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Una alternativa promisoria se ha encontrado para tratar una de las enfermedades epiteliales que más aqueja a la población mundial, se trata de los péptidos antimicrobianos que combaten la enfermedad del acné, desde el punto de vista dermatológico el acné es una enfermedad causada por la presencia de la bacteria comensal Propionibacterium acnés, la cual genera un problema de salud especialmente en la población joven que se ve afectada por secuelas de inflamación y pigmentación. El proceso infeccioso causado es reconocido por el sistema inmunológico generando la activación de péptidos antimicrobianos como respuesta complementaria del sistema inmune innato, estas biomoléculas se han constituido en posibles agentes terapéuticos en reemplazo de los fármacos convencionales que presentan efectos adversos en los pacientes y han generado resistencia bacteriana. En este documento se presenta una revisión de aspectos relevantes de la enfermedad, características de los péptidos antimicrobianos en general y los péptidos más relevantes que han demostrado actividad bactericida contra este patógeno. Abstract: A promising alternative has been found to treat one of the epithelial diseases that most afflicts the world population, it is the antimicrobial peptides that fight acne disease, from the dermatological point of view acne is a disease caused by the presence of the commensal bacterium Propionibacterium acnes, which generates a health problem especially in the young population that is affected by sequelae of inflammation and pigmentation. The infectious process caused is recognized by the immune system generating the activation of antimicrobial peptides as a complementary response of the innate immune system, these biomolecules have become potential therapeutic agents in replacement of conventional drugs that have adverse effects in patients and have generated resistance bacterial This document presents a review of relevant aspects of the disease, characteristics of antimicrobial peptides in general and the most relevant peptides that have demonstrated bactericidal activity against this pathogen. Maestría

La resistencia de los microorganismos a los antibióticos es un problema creciente de salud a nivel mundial, la búsqueda de nuevos antimicrobianos ha venido cobrando cada vez mayor relevancia y los péptidos antimicrobianos (PAMs), de origen natural o sintético, han surgido como una opción terapéutica promisoria. El péptido sintético [A331]-PfRif (321-340) derivado de una proteína de Plasmodium falciparum, es activo contra bacterias E. coli ML35 (CMI 25 µM) pero causa lisis de los glóbulos rojos desde 1,56 µM. Con el fin de identificar péptidos de secuencia corta y actividad selectiva contra bacterias, en este trabajo se evaluó la actividad antibacteriana de péptidos sintéticos cortos derivados de [A331]-PfRif (321-340). Se observó que los péptidos [A331]-PfRif (324-340), [A331]-PfRif (325-340) y [A331]-PfRif (327-340) de 17,16 y 14 residuos respectivamente, son activos contra bacterias E. coli y no son hemolíticos a concentraciones ≤200 µM. Por esto, su índice terapéutico incrementó significativamente (más de 200, 160 y 40 veces, respectivamente), en comparación con el del péptido original [A331]PfRif (321-340) (IT 0,083). El análisis informático y por dicroísmo circular sugiere que la capacidad de asumir una conformación α-helicoidal en ambientes que mimetizan las condiciones del entorno de membranas cargadas negativamente y la presencia de dos lisinas en la cara no polar de la hélice α anfipática son cruciales para la actividad y selectividad de estas secuencias. Aunque las tres secuencias muestran características interesantes, [A331]-PfRif (324-340): RKKKMKKALQYIKLLKE fue el producto de síntesis de mayor pureza, tiene el mayor IT, fue el único activo contra una cepa de P. aeruginosa y adicionalmente presentó efecto sinérgico con Gentamicina; por esto sugerimos a [A331]-PfRif (324-340) como un candidato potencial para su uso como antimicrobiano. Abstract: Microorganisms resistance to antibiotics is a growing problem of health worldwide, then the search for new antimicrobials has been gaining increasing relevance and antimicrobial peptides (AMPs), of natural or synthetic origin, have emerged as a therapeutic promising option. Synthetic peptide [A331]-PfRif (321-340) which is derived from a Plasmodium falciparum protein, is active against E. coli ML35 bacteria (25 μM MIC) but causes lysis of red blood cells from 1.56 μM. In order to identify peptides having short sequence and selective activity against bacteria, in this work, the antibacterial activity of short synthetic peptides derived from [A331]-PfRif (321-340) was evaluated. It was observed that the peptides [A331]-PfRif (324-340), [A331]-PfRif (325-340) and [A331]-PfRif (327-340) with 17, 16 and 14 residues respectively, are active against bacteria E. coli and are not hemolytic at concentrations ≤200 μM. For this, its therapeutic index increased significantly (more than 200, 160 and 40 times, respectively), compared to that of the original peptide [A331]-PfRif (321-340) (IT 0.083). Bioinformatic analysis and circular dichroism suggest that ability to assume α-helical conformation in environments mimic negatively charged membranes environmental conditions and presence of two lysines in the non-polar face of the amphipathic α-helix are crucial for the activity and selectivity of these sequences. Although the three sequences show interesting characteristics, peptide [A331]-PfRif (324-340): RKKKMKKALQYIKLLKE was the synthesis product having greater purity, highest IT, and the only with activity against a P. aeruginosa stain; additionally presented synergistic effect with Gentamicin. For this, we suggest that the peptide [A331]-PfRif (324-340) is a potential candidate for its use as an antimicrobial. Maestría

International audience; In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of argininerich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically > 10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.

Antimicrobial peptides (AMPs) attack bacterial membranes selectively, killing microbes at concentrations that cause no toxicity to the host cells. This selectivity is not due to interaction with specific receptors but is determined by the different lipid compositions of the membranes of the two cell types and by the peculiar physicochemical properties of AMPs, particularly their cationic and amphipathic character. However, the available data, including recent studies of peptide-cell association, indicate that this picture is excessively simplistic, because selectivity is modulated by a complex interplay of several interconnected phenomena. For instance, conformational transitions and self-assembly equilibria modulate the effective peptide hydrophobicity, the electrostatic and hydrophobic contributions to the membrane-binding driving force are nonadditive, and kinetic processes can play an important role in selective bacterial killing in the presence of host cells. All these phenomena and their bearing on the final activity and toxicity of AMPs must be considered in the definition of design principles to optimize peptide selectivity.

A novel amphipathic phenolic compound, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), that can be isolated from the Pacific oyster (Crassostrea gigas) has been found to protect human hepatocytes against oxidative stress. This study aims to establish a method for the measurement of DHMBA for industrial application. Liquid chromatography-tandem mass spectrometry using deuterated DHMBA as an internal standard and a polar end-capped ODS (Hypersil GOLD aQ) as the solid phase was validated. The limit of detection was 0.04 pmol (S/N = 5), and the limit of quantitation was 0.1 pmol (S/N = 10). The calibration curve was linear throughout the range of 0.1 - 16 pmol (r(2) = 0.9995). This method successfully quantified DHMBA in oysters from 11 sea areas in Japan. The results showed that the yield of DHMBA was variable from 9.8 to 58.8 μg g(-1) whole oyster meat wet weight but not affected by the seawater temperature. The proposed LC-MS/MS method is useful in quantitative studies for DHMBA and potentially for other amphipathic substances.

Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains, five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation.

Defense peptides are small amphipathic molecules that exhibit antimicrobial, antitumor, antiviral, and immunomodulatory properties. This review summarizes current knowledge on the mechanisms of antimicrobial activity of cationic and anionic defense peptides, indicating peptide-based as well as microbial cell-based factors affecting this activity. The peptide-based factors include charge, hydrophibicity, and amphipathicity, whereas the pathogen-based factors are membrane lipid composition, presence of sterols, membrane fluidity, cell wall components, and secreted factors such as extracellular proteinases. Since defense peptides have been considered very promising molecules that could replace conventional antibiotics in the era of drug-resistant pathogens, the issue of microbial resistance to antimicrobial peptides (AMPs) is addressed. Furthermore, selected approaches employed for optimization and de novo design of effective AMPs based on the properties recognized as important for the function of natural defense peptides are presented.

Methods are described for the design of amphipathic helical AMPs, to improve potency and/or increase selectivity with respect to host cells. One method is based on the statistical analysis of known helical AMPs to derive a sequence template and ranges of charge, hydrophobicity, and amphipathicity (hydrophobic moment) values that lead to broad-spectrum activity, but leaves optimization for selectivity to subsequent rounds of SAR determinations. A second method uses a small database of anuran AMPs with known potency (MIC values vs. E. coli) and selectivity (HC50 values vs. human erythrocytes), as well as the concept of longitudinal moment, to suggest sequences or sequence variations that can improve selectivity. These methods can assist in the initial design of novel AMPs with useful properties in vitro, but further development requires knowledge-based decisions and a sound prior understanding of how structural and physical attributes of this class of peptides affect their mechanism of action against bacteria and host cells.

International audience; Temporin-SHc (FLSHIAGFLSNLF(amide)) first isolated from skin extraction of the Tunisian frog Pelophylax saharica, which shows potent antimicrobial activity against Gram-positive bacteria and is highly active against yeasts and fungi without hemolytic activity at antimicrobial concentrations. The peptide adopts well-defined alpha-helical conformation when bound to SDS micelles. In this study, we explored the effects of residue at position 5 and the N-terminus hydrophobic character on the hydrophilic/polar face of temp-SHc, on its biological activities (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity and helicity). Antibacterial and hemolytic properties of temporin-SHc derivatives depend strongly on physicochemical properties. Therefore, slight decreasing amphipathicity together with hydrophobicity and helicity by the substitution Ile(5) -> Leu decreased antimicrobial potency approximately twofold without changing of hemolytic activity. It is noteworthy that a conservative amino acid substitution decreases the antimicrobial activity, underlining the differences between Leu/Ile side chains insertion into the lipid bilayer. While the modification of N-terminal hydrophobic character by four residue inversion decreased amphipathicity (twofold) of (4-1)L(5)temp-SHc and resulted in an increase in antibacterial activity against E. coli, E. faecalis and C. parapsilosis of at least fourfold, its therapeutic potential is limited by its drastic increase of hemolysis (LC50 = 2 mu M). We found that the percentage of helicity of temp-SHc analog is directly correlated to its hemolytic activity. Last, the hydrophobic N-terminal character is an important determinant of antimicrobial activity.

Bacteria have acquired extensive resistance mechanisms to protect themselves against antibiotic action. Today the bacterial membrane has become one of the “final frontiers” in the search for new compounds acting on novel targets to address the threat of multi-drug resistant (MDR) and XDR bacterial pathogens. β-Hairpin antimicrobial peptides are amphipathic, membrane-binding antibiotics that exhibit a broad range of activities against Gram-positive, Gram-negative, and fungal pathogens. However, most members of the class also possess adverse cytotoxicity and hemolytic activity that preclude their development as candidate antimicrobials. We examined peptide hydrophobicity, amphipathicity, and structure to better dissect and understand the correlation between antimicrobial activity and toxicity, membrane binding, and membrane permeability. The hydrophobicity, pI, net charge at physiological pH, and amphipathic moment for the β-hairpin antimicrobial peptides tachyplesin-1, polyphemusin-1, protegrin-1, gomesin, arenicin-3, and thanatin were determined and correlated with key antimicrobial activity and toxicity data. These included antimicrobial activity against five key bacterial pathogens and two fungi, cytotoxicity against human cell lines, and hemolytic activity in human erythrocytes. Observed antimicrobial activity trends correlated with compound amphipathicity and, to a lesser extent, with overall hydrophobicity. Antimicrobial activity increased with amphipathicity, but unfortunately so did toxicity. Of note, tachyplesin-1 was found to be 8-fold more amphipathic than gomesin. These analyses identify tachyplesin-1 as a promising scaffold for rational design and synthetic optimization toward an antibiotic candidate.

Antibacterial, membrane-lytic peptides belong to the innate immune system and host defense mechanism of a multitude of animals and plants. The largest group of peptide antibiotics comprises peptides which fold into an amphipathic α-helical conformation when interacting with the target. The activity of these peptides is thought to be determined by global structural parameters rather than by the specific amino acid sequence. This review is concerned with the influence of structural parameters, such as peptide helicity, hydrophobicity, hydrophobic moment, peptide charge and the size of the hydrophobic/hydrophilic domain, on membrane activity and selectivity. The potential of these parameters to increase the antibacterial activity and to improve the prokaryotic selectivity of natural and model peptides is assessed. Furthermore, biophysical studies are summarized which elucidated the molecular basis for activity and selectivity modulations on the level of model membranes. Finally, the knowledge about the role of peptide structural parameters is applied to understand the different activity spectra of natural membrane-lytic peptides.

The research described in this thesis focuses on synthetic modifications of the antibiotic peptide gramicidin S (GS). The aim of the research is the development of non__toxic analogs of GS using conformational and amphipathic changes induced by sugar amino acids (SAAs) and/or non__proteinogenic amino acids. The physical properties of the synthetic derivatives were studied using NMR, Molecular Modeling, Circular Dichroism, LC/MS, and X-ray techniques. Evaluation of the biological activity (antimicrobial activity and toxicity) of the peptides revealed that various synthetic derivatives appeared to be less toxic than GS. By the frequent use and misuse of antibiotics the amount of untreatable bacterial infections has increased. This research has contributed to the development of antibiotics for which resistance by bacteria should not easily occur.

Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties. © 2010 Elsevier Ltd. All rights reserved.

Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties. © 2010 Elsevier Ltd. All rights reserved.

The therapeutic index (TI) of peptide antibiotics is defined as the ratio of hemolytic to antimicrobial activity. We constructed the database of 51 non-homologous helical antimicrobial peptides, isolated from frogs, with associated therapeutic index values determined on E. coli and human erythrocytes. Sequence attributes calculated by the SPLIT 3.5 server (split.pmfst.hr/split) are used for developing relationships between peptide structure and its therapeutic index. Helical amphipathic segments and G, Q, H, D, E amino acid frequencies are positively correlated with TI. High preferences for transmembrane helix conformation are negatively correlated with TI. The composite predictor is developed as a specific combination of these sequence-based parameters. Corresponding log-log model for estimating the therapeutic index exhibits good correlation (r = 0.9) with experimental TI values.

An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, α-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the ‘sequence template’ approach which we have used to design potent artificial helical AMPs, to guide structure–activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.

0\. TITELBLATT UND INHALTSVERZEICHNIS 1\. EINLEITUNG 3 1.1. TRADITIONELLE ANTIBIOTIKA & ANTIBIOTIKARESISTENZ 3 1.2. ANTIMIKROBIELLE PEPTIDE: ENTDECKUNG & VORKOMMEN 4 1.3. KLASSIFIZIERUNG DER ANTIMIKROBIELLEN PEPTIDE 7 1.4. WIRKMECHANISMEN ANTIMIKROBIELLER PEPTIDE 8 1.5. FRAGE- UND ZIELSTELLUNG DER ARBEIT 14 2\. ERGEBNISSE 17 2.1. SYNTHESE & CHARAKTERISIERUNG DER HEXAPEPTIDE 17 2.2. BIOLOGISCHE AKTIVITÄTEN 19 2.3. CD-SPEKTROSKOPIE 22 2.4. HYDROPHOBIZITÄTSPARAMETER 25 2.5. FARBSTOFFFREISETZUNG AUS VESIKELN 29 2.6. BINDUNG AN LIPID A 32 2.7. ZELLPERMEABILISIERUNG VON ESCHERICHIA COLI-ZELLEN 34 3\. MATERIAL & METHODEN 39 3.1. MATERIALIEN 39 3.2. PEPTIDSYNTHESE & CHARAKTERISIERUNG 39 3.3. BIOLOGISCHE UNTERSUCHUNGEN 41 3.3.1. ANTIMIKROBIELLE AKTIVITÄT 41 3.3.2. ZELLPERMEABILISIERUNGSVERSUCH 42 3.3.3. HÄMOLYTISCHER TEST 42 3.4. BIOPHYSIKALISCHE UNTERSUCHUNGEN 43 3.4.1. CIRCULARDICHROISMUS MESSUNGEN 43 3.4.2. BINDUNGSUNTERSUCHUNGEN 43 3.4.3. FARBSTOFFFREISETZUNGSVERSUCHE 44 4\. DISKUSSION & EPILOG 46 4.1. DISKUSSION 46 4.2. EPILOG 54 5\. LITERATURVERZEICHNIS 56 6\. ANHANG 67 6.1. LISTE DER VERWENDETEN ABKÜRZUNGEN 67 6.2. AMINOSÄUREN / ÜBERSICHT 68 6.3. LIPID- / CHOLESTERINSTRUKTUREN 69 6.4. CHEMISCHE STRUKTUR VON EINEM LIPOPOLYSACCHARID 70 6.5. ZUSAMMENFASSUNG / SUMMARY 71 6.5.1. ZUSAMMENFASSUNG 71 6.5.2. SUMMARY 71 6.6. PUBLIKATIONEN 73 6.6.1. ARTIKEL 73 6.6.2. SYMPOSIUMSBEITRÄGE 73 6.7. CURRICULUM VITAE 74 6.8. DANKSAGUNG 76 6.9. EIDESSTATTLICHE ERKLÄRUNG 77, Die vorliegende Arbeit befaßt sich mit Struktur-Wirkungsbeziehungen von R-/W-reichen antimikrobiellen Hexapeptiden, welche von der Sequenz RRWWRF abgeleitet waren. Studien an Gram-positiven und �negativen Bakterien haben gezeigt, dass die cyclischen Analoga im Vergleich zu den linearen Peptiden eine hohe antimikrobielle Aktivität besaßen bei gleichzeitiger moderater Lyse der Erythrocyten. Farbstofffreisetzungsversuche aus Liposomen, welche die Lipidkomposition der Cytoplasmamembran der jeweiligen Zellspezies repräsentierten, zeigten, dass die Peptide in der Lage waren, neutrale und stark negativ geladene Lipiddoppelschichten zu permeabilisieren. Diese Befunde korrelierten mit den Ergebnissen aus den biologischen und biophysikalischen Untersuchungen, und es konnte geschlussfolgert werden, dass die antimikrobiellen Aktivitäten gegen Bacillus subtilis und die Hämolyse aufgrund von Peptid-Membraninteraktionen zu Stande kamen. Obwohl eine hohe Aktivität gegen Gram-negative Bakterien beobachtet werden konnte, zeigten Permeabilisierungsstudien an entsprechenden Liposomen keine Lyse durch die Peptide. Ergebnisse der Permeabilisierungsstudien in vivo ließen erkennen, dass es zu einer Permeabilisierung der äußeren und nur in sehr geringem Umfang der inneren Membran kommt. Alle Ergebnisse zusammengenommen zeigten deutlich, dass die antimikrobielle Aktivität der untersuchten Peptide Strukturdeterminanten folgt. Dabei ist die Ausbildung einer amphipathischen Struktur, wie sie durch NMR-Messungen bestätigt wurde, essentiell für die antimikrobielle Wirkung., In the present thesis the structure-function relationships of R-/W-rich antimicrobial hexapeptides that were derived from the lead sequence RRWWRF were investigated. Studies revealed that cyclic analogues were more active against Gram-positive and Gram-negative bacteria than the corresponding linear analogues, but displayed at the same time only moderate haemolytic activity. Dye release experiments with liposomes representing the phospholipid composition of the target bilayer of each cell species demonstrated permeabilizing activity of the hexapeptides towards neutral and highly negatively charged phospholipid bilayers. From the correlations with the data from the biological and biophysical experiments it is concluded that the observed antimicrobial activity against Bacillus subtilis and the haemolytic effect are based on peptide membrane interactions. Even though very high antimicrobial activity against Gram-negative cells was observed, no permeabilization of the corresponding liposomes by the peptides could be demonstrated. Permeability studies in vivo showed strong permeabilization of the outer and only weak permeabilization of the inner membrane. Taken together the studies indicate that antimicrobial activity is strongly determined by peptide structure. Furthermore, the formation of an amphipathic structure, as revealed by NMR-studies, is essential for the antimicrobial acticity of the hexapeptide analogues.

Protein amphipathic conformations, mainly alpha-helices and beta-strands, are believed to play an important role in protein folding, stability and function. The most popular method for characterizing such structures is the hydrophobic moment. We have analyzed the distribution of hydrophobic moment characteristics (peak magnitude, amphipathic indices and characteristic frequency) in a data bank containing several families of distant sequences multiply aligned by structural superposition. Sequence fragments were classified according to alpha-helix, beta-strand, non-alpha and non-beta conformations. This data bank provided an enhanced sample space compared with those previously reported in the literature. Precautions were taken to reduce over-representation of homologous sequences. Approximately 50% of all individual alpha-helices showed a hydrophobic moment peak in the expected position of the periodicity spectrum while only 38% of individual beta-strands fell in the expected range. False positives account for a surprisingly large 14 and 36% of the non-alpha and non-beta samples respectively. Conservation of hydrophobic moment characteristics and mainly the hydrophobic peak position in the expected periodicity range was examined in the multiple alignments of the distant sequences. Helices tend to conserve more frequently their hydrophobic moment than any other conformation and yet only 13% of all helical segments display such conservation in three-quarters or more of the familial sequences; the similar observation for beta-strands was even lower at 9%. Nonetheless, strongly hydrophobic positions within the structural segments were more conserved than expected.

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K+ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER > AMP >> ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC16:1PC, diC18:1PC, C16:0/C18:1PC, diC20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic–hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.