. The weak bioactivity of brevinin-1Ed was attributable to the unusual presence of an anionic amino acid in the middle of the helical hydrophilic face. This report contributes to world-wide investigations of the structure-activity relationships and evolutional diversification of anuran-skin antimicrobial peptides.     

Perturbation of peptide conformations induced in anisotropic environments

Reversed-phase high performance liquid chromatography (RP HPLC) has been found to be a convenient and powerful tool for the study of the secondary structure of peptides. Here, the ability of proline to perturb the secondary structures of peptides induced at aqueous-lipid interfaces and the induced conformation of polyproline peptides were investigated by means of RP HPLC. For these studies, four different complete sets of substitution analogues of model peptides expected to have specific induced conformations were used. In the first two studies, a single lysine was “walked” through two 18-residue polyproline sequences (one N-acetylated, the other not). In the remaining two studies, a proline was “walked” through two different sequences that had been found earlier to be induced into an α-helical conformation during RP HPLC (an 18-residue polyalanine sequence and the amphipathic 14-residue sequence Ac-LLKLLKKLLKKLKK-NH₂). Sixty-eight individual analogues were synthesized for this study and the effect of the respective substitutions on retention times was determined. The results are consistent with the concept that, upon interaction with the C-18 of the stationary phase during RP HPLC, polyproline is induced into a type II helical conformation, polyalanine into an α-helical conformation, and Ac-LLKLLKKLLKKLKK-NH₂ into an amphipathic α-helical array. In an extension of this study, the antimicrobial activities of Ac-LLKLLKKLLKKLKK-NH₂ and its 18 proline substitution analogues were found to be inversely correlated with their RP HPLC retention times.     

Interaction of staphylococcal delta-toxin and synthetic analogues with erythrocytes and phospholipid vesicles. Biological and physical properties of the amphipathic peptides

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.