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. 2007 Sep;16(9):1969–1976. doi: 10.1110/ps.072966007

Structure–activity study of the antibacterial peptide fallaxin

Sandra L Nielsen 1, Niels Frimodt-Møller 2, Birthe B Kragelund 3, Paul R Hansen 1
PMCID: PMC2206974  PMID: 17766389

Abstract

Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure–activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N- and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, (VISA), methicillin-susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 μM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity 〈H〉, mean hydrophobic moment 〈μH〉, and α-helicity. We were able to group the active and inactive analogs according to mean hydrophobicity 〈H〉 and mean hydrophobic moment 〈μH〉. Far-UV CD-spectroscopy experiments on fallaxin and several analogs in buffer, in TFE, and in membrane mimetic environments (small unilamellar vesicles) indicated that a coiled-coil conformation could be an important structural trait for antibacterial activity. This study provides data that support fallaxin analogs as promising lead structures in the development of new antibacterial agents.

Keywords: alanine scan, antibacterial activity, coiled-coil conformation, fallaxin, solid-phase peptide synthesis

Multicellular organisms produce antimicrobial peptides (AMPs) that protect them against pathogens. These peptides, also known as host-defense peptides, are an integral part of the inherent immune response in humans and various living organisms (Hancock and Chapple 1999; Boman 2003; Yount and Yeamann 2004). AMPs show activity against a wide range of pathogens such as Gram-positive and Gram-negative bacteria, fungi, parasites, and viruses (Zasloff 2002; Hancock and Sahl 2006). In 1981, Boman succeeded in isolating the first antimicrobial peptide, cecropin, from the nocturnal moth Hyalophora cecropia (Steiner et al. 1981). Today more than 900 different peptides have been isolated from different eukaryotic organisms. A complete list can be found in the Antimicrobial Sequence Database (AMSDb; http://www.bbcm.univ.trieste.it/∼tossi/amsdb.html; accessed April 2007). AMPs are generally between six and 50 amino acid residues in length of which 40%–60% are hydrophobic. They possess a net positive charge between +2 and +7. Many of these peptides are unstructured in water but adopt amphipathic structures upon interaction with biological membranes or membrane-mimicking environments (Yeaman and Yount 2003; Brogden 2005).

In recent years, the rapid increase in multiresistant bacteria has prompted a growing interest in antimicrobial peptides as an alternative to conventional antibiotics. In contrast to conventional antibiotics, which work by interfering with a specific biochemical reaction within the cell, antimicrobial peptides are thought to disrupt the bacterial membrane in a non-receptor-mediated fashion. The exact mechanism of action has not yet been fully described.

The antibacterial peptide fallaxin, a 25-amino-acid-long C-terminal amide: GVVDILKGAAKDIAGHLASKVMNKL-NH2, was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax (Rollins-Smith et al. 2005). Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. At concentrations below 200 μM, the peptide shows no activity toward the Gram-positive bacterium Staphylococcus aureus or the yeast Candida albicans. Fallaxin shows almost no toxicity toward erythrocytes, with a hemolytic activity of HC50 > 200 μM (Rollins-Smith et al. 2005).

Here, we report a structure–activity study of fallaxin, including a complete alanine scan of fallaxin as well as N- and C-terminal truncated analogs, with a total of 66 peptides analyzed. The peptides were tested for antibacterial activity against methicillin-resistant S. aureus (MRSA), vancomycin-intermediate resistant S. aureus (VISA), methicillin-susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. The cytotoxicity of fallaxin and its alanine analogs against human erythrocytes was assessed in a hemolytic activity assay. Intrinsic structure as well as induced structure by TFE and by the presence of small unilamellar vesicles were investigated by far-UV CD spectroscopy. Finally, we correlate activity with structure and with various structural parameters such as charge, hydrophobicity 〈H〉, mean hydrophobic moment 〈μH〉, and helicity.

Results and Discussion

Antibacterial activity of fallaxin and analogs

In this study, fallaxin inhibited the growth of the Gram-negative bacteria E. coli and K. pneumoniae with relatively low potency (MIC-values of 100 μM). Fallaxin showed no antibacterial activity against the Gram-negative bacterium P. aeruginosa and the Gram-positive bacteria MRSA, VISA, and MSSA at concentrations below 100 μM (▶). Rollins-Smith et al. (2005) found moderate fallaxin activity against E. coli, P. aeruginosa, and K. pneumoniae, with MIC-values of 40 μM, 80 μM, and 80 μM, respectively. As the assays are sensitive to environmental parameters and since Rollins-Smith et al. (2005) used different bacterial strains from this study, the divergent results are most likely to be related to these matters. We found the hemolytic activity of fallaxin to be 0% at a peptide concentration of 50 μM, which is in full accordance with Rollins-Smith et al. (2005).

Table 1.

Activity and structural parameters of fallaxin and alanine scan of fallaxin

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Ala-scan of fallaxin

In order to assess the role of each individual amino acid residue of fallaxin in antimicrobial activity, we performed an alanine scan in which each residue was systematically replaced by the small and neutral amino acid Ala. Any naturally occurring Ala was replaced by Leu. From the results shown in ▶ it is clear that substituting specific positions has a great impact on the antimicrobial activity of fallaxin. Generally, the set of analogs were all more active against the Gram-negative bacteria E. coli, K. pneumoniae, and P. aeruginosa (MIC values 3.13 → 100 μM) than against the Gram-positive bacteria MSSA, MRSA, and VISA (MIC values 25 → 100 μM), with the A → L analogs being the most potent. We define a fallaxin analog as active when the analog displays a MIC value of 25 μM or better against at least one bacterial strain.

In agreement with previous studies on AMPs, the antibacterial activity of fallaxin was shown to depend on the overall charge. A substitution of Asp4 or of Asp12 with the electrically neutral Ala (FA4 and FA12) resulted in increased antibacterial activity. FA12 showed the best activity toward E. coli and K. pneumoniae, with observed MIC values of 3.13 and 25 μM compared to that of fallaxin of 100 μM. In contrast, replacing the positively charged Lys with Ala in positions FA7, FA11, FA20, and FA24 resulted in loss of activity compared to fallaxin. These results can be explained by an increased or decreased electrostatic attraction between the positively charged peptides and negatively charged bacterial membranes, respectively. Only one substitution, His16 with Ala16, had no observable effect on either the antibacterial or the hemolytic activity.

Replacing Ala with the more hydrophobic Leu (FL9, FL10, FL14, and FL18) resulted in an improved antibacterial activity toward the Gram-positive bacterial strains MRSA, MSSA, and VISA. FL9 and FL10 showed the best overall activity (broad-spectrum), whereas FL14 showed the second best activity toward E. coli with a MIC-value of 6.25 μM. In line with this, when Leu was replaced with Ala (FA6, FA17, and FA25), the antibacterial activity decreased compared to that of fallaxin, indicating altogether that Leu is very important for antibacterial activity. Interestingly, all Leu residues are located on the hydrophobic face of an ideal helix. A similar observation was made when replacing the hydrophobic aliphatic amino acids Val and Ile (FA2, FA3, FA13, and FA21). Substitution of Ala residues, which is known to possess a higher intrinsic propensity for helix formation (Muñoz and Serrano 1994), with the lower intrinsic-helix-propensity residue Leu, would be assumed to destabilize helix formation. However, since these substitutions (A → L) result in a higher antibacterial activity and since the opposite is the case for the L → A, V → A, and I → A substitutions, this suggests that higher-ordered structures, in terms of coiled-coils, could be implicated in the membrane active state of the peptides.

Substituting either Gly8 or Gly15 with Ala resulted in an improved antibacterial activity. This was expected in terms of helix propensity. Gly in internal positions of a helix will destabilize helix formation as a result of the greater conformational freedom. The two glycines are located on the opposite face of the leucine residues. Replacing Gly with Ala increases helix stability and probability for “leucine-zipper” formation, leading to increased activity. The opposite effect was observed when Gly1 was replaced. We speculate that this result may have to do with helix capping destabilization.

C- and N-terminus truncations

We synthesized 19 N-terminal truncated analogs and 19 C-terminal truncated analogs. Unfortunately, all the truncated analogs showed MIC values >100 μM for all bacterial strains tested. Although, Rollins-Smith et al. (2005) found fallaxin 1–22 to be inactive against E. coli and S. aureus at concentrations up to 160 μM, the lack of any activity or selectivity for either bacterial or mammalian cells was quite surprising to us. Our result may be explained by the RP-HPLC retention times, mean hydrophobicity 〈H〉, and mean hydrophobic moment of these peptides as described in the sections below.

Hemolytic activity of fallaxin and Ala-scan analogs

The hemolytic activity was determined against human erythrocytes (ORh+). The obtained results from the hemolytic assay are listed in ▶.

We noted a correlation between antibacterial activity and hemolytic activity. The most active analogs—FL9, FL10, FA12, FL14, and FA18—also showed the highest hemolytic activity (▶). The alanine-scan revealed a few analogs with improved antibacterial activity, without them being hemolytic. The peptide analogs FA4 and FA19 showed an activity toward E. coli of 50 and 25 μM, respectively, and a hemolytic activity of 1% and 2%, respectively. The data show that antibacterial activity is difficult to dissociate from hemolytic activity, and this fact has also been reported for numerous other antibacterial peptides including indolicidin (Ryge et al. 2004) and melittin (Blondelle and Houghten 1991).

Mean hydrophobicity 〈H〉 and mean hydrophobic moment 〈μH〉

Eisenberg's consensus scale was used to calculate both the mean hydrophobicity 〈H〉 and mean hydrophobic moment 〈μH〉 (Eisenberg 1984). The mean hydrophobicity 〈H〉 is a measure of the amino acid residue relative affinities for hydrophobic phases. The hydrophobic moment 〈μH〉 is a measure of the amphiphilicity or asymmetry of hydrophobicity of a segment of a polypeptide chain. We observed that fallaxin analogs with 〈H〉 ranging from −0.0256 to −0.0088 and with 〈μH〉 ranging from 0.00766 to 0.1164 showed MIC values of 25 μM or better against at least one bacterial strain. This is true for FA8, FL9, FL10, FA12, FL14, and FA15, the exceptions being FA1 (〈H〉 = −0.00256 and 〈μH〉 = 0.1020) and FA12 (〈H〉 = 0.0096 and 〈μH〉 = 0.0766). Hence, the analogs FA2–FA7, FA11, FA13, FA16, FA17, and FA20–FA25 showed an antibacterial activity worse than 25 μM.

The mean hydrophobicity and mean hydrophobic moment of the C- and N-terminal truncated fallaxin analogs were outside the above range. We noticed that the active fallaxin analogs all had RP-HPLC retention times >15.2 min, which all were higher than those of the truncated fallaxin analogs.

Our results show that the mean hydrophobicity and mean hydrophobic moment alone cannot be used as a general tool to predict the antibacterial activity of a peptide. These parameters are useful only when comparing analogs derived from the same peptide. Although experimental conditions may vary, the RP-HPLC retention time seems to be a better parameter for prediction of antibacterial activity. This is illustrated in ▶. The charge at pH 7.0, 〈H〉, and 〈μH〉 of the truncated analogs FCt 1–11, FCt1–16, and FNt17–25 are comparable with the antibacterial peptides, anoplin, A7-Anoplin (Ifrah et al. 2005), and I6A8L15I17-M2a (Wieprecht et al. 1997). However, the antibacterial activities are very different.

Table 2.

Comparison of different peptides with respect to charge, 〈H〉, 〈μ〉 , MIC-activity (μM), Rt

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Conformational studies

The secondary structure of fallaxin and analogs was investigated using far-UV CD spectroscopy. Four different solvents at pH 7.0 were used: (1) phosphate buffered saline (PBS); (2) 50% trifluoroethanol (TFE; cosolvent known to stabilize secondary structures) in PBS, and two different lipid-vesicle suspensions (SUVs) in PBS; (3) DMPC/DMPG (3:1) mimicking a bacterial membrane; and (4) DMPC mimicking a mammalian membrane.

Far-UV CD experiments on fallaxin and several analogs (FA1, FA8, FL9, FA12, and FL14) in aqueous buffer all revealed a highly unfolded structure, most likely monomeric, with little intrinsic helicity (▶). These analogs were all soluble in PBS buffer.

Table 3.

Conformational studies, calculated α-helical content, and θ222/θ208 -ratios of fallaxin and a selection of Ala-scan analogs

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The far-UV CD spectra of fallaxin and Ala-scan analogs in 50% TFE revealed a characteristic α-helical structure with double minimas at 208 nm and 222 nm, corresponding to the π–π* and n–π* transitions. The helical content of fallaxin was calculated to be 53%, whereas all analogs, with the exception of FA25, had a larger helical content of 61%–96%. Attempts to correlate the calculated helical content with antibacterial and hemolytic activities of the analogs were inconclusive. The results are summarized in ▶.

Experiments on fallaxin and analogs FA1, FA8, FL9, FA12, and FL14 in DMPC and in DMPC/DMPG (3:1) all revealed a conformational change from interaction with the model membranes. Compared to the experiments run in TFE, the resulting far-UV CD-spectra of almost all analogs revealed a decreased negative band intensity at 208 nm (π–π*) and an increased negative band intensity at 222 nm (n–π*) in both vesicle suspensions. The ratio of the molar ellipticities at 222 nm and 208 nm ([θ]222/[θ]208) has been used in several studies to evaluate the presence of coiled-coil helices (Ladokhin and White 1999; Choy et al. 2003). According to the literature, a value below 1 (∼0.80) is characteristic for a noninteracting α-helix, whereas a value above 1 (∼1.04) indicates a two-stranded coiled-coil (Cooper and Woody 1990). The most significant changes were observed for two of the most active analogs, FL9 and FL14 (MICE.coli-value of 12.5 μM). These analogs had a ratio value of 1.19 and 0.99 in DMPC/DMPG and 1.04 and 0.96 in DMPC, respectively, indicative of coiled-coil conformation. The observed [θ]222/[θ]208-ratio of 0.81 and 0.78 in the TFE experiments indicates a characteristic noninteracting α-helical structure in accordance with the CD spectrum (▶). [θ]222/[θ]208-ratios for analogs FA1, FA8, FL9, FA12, FL14, and fallaxin are listed in ▶. As previously described, the analogs in which an alanine residue had been substituted for a leucine (FL analogs) displayed an increased antibacterial activity toward the Gram-positive bacteria. The cell wall of the Gram-positive bacteria primarily consists of a thick hydrophilic peptidoglycan layer covering a hydrophobic phospholipid membrane. In order to successfully penetrate the peptidoglycan layer and reach the phospholipid membrane, the coiled-coil structure is favored, thus enabling the peptides to shield their individual hydrophobic side chains. Helical wheel projections of FL14 and FL9 reveal a more pronounced leucine-zipper motif compared to both fallaxin, FA1, FA8, and FA12. However, FA12 also showed an increased antibacterial activity toward the Gram-positive bacteria MSSA, MRSA, and VISA. These results may be explained by another mechanism, probably driven by electrostatic interactions.

Figure 1.

Figure 1.

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Structure analysis of fallaxin analogs by far-UV CD spectroscopy. Shown are the effects of different solvents/lipids on the far-UV CD spectra of (A) FA1, (B) FA12, and (C) FL14. The far-UV CD spectra were recorded on peptide concentrations of 25 μM in four different solvents/lipids, all at pH 7.0. (Black lines) 10 mM phosphate buffered saline (PBS); (green lines) 50% trifluoroethanaol (TFE) in 10 mM PBS; (blue lines) 4 mM DMPC in 10 mM PBS; (pink lines) 4 mM DMPC/DMPG (3:1) in 10 mM PBS. The calculated α-helix content and [θ]222/[θ]208 ratios are listed in ▶.

The analogs FA1 and FA8 contain approximately the same percentage of α-helix in 50% TFE, are equally hydrophobic (〈H〉 = −0.0256), and have an almost identical mean hydrophobic moment (〈μH〉 = 0.1020 and 0.1019). Therefore, it is interesting that these peptides do not display the same antibacterial activity. FA8 has a MIC-value toward E. coli of 25 μM, whereas FA1 as the least active of the investigated analogs has a MICE.coli-value >100. Compared to FA8 and to the rest of the analogs, FA1 is least likely to form a higher-ordered structure, as [θ]222/[θ]208 values of 0.91 and 0.94 are observed in DMPC and DMPC/DMPG, indicating more monomeric α-helix structure. This could also explain why FA1 does not show antibacterial activity at concentrations below 100 μM. As described above, the involvement of higher-ordered structures was also indicated from the antibacterial activities (MIC-values).

Conclusion

In this paper, we present a structure–activity study of the antibacterial peptide fallaxin. The set of C- and N-terminal truncated analogs of fallaxin all showed an antibacterial activity of >100 μM. We therefore conclude that the full fallaxin peptide 1–25 is required to display antibacterial activity. The alanine scan identified seven analogs (FA8, FL9, FL10, FA12, FL14, FA15) that displayed MIC-values of 25 μM or better against at least one of the bacterial strains tested. Generally, the potent analogs were more active against Gram-negative bacteria than Gram-positive bacteria. The compounds with the best MIC-activity also showed the highest hemolytic activity and longest HPLC-retention times. Our best candidate was FA12, which displayed MIC-values of 3.12, 25, 25, and 50 μM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. We were able to group the active and inactive analogs according to mean hydrophobicity and mean hydrophobic moment. The majority of the active fallaxin analogs had a mean hydrophobicity ranging from −0.0256 to −0.0088 and a hydrophobic moment ranging from 0.00766 to 0.1164. The less active analogs had values outside this range. Our results show that the mean hydrophobicity and mean hydrophobic moment alone cannot be used as a general tool to predict antibacterial activity of a peptide. The RP-HPLC-retention time is a better parameter for prediction of antibacterial activity.

The secondary structure of fallaxin and a selection of analogs, active and inactive (FA1, FA8, FL9, FA12, and FL14) were investigated in four different solvents—phosphate buffered saline, TFE, and two vesicle suspensions. Results from these experiments indicate that a coiled-coil conformation could be an important structural trait for antibacterial activity.

Data obtained during this study support fallaxin analogs as promising lead structures in the development of new antibacterial agents.

Materials and Methods

Chemicals and materials

The chemicals and materials used were TentaGel S RAM resin (loading 0.24 meq/g) from RAPP Polymers; TFE from ACROS; DIEA, α-cyano-p-hydroxycinnamic acid and MTT from Aldrich; HATU and protected amino acids from PerSeptive Biosystems, Novabiochem, Molekula, and Bachem; phenol, piperidine, anhydrous ampicillin, and TIS from Fluka; L-α-amino-butanoic acid, BSA, Triton X-100, ACTH, and Substance P from Sigma; phenylisothiocyanate from Pierce; and TFA from Merck. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in chloroform and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) in chloroform:methanol:water were purchased from Avanti Polar Lipids (Alabaster). All starting chemicals were used without further purification. Sterile 96-well polypropylene plates were from COSTAR, Corning Incorporated (Corning); sterile 96-well polystyrene microtiter plates were from Nunc; Mueller-Hinton broth was from Fluka. Freshly drawn human erythrocytes in CPD (citrate-phosphatedextrose) buffer were obtained from the Copenhagen University Hospital blood bank (Copenhagen).

HPLC, MALDI TOF-MS, and amino acid analysis (AAA)

Analytical HPLC was performed using a Waters C18-reverse-phase column (Symmetry C18, 5 μm, 4.6 mm × 250 mm, Part No. WAT054275; Waters Corp.) on a Waters 600E system. Preparative HPLC was done on a Vydac C18-reverse-phase column (10–15 μm, 22 mm × 250 mm, Part No. 218TP101522; VYDAC). Samples were chromatographed as described by Ifrah et al. (2005). MALDI-TOF-MS was done on a VG Tof Spec E Fisons instrument (Fisons Instruments), using α-cyano-p-hydroxycinnamic acid as matrix. Substance P and ACTH were used as calibrants. Amino acid analysis was performed on a Waters PicoTag analyzer as described (Ryge et al. 2004).

Peptide synthesis, purification, and characterization

Peptide syntheses were performed using standard Fmoc chemistry procedures on Tenta Gel S RAM resin. Protected amino acids were coupled in fourfold excess employing a protocol using HATU/DIEA (1:1.5) activation as described by Ryge et al. (2004). Cleavage and deprotecting were carried out using reagent L: TFA/TIS/DTT/H2O (88:2:5:5) for 2 h. The final workup was performed as described by Ifrah et al. (2005). The peptide products were purified by preparative HPLC and lyophilized, and the masses were verified by MALDI TOF-MS. The concentration of stock solutions was determined by amino acid analysis. Stock solutions were used for antibacterial testing, hemolytic activity studies, and CD spectroscopy analysis.

Antibacterial and hemolytic activity

The strains used for determining antibacterial activity included the six American Type Culture Collection (ATCC) strains—E. coli ATCC 25922; K. pneumoniae ATCC 700603; P. aeruginosa ATCC 27853; vancomycin-intermediate-resistant S. aureus ATCC 700699MU50; methicillin-resistant S. aureus ATCC 29213; and methicillin-susceptible S. aureus ATCC 33591. The minimal inhibitory concentration (MIC) of each peptide was determined using a broth microdilution assay modified from the method of Hancock (http://www.cmdr.ubc.ca/bobh/methods.php; accessed April 2007).

Freshly drawn human erythrocytes, in citrate-dextrose-phosphate (CDP) buffer, from Copenhagen University Hospital were used to determine the hemolytic activity as described by Ryge and Hansen (2005).

CD spectroscopy

Far-UV-CD spectra were recorded at room temperature on a Jasco J810 spectropolarimeter. Peptides were dissolved to a final concentration of 25 μM in either 10 mM phosphate buffer (pH 7.0), 10 mM phosphate buffer (pH 7.0) containing 50% TFE, 4 mM DMPC in 10 mM phosphate buffer (pH 7.0), or 4 mM DMPC/DMPG (3:1) in 10 mM phosphate buffer (pH 7). Scans between 290 nm and 190 nm, with an average of five scans per experiment were averaged from recording in a 0.1-cm cell. Following baseline correction, the percentage α-helicity of the peptides was determined as described by Konno et al. (2001) and McLean et al. (1991). Small unilamellar vesicles (SUVs) were prepared as described by Gad et al. (E.A. Gad, J.G. Olsen, K. Teilum, J.A. Martial, I. Struman, and B.B. Kragelund, unpubl.).

Acknowledgments

We thank Frank Hansen and Jette Petersen for excellent technical assistance. Professor Morten Dziegel, Copenhagen University Hospital blood bank, is thanked for supplying blood. S.L.N. greatly acknowledges a scholarship from Novo-Nordisk/Novozymes. This work was supported by the J.S. Foundation and the Aase and Ejnar Danielsens Foundation to P.R.H., and from the Danish National Science Counsil to B.B.K. (#2140604).

A preliminary account of this work will be presented at the 20th American Peptide Symposium, Montreal, Canada, 2007.

Footnotes

Reprint requests to: Paul Robert Hansen, Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark; e-mail: prh@life.ku.dk; fax: 45-35332398.

Abbreviations: ACTH, adrenocorticotropic hormone; ATCC, American type culture collection; Boc, tert-butyloxycarbonyl; BSA: bovine serum albumin; CFU, colony forming unit; CPD, citrate-phosphate-dextrose; DIEA, diisopropylethylamine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; DTT: dithiothrethiol; Fmoc: 9-fluorenylmethoxy; 〈H〉, mean hydrophobicity; HC50: the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37°C; 〈μH〉, mean hydrophobic moment; HATU, (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate); HOBt, 1-hydroxybenzotriazole; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MIC, minimum inhibitory concentration; NMP, N-methyl-2-pyrrolidone; SUV, small unilamellar vesicles; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TIS, triisopropylsilane; Trt, triphenylmethyl.

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