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The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The amphibian host-defense peptide caerin 1.8 [(1)GLFKVLGSV(10)AKHLLPHVVP(20)VIAEKL(NH2)] inhibits fibril formation of amyloid β 1-42 [(1)DAEFRHDSG(10)YEVHHQKLVF(20)FAEDVGSNKG(30)AIIGLMVGGV(40)VIA] [Aβ42] (the major precursor of the extracellular fibrillar deposits of Alzheimer's disease). Some truncated forms of caerin 1.8 also inhibit fibril formation of Aβ42. For example, caerin 1.8 (1-13) [(1)GLFKVLGSV(10)AKHL(NH2) and caerin 1.8 (22-25) [KVLGSV(10)AKHLLPHVVP(20)VIAEKL(NH2)] show 85% and 75% respectively of the inhibition activity of the parent caerin 1.8. The synthetic peptide KLVFFKKKKKK is a known inhibitor of Aβ42 fibril formation, and was used as a standard in this study. Caerin 1.8 is the more effective fibril inhibitor. IC50 values (± 15%) are caerin 1.8 (75 μM) and KLVFFKKKKKK (370 μM). MALDI mass spectrometry shows the presence of a small peak corresponding to a protonated 1:1 adduct [caerin 1.8/Aβ42]H(+). Molecular dynamics simulation suggests that both hydrogen bonding and hydrophobic interactions between Aβ42 and caerin 1.8 facilitate the formation of a 1:1 complex in water. Fibril formation from Aβ42 has been proposed to be based around the (16)KLVF(20)F region of Aβ42; this region in the 1:1 complex is partially blocked from attachment of a further molecule of Aβ42., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The major mode of transmission of the human immunodeficiency virus (HIV) is by sexual intercourse. In the effort to halt the spread of HIV, one measure that holds great promise is the development of effective microbicides that can prevent transmission. Previously we showed that several amphibian antimicrobial peptides (AMPs) completely inhibit HIV infection of T cells while maintaining good viability of the T cell targets. These peptides also inhibited the transfer of HIV by dendritic cells (DCs) to T cells when added up to 8h after virus exposure. Here we report on the anti-HIV activity of 18 additional structurally related caerin 1 family peptides in comparison with our previous best candidate caerin 1.9. Nine peptides were equally effective or more effective in the inhibition of T cell infection and disruption of the HIV envelope as caerin 1.9. Of those nine peptides, three peptides (caerin 1.2, caerin 1.10, and caerin 1.20) exhibited excellent inhibition of HIV infectivity at low concentrations (12-25μM) and limited toxicity against target T cells and endocervical epithelial cells. There was a direct correlation between the effectiveness of the peptides in disruption of the viral envelope and their capacity to inhibit infection. Thus, several additional caerin 1 family peptides inhibit HIV infection have limited toxicity for vaginal epithelial cells, and would be good candidates for inclusion in microbicide formulations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Calmodulin (CaM) is a ubiquitous protein in nature and plays a regulatory role in numerous biological processes, including the upregulation of nitric oxide (NO) synthesis in vivo. Several peptides that prevent NO production by interacting with CaM have been isolated in the cutaneous secretions of Australian amphibians, and are thought to serve as a defense mechanism against predators. In this work, we probe the mechanism by which three of these peptides, namely, caerin 1.8, dahlein 5.6, and a synthetic modification of citropin 1.1, interact with CaM to inhibit NO signaling. Isothermal titration calorimetry was used to determine thermodynamic parameters of the binding interactions and revealed that all the peptides bind to CaM in a similar fashion, with the peptide encapsulated between the two lobes of CaM. Ion mobility-mass spectrometry was used to investigate the changes in collision cross section that occur as a result of complexation, providing additional evidence for this binding mode. Finally, nuclear magnetic resonance spectroscopy was used to track chemical shift changes upon binding. The results obtained confirm that these complexes adopt canonical collapsed structures and demonstrate the strength of the interaction between the peptides and CaM. An understanding of these molecular recognition events provides insights into the underlying mechanism of the amphibian host-defense system.

A previous review summarized research prior to 2004 carried out on the bioactive host-defense peptides contained in the skin secretions of Australian anurans (frogs and toads). This review covers the extension of that research from 2004 to 2012, and includes membrane-active peptides (including antibacterial, anticancer, antifungal and antiviral peptides) together with the mechanisms by which these peptides interact with model membranes, peptides that may be classified as "neuropeptides" (including smooth muscle active peptides, opioids and immunomodulators) and peptides which inhibit the formation of nitric oxide from neuronal nitric oxide synthase. The review discusses the outcome of cDNA sequencing of signal-spacer-active peptides from an evolutionary viewpoint, and also lists those peptides for which activities have not been found to this time., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Anuran peptides which contain His, including caerin 1.8 (GLFKVLGSVAKHLLPHVVPVIAEKL-NH(2)), caerin 1.2 (GLLGVLGSVAKHVLPHVVPVIAEHL-NH(2)), Ala(15) maculatin 1.1 (GLFGVLAKVAAHVVAIEHF-NH(2)), fallaxidin 4.1 (GLLSFLPKVIGHLIHPPS-OH), riparin 5.1 (IVSYPDDAGEHAHKMG-NH(2)) and signiferin 2.1 (IIGHLIKTALGMLGL-NH(2)), all form MMet(2+) and (M + Met(2+)-2H(+))(2+) cluster ions (where Met is Cu, Mg and Zn) following electrospray ionisation (ESI) in a Waters QTOF 2 mass spectrometer. Peaks due to Cu(II) complexes are always the most abundant relative to other metal complexes. Information concerning metal(2+) connectivity in a complex has been obtained (at least in part) using b and y fragmentation data from ESI collision-induced dissociation tandem mass spectrometry (CID MS/MS). Theoretical calculations, using AMBER version 10, show that MCu(2+) complexes with the membrane active caerin 1.8, Ala(15) maculatin 1.1 and fallaxidin 4.1 are four-coordinate and approximating square planar, with ligands including His and Lys, together with the carbonyl oxygens of particular backbone amide groups. When binding can occur through two His, or one His and one Lys, the His/Lys ligand structure is the more stable for the studied systems. The three-dimensional (3D) structures of the complexes are always different from the previously determined structures of the uncomplexed model peptides (using 2D nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking solvents like trifluoroethanol/water)., (Copyright © 2011 John Wiley & Sons, Ltd.)

The increasing prevalence of antibiotic-resistant bacteria is becoming a public health crisis. Antimicrobial peptides (AMPs) are a promising solution, because bacterial resistance is less likely. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a versatile and valuable technique for investigation of these peptides. This article looks at the different approaches to the interpretation of QCM-D data, showing how to extract the maximum information from the data. Five AMPs of diverse charge, length and activity are used as case studies: caerin 1.1 wild-type, two caerin 1.1 mutants (Gly15Gly19-caerin 1.1 and Ala15Ala19-caerin 1.1), aurein 1.2 and oncocin. The interaction between the AMP and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane is analysed inter alia using frequency-dissipation plots (∆f-∆D plots) to ascertain the mechanism of action of the AMP. The ∆f-∆D plot can then be used to provide a fingerprint for the AMP-membrane interaction. Building up a database of these fingerprints for all known AMPs will enable the relationship between AMP structure and membrane activity to be better understood, hopefully leading to the future development of antibiotics without bacterial resistance.

Citropin 1.1, maculatin 1.1, and caerin 1.1 are short antibacterial cationic peptides from the skin glands of the Australian tree frog Litoria species. Several analogues have been synthesized to give a better insight into the relationship between the structure of the peptides and their antibacterial and haemolytic activity. Binding studies using a surface plasmon resonance (SPR) biosensor together with a vesicle-capture sensor chip have been used to investigate selectivity of the peptides and their analogues for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, as well as for vesicles made from lipid extracts from Escherichia coli and bovine brain. Data obtained for membrane selectivity using natural lipid extracts show better correlation with minimum inhibitory concentration (MIC) values against Gram-positive bacteria and haemolytic activity than that obtained using synthetic DMPG and DMPC. Electron microscopy and membrane leakage studies using Gram-positive bacteria gave further insight into the membrane disruption properties of the peptides. For maculatin 1.1, it was found that the central proline residue, which is responsible for a bend in the alpha-helical structure, is essential not only for the antibacterial activity but also for binding, and perturbation of membranes. The caerin analogues showed only small variations in their MIC values and membrane binding. In contrast, for citropin 1.1, the analogue replacing the aspartate with a lysine showed the lowest MIC against Gram-positive bacteria and best membrane binding to E. coli lipid extracts, coinciding with an increased hydrophobic moment of the peptide. These data give further insight into these antimicrobial natural products, toward the development and evaluation of these and other analogues as potential antibiotics.

The solution structure of fallaxidin 4.1a, a C-terminal amidated analogue of fallaxidin 4.1, a cationic antimicrobial peptide isolated from the amphibian Litoria fallax, has been determined by nuclear magnetic resonance (NMR). In zwitterionic dodecylphosphocholine (DPC) micelles, fallaxidin 4.1a adopted a partially helical structure with random coil characteristics. The flexibility of the structure may enhance the binding and penetration upon interaction with microbial membranes. Solid-state (31)P and (2)H NMR was used to investigate the effects of fallaxidin 4.1a on the dynamics of phospholipid membranes, using acyl chain deuterated zwitterionic dimyristoylphosphatidylcholine (DMPC-d(54)) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In DMPC-d(54) vesicle bilayers, fallaxidin 4.1a caused a decrease in the (31)P chemical shift anisotropy (CSA), and a decrease in deuterium order parameters from the upper acyl chain region, indicating increased lipid motion about the phosphate headgroups. Conversely, for DMPC-d(54)/DMPG, two (31)P CSA were observed due to a lateral phase separation of the two lipids and/or differing headgroup orientations in the presence of fallaxidin 4.1a, with a preferential interaction with DMPG. Little effect on the deuterated acyl chain order parameters was observed in the d(54)-DMPC/DMPG model membranes. Real time quartz crystal microbalance analyses of fallaxidin 4.1a addition to DMPC and DMPC/DMPG supported lipid bilayers together with the NMR results indicated transmembrane pore formation in DMPC/DMPG membranes and peptide insertion followed by disruption at a threshold concentration in DMPC membranes. The different interactions observed with "mammalian" (DMPC) and "bacterial" (DMPC/DMPG) model membranes imply fallaxidin 4.1a may be a useful antimicrobial peptide, with preferential cytolytic activity toward prokaryotic organisms at low peptide concentrations (<5 microM).

Positive and negative ion electrospray mass spectrometry together with Edman sequencing (when appropriate) has been used to sequence the host-defence peptides secreted from skin glands of the tree frog Litoria peronii. The peptide profiles are different in winter and summer. In winter, the frog produces small amounts of the known caerin 1.1 [GLLSVLGSVAKHVLPHVVPVIAEHL-NH(2)] (a wide-spectrum antibiotic) and caerin 2.1 [GLVSSIGRALGGLLADVVKSKQPA-OH], a narrow-spectrum antibiotic and an inhibitor of neuronal nitric oxide synthase. The major peptides produced throughout the year are the pGlu-containing peroniins 1.1 to 1.5 (e.g. peroniin 1.1 [pEPWLPFG-NH(2)], a smooth muscle contractor from 10(-7) M), and caerulein [pEQDY(SO(3)H)TGWMDF-NH(2)], a known and potent smooth muscle contractor from 10(-10) M. There are also some precursors to the peroniin 1 peptides, only detected in the skin secretion in summer, which are inactive and appear to be all (or part) of the spacer peroniin 1 peptides, e.g. peroniin 1.1b [SEEEKRQPWLPFG-NH(2)]. There are three members of the Litoria peronii Group of tree frogs classified in Australia, namely, L. peronii, L. rothii and L.tyleri. A comparison of the skin peptide profiles of L. peronii with those reported previously for L. rothii suggests that either these two species of tree frog are not as closely related as determined previously on morphological grounds, or that skin peptide divergence in tree frogs of this Group is more extensive than in others that have been studied., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

The glandular skin secretion of the Eastern Dwarf Tree Frog Litoria fallax contains nine peptides named fallaxidins. The sequences of these peptides were elucidated using a combination of positive and negative electrospray mass spectrometry together with Edman sequencing. Among these peptides are: (i) fallaxidins 1.1 and 2.1 which have the sequences YFPIPI-NH2 and FWPFM-NH2. The activities of these peptides are unknown, but it has been shown that they are not smooth muscle active, opioids or antimicrobially active, nor do they effect proliferation of lymphocytes; (ii) two weakly active antibiotics, fallaxidins 3.1 and 3.2 (e.g. fallaxidin 3.1, GLLDLAKHVIGIASKL-NH2), and a moderately active antibiotic fallaxidin 4.1 (GLLSFLPKVIGVIGHLIHPPS-OH). Fallaxidin 4.1 has an unusual sequence for an antibiotic, containing three Pro residues together with a C-terminal CO2H group. cDNA cloning has confirmed the identity of the nine isolated peptides from L. fallax, together with five additional peptides not detected in the peptide profile. The pre-regions of the nine preprofallaxidins are conserved and similar to those of the caerin peptides from L. caerulea and L. splendida, suggesting that the fallaxidin and caerin peptides, although significantly different in sequence, originated from a common ancestor gene.

Skin secretions of numerous Australian tree frogs contain antimicrobial peptides that form part of the host defense mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al. (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as novel antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1, which exist as random coil in aqueous solution but have alpha-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC ( d 54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid headgroups by deuterium and (31)P NMR, respectively. In this report we demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterized using surface plasmon resonance (SPR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides has significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface interaction for aurein 1.2 and pore formation rather than membrane lysis by the longer peptides.

The skin secretion of the Dainty Green Tree Frog Litoria gracilenta contains 16 peptides, which protect the animal from predators, both large and small. A combination of negative and positive ion electrospray mass spectrometry together with Lys-C enzymic digest and Edman sequencing identifies three new wide-spectrum caerin 1 antibiotics, namely Caerin 1.17 [GLFSVLGSVAKHLLPHVAPIIAEKL-NH2], Caerin 1.18 [GLFSVLGSVAKHLLPHVVPVIAEKL-NH2], and Caerin 1.19 [GLFKVLGSVAKHLLPHVAPIIAEKL-NH2], and a narrow spectrum antibiotic Caerin 3.5 [GLWEKVKEKANELVSGIVEGVK-NH2].

Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs.

Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the so-called carpet model and the pore-forming model. We report here on the lytic activity of antibiotic peptides from Australian tree frogs, maculatin 1.1, citropin 1.1, and aurein 1.2, on POPC or POPC/POPG model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide/lipid mol ratio necessary to induce 50% of probe leakage was smaller for maculatin compared with aurein or citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside of single giant unilamellar vesicles is observed after peptide exposure. We conclude that maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight markers). Conversely, citropin and aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, to study the relevance of the proline residue on the membrane-action properties of maculatin, the same experimental approach was used for maculatin-Ala and maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide/lipid mol ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of maculatin-Ala and maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring maculatin sequence. As observed for maculatin, the lytic action of Maculatin-Ala and maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.

Antimicrobial peptides, isolated from the dorsal glands of Australian tree frogs, possess a wide spectrum of biological activity and some are specific to certain pathogens. These peptides have the capability of disrupting bacterial membranes and lysing lipid bilayers. This study focused on the following amphibian peptides: (1) aurein 1.2, a 13-residue peptide; (2) citropin 1.1, with 16 residues; and (3) maculatin 1.1, with 21 residues. The antibiotic activity and structure of these peptides have been studied and compared and possible mechanisms by which the peptides lyse bacterial membrane cells have been proposed. The peptides adopt amphipathic alpha-helical structures in the presence of lipid micelles and vesicles. Specifically 15N-labelled peptides were studied using solid-state NMR to determine their structure and orientation in model lipid bilayers. The effect of these peptides on phospholipid membranes was determined by 2H and 31P solid-state NMR techniques in order to understand the mechanisms by which they exert their biological effects that lead to the disruption of the bacterial cell membrane. Aurein 1.2 and citropin 1.1 are too short to span the membrane bilayer while the longer maculatin 1.1, which may be flexible due to the central proline, would be able to span the bilayer as a transmembrane alpha-helix. All three peptides had a peripheral interaction with phosphatidylcholine bilayers and appear to be located in the aqueous region of the membrane bilayer. It is proposed that these antimicrobial peptides have a "detergent"-like mechanism of membrane lysis.

Caerin 1.1 is a potent broad-spectrum antibacterial peptide isolated from a number of Australian frogs of the Litoria genus. In membrane-like media, this peptide adopts two alpha-helices, separated by a flexible hinge region bounded by Pro15 and Pro19. Previous studies have suggested that the hinge region is important for effective orientation of the two helices within the bacterial cell membrane, resulting in lysis via the carpet mechanism. To evaluate the importance of the two Pro residues, they were replaced with either Ala or Gly. The antibacterial activity of these two peptides was tested, and their three-dimensional structures were determined using two-dimensional NMR spectroscopy and restrained molecular dynamics calculations. The resulting structures indicate that the central hinge angle decreases significantly upon replacement of the Pro residues with Gly and to a further extent with Ala. This trend was mirrored by a corresponding decrease in antibiotic activity, further exemplifying the necessity of the hinge in caerin 1.1 and related peptides. In a broader context, the use of Pro, Gly, and Ala variants of caerin 1.1 has enabled the relationship between conformational flexibility and activity to be directly investigated in a systematic manner.

The caerin 1 peptides are among the most powerful of the broad-spectrum antibiotic amphibian peptides. Caerin 1.1 has previously been shown to form an amphipathic helix-bend-helix structure in aqueous trifluoroethanol (H. Wong, J. H. Bowie, and J. A. Carver European Journal Biochemistry, 1997, Vol. 247, pp. 545-557) and structure-activity relationship studies indicate that both helices are required for activity, as well as flexibility in the bend region connecting the two. The structure of caerin 1.1 in dodecylphosphocholine micelles was investigated and shown to be very similar to that determined in aqueous trifluoroethanol. Caerin 1.4, which is identical to caerin 1.1, but with serine residues replacing Val5 and Gly7, is less active than caerin 1.1 against most bacterial species but has improved activity against Escherichia coli and Micrococcus luteus. The solution NMR structure of caerin 1.4 was determined in both aqueous trifluoroethanol and dodecylphosphocholine micelles, and was shown to be similar to caerin 1.1. It was concluded that differences in the hydrophobicity and hydrophilic angle of the first helix are probably responsible for the different spectra of antibacterial activity. The similarity of the structures calculated in aqueous trifluoroethanol and dodecylphosphocholine micelles suggests that, for caerin 1.1 and 1.4, these solvent systems are equally as good at representing a membrane environment., (Copyright 2003 Wiley Periodicals, Inc.)

A large number of bioactive peptides have been isolated from amphibian skin secretions. These peptides have a variety of actions including antibiotic and anticancer activities and the inhibition of neuronal nitric oxide synthase. We have investigated the structure-activity relationship of citropin 1.1, a broad-spectrum antibiotic and anticancer agent that also causes inhibition of neuronal nitric oxide synthase, by making a number of synthetically modified analogues. Citropin 1.1 has been shown previously to form an amphipathic alpha-helix in aqueous trifluoroethanol. The results of the structure-activity studies indicate the terminal residues are important for bacterial activity and increasing the overall positive charge, while maintaining an amphipathic distribution of residues, increases activity against Gram-negative organisms. Anticancer activity generally mirrors antibiotic activity suggesting a common mechanism of action. The N-terminal residues are important for inhibition of neuronal nitric oxide synthase, as is an overall positive charge greater than three. The structure of one of the more active synthetic modifications (A4K14-citropin 1.1) was determined in aqueous trifluoroethanol, showing that this peptide also forms an amphipathic alpha-helix.

Collision-induced fragmentations of deprotonated maculatin 1 peptides provide significant sequencing information. When the peptide lacks those residues which can fragment through their alpha side chains (e.g. Thr, Ser, Glu and Gln in this study) the basic alpha and beta' backbone cleavages occur from the [Mbond;H](-) anion. When Thr, Ser, Glu and Gln are present, the ease of side-chain fragmentation of these residues is: Thr (loss of MeCHO) > Ser (CH(2)O) > Glu (H(2)O) > Gln (NH(3)). When one of more of these residues is (are) present, the alpha and beta' cleavages often occur from a fragment rather than the [Mbond;H](-) anion, e.g. for Thr, the [(Mbond;H)(-)bond;MeCHO](-) anion. These four residues also initiate gamma backbone cleavage reactions. The relative abundances of peaks resulting from gamma cleavage are Glu > Ser = Thr > Gln for maculatin 1 spectra. An unusual Gln19/Ile17 cyclisation/cleavage reaction occurs in maculatin spectra: the peptide [Mbond;H](-) anion must adopt a helical conformation in order for these two groups to interact. Analogous fragmentations have been reported previously in the negative ion spectra of the caerin 1 peptides., (Copyright 2003 John Wiley & Sons, Ltd.)

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.

Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, alpha-helical structure in solution. Its orientation and conformation when incorporated to pre-formed DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicles was determined using polarised Fourier transform infrared-attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of 70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35 degrees to the membrane normal and with a predominant alpha-helical structure. These results suggest that maculatin 1.1 acts through a pore-forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and alpha-helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46 degrees) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with alpha-helix, beta-structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram-positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions.

The collision induced spectra of [M - H](-) anions from of caerin 1 peptides and some synthetic modifications show the usual alpha, beta and beta' backbone cleavages together with Ser (epsilon,gamma) and Glu (gamma) cleavages which break the peptide backbone in the vicinity of those residues. All of these cleavages require the peptide backbone to be flexible. There is also a backbone cleavage of a type not observed before. This cleavage involves nucleophilic attack of the carboxylate anion of the Glu23 side chain at the backbone CH of Ile 21. We propose that this cleavage requires the caerin peptide to be in an alpha helical conformation (the 3D structure that this peptide adopts in solution) in order that the interacting groups are held in close proximity., (Copyright 2002 John Wiley & Sons, Ltd.)

The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.

Nineteen citropin peptides are present in the secretion from the granular dorsal glands of the Blue Mountains tree-frog Litoria citropa; 15 of these peptides are also present in the secretion from the submental gland. Two major peptides, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), citropin 1.2 (GLFDIIKKVASVVGGL-NH2) and a minor peptide, citropin 1.3 (GLFDIIKKVASVIGGL-NH2) are wide-spectrum antibacterial peptides. The amphibian has an endoprotease which deactivates these membrane-active peptides by removing residues from the N-terminal end: loss of three residues gives the most abundant degradation products. The solution structure of the basic peptide citropin 1.1 has been determined by NMR spectroscopy [in a solvent mixture of trifluoroethanol/water (1 : 1)] to be an amphipathic alpha-helix with well-defined hydrophobic and hydrophilic regions. The additional four peptides produced by the dorsal glands are structurally related to the antibacterial citropin 1 peptides but contain three more residues at their C-terminus [e.g. citropin 1.1.3 (GLFDVIKKVASVIGLASP-OH)]. These peptides show minimal antibacterial activity; their role in the amphibian skin is not known.

A combination of electrospray mass spectrometry, Lys-C digest/mass spectrometry and automated Edman sequencing provides the amino acid sequences of nineteen citropin peptides isolated from the granular dorsal and submental glands of the Blue Mountains tree frog Litoria citropa. Citropin 1.1 [Gly Leu Phe Asp Val Ile Lys Lys Val Ala Ser Val Ile Gly Gly Leu (NH(2))] and citropin 1.2 [Gly Leu Phe Asp Ile Ile Lys Lys Val Ala Ser Val Val Gly Gly Leu (NH(2))] are the two major skin peptides: both show significant wide-spectrum antibacterial activity., (Copyright 1999 John Wiley & Sons, Ltd.)

Tadpoles of the Magnificent Tree Frog Litoria splendida produce host defence peptides early in their development and well before metamorphosis. Peptides were identified and characterized using high performance liquid chromatography and electrospray mass spectrometry. No host defence peptides were identified in the eggs. The neuropeptide caerulein was detected 10 d after egg deposition, and the antibiotic peptides caerin 1.1, caerin 1.6 and caerin 3.1 first appeared at 14 d. The concentration of peptides increases with the onset of metamorphosis at 84 d, when the host-defence peptide profile is the same as that of the adult.

Six peptides have been isolated and characterized from the dorsal glands of the tree frog Litoria genimaculata. One of these is the known hypotensive peptide caerulein; the others have been named maculatins. The amino acid sequences of the maculatin peptides have been determined using a combination of fast atom bombardment mass spectrometry and automated Edman sequencing. Four of the maculatin peptides show antibiotic activity, with maculatin 1.1 [GLFGVLAKVAAHVVPAIAEHF(NH2)] showing the most pronounced activity, particularly against gram-positive organisms. Maculatin 1.1 resembles the known caerin 1 antibiotic peptides, except that four of the central amino acid residues (of the caerin 1 system) are missing in maculatin 1.1. A comparison of the antibiotic activity of maculatin 1.1 with those of caerin 1.1 is reported.

The skin glands of the tree frog Litoria chloris contain a variety of peptides including four antibacterial peptides of the caerin 1 family. Two of these, caerins 1.6 and 1.7, are also present in the related species Litoria xanthomera. The other two peptides, caerins 1.8 and 1.9, are new. Their sequences are: GLFKVLGSVAKHLLPHVVPVIAEKL-NH2 [Caerin 1.8] and GLFGVLGSIAKHVLPHVVPVIAEKL-NH2 [Caerin 1.9]. Comparison of the skin peptide profiles of L. chloris and L. xanthomera confirms that these species are more closely related to each other than to any other species of the genus Litoria that we have studied. A comparison is made of the antibiotic activities of nine members of the caerin 1 family of peptides isolated from tree frogs of the genus Litoria.

Electrospray mass spectrometry and automated Edman sequencing provides the structures of two new caerin 1 antimicrobial peptides from the skin glands of the Australian tree frog Litoria chloris. These are: caerin 1.8 Gly Leu Phe Lys Val Leu Gly Ser Val Ala Lys His Leu Leu Pro His Val Val Pro Val Ile Ala Glu Lys Leu (NH2), and caerin 1.9, Gly Leu Phe Gly Val Leu Gly Ser Ile Ala Lys His Val Leu Pro His Val Val Pro Val Ile Ala Glu Lys Leu (NH2).

Caerin 1.1 is one of the major antimicrobial peptides isolated from the skin of the Australian green tree frog, Litoria splendida. Two-dimensional 1H-1H and 1H-13C NMR spectroscopy in trifluoroethanol/H2O (50:50, by vol.) have been used to assign the 1H and 13C-NMR spectra of this 25-amino-acid peptide. From an examination of these data, and using distance geometry and molecular dynamics calculations, the solution conformation of caerin 1.1 has been determined. The peptide adopts two well-defined helices from Leu2 to Lys11 and from Val17 to His24 separated by a region of less-defined helicity and greater flexibility. Overall, the peptide has a distinct amphipathic charge distribution. The solution structure of caerin 1.1 is compared with activity data against a variety of micro-organisms for the parent peptide and some naturally occurring and synthetic variants of caerin 1.1. The structural and activity data are consistent with caerin 1.1 interacting with membranes in a similar manner to other antimicrobial peptides, i.e. via a carpet-like mechanism whereby the individual peptides aggregate in a helical manner and orient themselves parallel to the membrane in a sheet-like arrangement [Shai, Y. (1995) Trends Biochem. Sci. 20, 460-464].

The secretion of the skin glands of the 'orange-thighed frog' Litoria xanthomera contains seven peptides. One of these is the know hypotensive peptide caerulein. Two new peptides, caerin 1.6 [GLFSVLGAVAKHVLPHVVPVIAEKL(NH2)], and caerin 1.7 [GLFKVLGSVAKHLLPHVAPVIAEKL(NH2)] show antibacterial properties. Two other peptides lack the first two amino acid residues of caerins 1.6 and 1.7 and show no antibacterial activity. The identification of the peptides in Litoria xanthomera confirms that this species is related to Litoria caerula, Litoria gilleni and Litoria splendida but not as closely as those three species are related to each other.

Mass spectrometric sequencing, enzymic digestion and Edman degradation provide the structures of the two antimicrobial peptides from the skin glands of the Australian tree frog Litoria xanthomera as:- Gly Leu Phe Ser Val Leu Gly Ala Val Ala Lys His Val Leu Pro His Val Val Pro Val Ile Ala Glu Lys Leu (NH2) (caerin 1.6), and Gly Leu Phe Lys Val Leu Gly Ser Val Ala Lys His Leu Leu Pro His Val Ala Pro Val Ile Ala Glu Lys Leu (NH2) (caerin 1.7).