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Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates in vivo ., Competing Interests: The authors SK, AS, and IK, are employers of the company Enamine Co, Ltd. SA, OB, and IK are co-founders of the company Lumobiotics GmbH. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Afonin, Koniev, Préau, Takamiya, Strizhak, Babii, Hrebonkin, Pivovarenko, Dathe, le Noble, Rastegar, Strähle, Ulrich and Komarov.)

The influence of several antimicrobial trivalent cyclic hexapeptides on the mixing behavior of bilayer lipid membranes containing phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) with varying composition was studied using DSC and ITC. The peptides contained three arginines and three aromatic amino acids and had different sequences. All of them induce clustering of PG-rich clusters with bound peptides after binding. In a previous publication we could show that a correlation between clustering efficacy and the antimicrobial activity of the peptides exists (S. Finger et al., Biochim. Biophys. Acta 1848 (2015) 2998-3006). In the current study we investigated whether the non-ideality of the lipid mixture had any effect on the clustering efficacy and the critical peptide/lipid clustering ratio. We could show that for PG/PE membranes containing 1:1 M ratios and lipids with equal or unequal chain lengths, the amount of clustered PG depended only slightly on the absolute chain length and on the chain length difference between PG and PE. Much larger differences were observed when the PG/ PE mixing ratio was changed. In mixtures of DPPG/DPPE with high PG content, the amount of clustered PG per added peptide was much higher than in PE-rich mixtures. The ITC experiments showed that the critical peptide/lipid ratio for cluster formation is also strongly dependent on the PG/PE ratio in the mixture. In the PG/PE 3:1 mixture, the formation of clusters with bound peptide is much more likely than for mixtures with less PG. For 1:1 and 1:3 lipid mixtures, the critical peptide/lipid ratio for demixing is between 0.002 and 0.004. Therefore, even in these mixtures clustering occurs way below charge saturation of the PG in the mixture and the PG-rich clusters are not charge compensated either. The peptide concentration necessary for inducing clustering amounts to ~8 μM, a value well within the range of minimal inhibitory concentration values observed for the cyclic peptides studied here. Our results show that not only the structure of the cyclic peptide influences the clustering efficacy but also the mixing behavior of the lipids in the bilayers has an influence on the amount of clustering induced by binding of cyclic peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) has a rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. Its detailed mode of action has, however, remained elusive. In contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates to the cytoplasm. Using a combination of proteome analysis, fluorescence microscopy, and membrane analysis we show that cWFW instead triggers a rapid reduction of membrane fluidity both in live Bacillus subtilis cells and in model membranes. This immediate activity is accompanied by formation of distinct membrane domains which differ in local membrane fluidity, and which severely disrupts membrane protein organisation by segregating peripheral and integral proteins into domains of different rigidity. These major membrane disturbances cause specific inhibition of cell wall synthesis, and trigger autolysis. This novel antibacterial mode of action holds a low risk to induce bacterial resistance, and provides valuable information for the design of new synthetic antimicrobial peptides.

The development of antimicrobial peptides as new class of antibiotic agents requires structural characterisation and understanding of their diverse mechanisms of action. As the cyclic hexapeptide cWFW (cyclo(RRRWFW)) does not exert its rapid cell killing activity by membrane permeabilisation, in this study we investigated alternative mechanisms of action, such as peptide translocation into the cytoplasm and peptide interaction with components of the phospholipid matrix of the bacterial membrane. Using fluorescence microscopy and an HPLC-based strategy to analyse peptide uptake into the cells we could confirm the cytoplasmic membrane as the major peptide target. However, unexpectedly we observed accumulation of cWFW at distinct sites of the membrane. Further characterisation of peptide-membrane interaction involved live cell imaging to visualise the distribution of the lipid cardiolipin (CL) and isothermal titration calorimetry to determine the binding affinity to model membranes with different bacterial lipid compositions. Our results demonstrate a distribution of the cyclic peptide similar to that of cardiolipin within the membrane and highly preferred affinity of cWFW for CL-rich phosphatidylethanolamine (POPE) matrices. These observations point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cWFW which is neither based on membrane permeabilisation nor translocation into the cytoplasm but rather on preferred partitioning into particular lipid domains. As the phospholipids POPE/CL play a key role in the dynamic organisation of bacterial membranes we discuss the consequences of this peptide-lipid-interaction and outline the impact on antimicrobial peptide research.

Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.

Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Antibiotics are mandatory additives in semen extenders to control bacterial contamination. The worldwide increase in resistance to conventional antibiotics requires the search for alternatives not only for animal artificial insemination industries, but also for veterinary and human medicine. Cationic antimicrobial peptides are of interest as a novel class of antimicrobial additives for boar semen preservation. The present study investigated effects of two synthetic cyclic hexapeptides (c-WFW, c-WWW) and a synthetic helical magainin II amide derivative (MK5E) on boar sperm during semen storage at 16 °C for 4 days. The standard extender, Beltsville Thawing Solution (BTS) containing 250 µg/mL gentamicin (standard), was compared to combinations of BTS with each of the peptides in a split-sample procedure. Examination revealed peptide- and concentration-dependent effects on sperm integrity and motility. Negative effects were more pronounced for MK5E than in hexapeptide-supplemented samples. The cyclic hexapeptides were partly able to stimulate a linear progressive sperm movement. When using low concentrations of cyclic hexapeptides (4 µM c-WFW, 2 µM c-WWW) sperm quality was comparable to the standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. In order to investigate the interaction of peptides with the membrane, electron spin resonance spectroscopic measurements were performed using spin-labeled lipids. C-WWW and c-WFW reversibly immobilized an analog of phosphatidylcholine (PC), whereas MK5E caused an irreversible increase of PC mobility. These results suggest testing the antimicrobial efficiency of non-toxic concentrations of selected cyclic hexapeptides as potential candidates to supplement/replace common antibiotics in semen preservation.

The transport of bioactive compounds to the site of action is a great challenge. A promising approach to overcome application-related problems is the development of targeting colloidal transport systems, such as micelles which are equipped with uptake mediating moieties. Here, we investigated a set of novel lipopeptides which exhibit a surfactant-like structure due to attachment of two palmitoyl chains to the N-terminus of cationic or anionic amino acid sequences. We analyzed the association behavior of these lipopeptides by using 5(6)-carboxyfluorescein (CF)-labeled derivatives as a fluorescent probe and different spectroscopic methods such as fluorescence anisotropy and fluorescence correlation spectroscopy (FCS). The photophysical properties as well as the diffusion and rotational movements of the CF-labeled lipopeptides were exploited to determine the cmc and the size of the micelles consisting of lipopeptides. We could distinguish cationic and anionic lipopeptides by their association behavior and by studying the interactions with mouse brain capillary endothelial cells (b.end3). The cationic derivatives turned out to be very strong surfactants with a very low cmc in the micromolar range (0.5-14 μM). The unique combination of micelle-forming property and cell-penetrating ability can pave the road for the development of a novel class of efficient drug carrier systems.

Transglutaminase-1 (TG1)-deficient autosomal-recessive congenital ichthyosis (ARCI) is a rare and severe genetic skin disease caused by mutations in TGM1. It is characterized by collodion babies at birth, dramatically increased transepidermal water loss (TEWL), and lifelong pronounced scaling. The disease has a tremendous burden, including the problem of stigmatization. Currently, no therapy targeting the molecular cause is available, and the therapeutic situation is deplorable. In this study, we developed the basis for a causative therapy aiming at the delivery of the enzyme to the inner site of the keratinocytes' plasma membrane. We prepared sterically stabilized liposomes with encapsulated recombinant human TG1 (rhTG1) and equipped with a highly cationic lipopeptide vector to mediate cellular uptake. The liposomes overcame the problems of insufficient cutaneous delivery and membrane penetration and provided excellent availability and activity of rhTG1 in primary keratinocytes. To demonstrate the general feasibility of this therapeutic approach in a humanized context, we used a skin-humanized mouse model. Treatment with rhTG1 liposomes resulted in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin: in situ monitoring showed a restoration of TG1 activity, and cholesterol clefts vanished ultrastructurally. Measurement of TEWL revealed a restoration of epidermal barrier function. We regard this aspect as a major advance over available nonspecific approaches making use of, for example, retinoid creams. We conclude that this topical approach is a promising strategy for restoring epidermal integrity and barrier function and provides a causal cure for individuals with TG1 deficiency., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Tryptophan and arginine-rich cyclic hexapeptides of the type cyclo-RRRWFW combine high antibacterial activity with rapid cell killing kinetics, but show low toxicity in human cell lines. The peptides fulfil the structural requirements for membrane interaction such as high amphipathicity and cationic charge, but membrane permeabilisation, which is the most common mode of action of antimicrobial peptides (AMPs), could not be observed. Our current studies focus on elucidating a putative membrane translocation mechanism whereupon the peptides might interfere with intracellular processes. These investigations require particular analytical tools: fluorescent analogues and peptides bearing appropriate reactive groups were synthesized and characterized in order to be used in confocal laser scanning microscopy and HPLC analysis. We found that minimal changes in both the cationic and hydrophobic domain of the peptides in most cases led to significant reduction of antimicrobial activity and/or changes in the mode of action. However, we were able to identify two modified peptides which exhibited properties similar to those of the cyclic parent hexapeptide and are suitable for subsequent studies on membrane translocation and uptake into bacterial cells.

Magnetic resonance imaging (MRI) provides the opportunity of tracking cells in vivo. Major challenges in dissecting cells from the recipient tissue and signal sensitivity constraints albeit exist. In this study, we aimed to tackle these limitations in order to study inflammation in autoimmune encephalomyelitis. We constructed a very small dual-tunable radio frequency (RF) birdcage probe tailored for (19)F (fluorine) and (1)H (proton) MR mouse neuroimaging. The novel design eliminated the need for extra electrical components on the probe structure and afforded a uniform -field as well as good SNR. We employed fluorescently-tagged (19)F nanoparticles and could study the dynamics of inflammatory cells between CNS and lymphatic system during development of encephalomyelitis, even within regions of the brain that are otherwise not easily visualized by conventional probes. (19)F/(1)H MR Neuroimaging will allow us to study the nature of immune cell infiltration during brain inflammation over an extensive period of time.

The activity of cyclo-RRRWFW (c-WFW) against Escherichia coli has been shown to be modulated by the aromatic motif and the lipopolysaccharides (LPS) in the bacterial outer membrane. To identify interaction sites and to elucidate the mode of c-WFW action, peptides were synthesized by the replacement of tryptophan (W) with analogs having altered hydrophobicity, dipole and quadrupole moments, hydrogen-bonding ability, amphipathicity, and ring size. The peptide activity against Bacillus subtilis and erythrocytes increased with increasing hydrophobicity, whereas the effect on E. coli revealed a more complex pattern. Although they had no effect on the E. coli inner membrane even at concentrations higher than the MIC, peptides permeabilized the outer membrane according to their antimicrobial activity pattern, suggesting a major role of LPS in peptide transport across the wall. For isothermal titration calorimetry (ITC) studies of peptide-lipid bilayer interaction, we used POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline), either alone or in mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), to mimic the charge properties of eukaryotic and bacterial membranes, respectively, as well as in mixtures with lipid A, rough LPS, and smooth LPS as models of the outer membrane of E. coli. Peptide accumulation was determined by both electrostatic and hydrophobic interactions. The susceptibility of the lipid systems followed the order of POPC-smooth LPS >> POPC-rough LPS > POPC-lipid A = POPC-POPG > POPC. Low peptide hydrophobicity and enhanced flexibility reduced binding. The influence of the other properties on the free energy of partitioning was low, but an enhanced hydrogen-bonding ability and dipole moment resulted in remarkable variations in the contribution of enthalpy and entropy. In the presence of rough and smooth LPS, the binding-modulating role of these parameters decreased. The highly differentiated activity pattern against E. coli was poorly reflected in peptide binding to LPS-containing membranes. However, stronger partitioning into POPC-smooth LPS than into POPC-rough LPS uncovered a significant role of O-antigen and outer core oligosaccharides in peptide transport and the permeabilization of the outer membrane and the anti-E. coli activity of the cyclic peptides.

We utilized epifluorescence microscopy to investigate the morphological changes in labeled lipid bilayers supported on quartz surfaces (SLBs) induced by the interaction of cationic antimicrobial peptides with the lipid membranes. The SLBs were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and mixtures thereof as well as from Escherichia coli lipid extract. We succeeded in the preparation of POPG and POPG-rich SLBs without the necessity to use fusogenic agents such as calcium by using the Langmuir-Blodgett/Langmuir-Schaefer transfer method. The adsorption of the peptides to the SLBs was initially driven by electrostatic interactions with the PG headgroups and led to the formation of lipid protrusions bulging out from the lipid layer facing the bulk, originating particularly from domain boundaries and membrane defects. The shape, size, and frequency of the lipid protrusions are mainly controlled by the peptide macroscopic properties and the membrane composition. A restructuring of the lipid protrusions into other structures can also occur over time.

Early studies of immobilized peptides mainly focused upon the relationship between structural properties and the activity of soluble and surface-tethered sequences. The intention of this study was to analyze the influence of immobilization parameters upon the activity profile of peptides. Resin beads (TentaGel S NH(2), HypoGel 400 NH(2), and HypoGel 200 NH(2)) with polyethylene glycol spacers of different lengths were rendered antimicrobial by linkage of an amphipathic model KLAL peptide and magainin-derived MK5E. Standard solid-phase peptide synthesis, thioalkylation, and ligation strategies were used to immobilize the peptides at the C and N termini and via different side-chain positions. Depending upon the resin capacity and the coupling strategies, peptide loading ranged between 0.1 and 0.25 micromol/mg for C-terminally and around 0.03 micromol/mg for N-terminally and side-chain-immobilized peptides. Tethering conserved the activity spectra of the soluble peptides at reduced concentrations. The resin-bound peptides were antimicrobial toward Escherichia coli and Bacillus subtilis in the millimolar range compared to the results seen with micromolar concentrations of the free peptides. B. subtilis was more susceptible than E. coli. The antimicrobial activity distinctly decreased with reduction of the spacer length. Slight differences in the antimicrobial effect of KLAL and MK5E bound at different chain positions on TentaGel S NH(2) suggest that the activity is less dependent upon the position of immobilization. Soluble KLAL was active toward red blood cells, whereas MK5E was nonhemolytic at up to about 400 microM. Resin-induced hemolysis hampered the determination of the hemolytic effect of the immobilized peptides. TentaGel S NH(2)-bound peptides enhanced the permeability of the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline) and mixed POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG) bilayers used to model the charge properties of the biological targets. The results suggest that surface immobilization of the cationic amphipathic antimicrobial peptides does not influence the membrane-permeabilizing mode of action. Peptide insertion into the target membrane and likely the exchange of membrane-stabilizing bivalent cations contribute to the antimicrobial effect. In conclusion, reasonable antimicrobial activity of surface-bound peptides requires the optimization of the coupling parameters, with the length of the spacer and the amount of target-accessible peptide being the most important factors.

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.

Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10(-13) m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.

The linear sequence KLAL (KLALKLALKALKAALKLA-NH(2)) and its corresponding d,l-isomers k(9)a(10)-KLAL (KLALKLALkaLKAALKLA-NH(2)) and l(11)k(12)-KLAL (KLALKLALKAlkAALKLA-NH(2)) are model compounds for potentially amphipathic alpha-helical peptides which are able to bind to membranes and to increase the membrane permeability in a structure- and target-dependent manner (Dathe and Wieprecht, 1999) We first studied the secondary structure of KLAL and its analogs bound to the air/water using infrared reflection absorption spectroscopy. For the peptide films the shape and position of the amide I and amide II bands indicate that the KLAL adopts at large areas per molecule an alpha-helical secondary structure, whereas at higher surface pressures or smaller areas it converts into a beta-sheet structure. This transition could be observed in the compression isotherm as well as during the adsorption at the air/water interface from the subphase as a function of time. The secondary structures are essentially orientated parallel to the air/water interface. The analogs with d-amino acids in two different positions of the sequence, k(9)a(10)-KLAL and l(11)k(12)-KLAL, form only beta-sheet structures at all surface pressures. The observed results are interpreted using a comparison of hydrophobic moments calculated for alpha-helices and beta-sheets. The differences between the hydrophobic moments calculated using the consensus scale are not large. Using the optimal matching hydrophobicity scale or the whole-residue hydrophobicity scale the beta-sheet even has the larger hydrophobic moment.

To investigate structural features modulating the biological activity of cupiennin 1 peptides from the spider Cupiennius salei, three truncated cupiennin 1d analogs were synthesized. The fact that their growth inhibiting effect on Gram-negative and Gram-positive bacteria, their lytic activity with human red blood cells and their insecticidal effect on Drosophila melanogaster correlates with structural properties shows that the hydrophobic N-terminal chain segment includes the major determinants of structure and activity. The polar C-terminus seems to modulate peptide accumulation at negatively charged cell surfaces via electrostatic interactions and has no important effect on the peptides' amphipathic secondary structure.

A new family of antimicrobial peptides was isolated from the venom of Cupiennius salei. The peptides were purified to homogeneity, and the sequence of cupiennin 1a was determined by Edman degradation: GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH(2). The amino acid sequences of cupiennin 1b, c, and d were obtained by a combination of sequence analysis and mass spectrometric measurements of comparative tryptic peptide mapping. All peptides consist of 35 amino acid residues and are characterized by a more hydrophobic N-terminal chain region and a C terminus composed preferentially of polar and charged residues. The total charge of all cupiennins calculated under physiological conditions is +8, and their C terminus, formed by a glutamic acid residue, is amidated. Conformational studies of the peptides revealed a high helix forming potential. Antimicrobial assays on bacteria with cupiennin 1a, 1d, and synthesized cupiennins 1a* and 1d* showed minimal inhibitory concentrations for bacteria in the submicromolar range. Their lytic effect on human red blood cells was lower by a factor of 8 to 14 than the highly hemolytic melittin. Cupiennin 1a, 1b, 1d, 1a*, and 1d* showed pronounced insecticidal activity. The immediate biological effects and the structural properties of the isolated cupiennins indicate a membrane-destroying mode of action on prokaryotic as well as eukaryotic cells.

Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Starting from the sequences of magainin 2 analogs, peptides with slightly increased hydrophobic moment (mu) but retained other structural parameters were designed. Circular dichroism investigations revealed that all peptides adopt an alpha-helical conformation when bound to phospholipid vesicles. Analogs with increased mu were considerably more active in permeabilizing vesicles mainly composed of zwitterionic lipid. In addition, the antibacterial and hemolytic activities of these analogs were enhanced. Correlation of permeabilization and binding indicated that the activity increase is predominantly caused by an increased membrane affinity of the peptides due to strengthened hydrophobic interactions.

The hydrophobicity (H), hydrophobic moment (mu) and the angle subtended by the positively charged helix face (phi) of a set of model and magainin 2 amide peptides with conserved charge and helix propensity have been shown to be effective modulators of antibacterial and haemolytic activity. Except peptides of low hydrophobicity which are inactive, changing the parameters has little influence on the activity against Gram-negative bacteria, thus revealing the dominance of electrostatic interactions for the effect. However, the increase of H, mu and phi substantially enhances haemolytic and Gram-positive antibacterial activity and is related to a reduction of peptide specificity for Gram-negative bacteria.

Based on neural network calibration the confidence intervals of aromaticity determination from infrared reflectance spectra of raw brown coals were estimated by means of the bootstrap method, a simplified Monte Carlo Simulation. The standard deviations and the confidence intervals were estimated to characterise the analysis error.It is shown that confidence intervals of non-linear analysis methods like Back Propagation Neural Networks (BPNN) can be estimated by the bootstrap method. The estimated confidence intervals of the calibration confirm the analysis by BPNN.