nNOS inhibition, antimicrobial and anticancer activity of the amphibian skin peptide, citropin 1.1 and synthetic modifications. The solution structure of a modified citropin 1.1.

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.     

Design, synthesis and antibacterial activity of cecropin-like model peptides

In order to investigate structure-activity relationships of cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of cecropins and the design of the model peptides were based on Chou-Fasman calculations. The peptides were synthesized by solid-phase methods and purified by reverse-phase liquid-chromatography on C18-silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N-terminal segment that is connected to a hydrophobic helical C-terminal segment by a flexible non-helical hinge region.     

Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens

Peptides derived from the N-terminal domain that comprises an amphipathic alpha-helix in human lactoferrin (LFh 18-31 and LFh 20-38) and bovine lactoferrin (LFb 17-30 and LFb 19-37) were chemically synthesised. Since many positively charged amphipathic alpha-helices contain antimicrobial activity, the peptides were tested for their antimicrobial activity against various oral pathogens. Both peptides from bovine lactoferrin had more potent antimicrobial activities than the human equivalents. Peptide LFb 17-30, containing the largest number of positively charged amino acids, showed the highest antimicrobial activity to both Gram-positive and Gram-negative bacteria. Since native lactoferrin molecules had no killing activity, release of these peptides from the native protein should be investigated to explore the use in oral care products.     

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure–activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC₅₀ = 1.56–3.13 μM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 μM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides. Zi-gang Tian and Tian-tang Dong contributed equally to this paper     

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear alpha-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.     

Influence of lipids on membrane assembly and stability of the potassium channel KcsA

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two-dimensional 1H-nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long alpha-helix containing eight turns along nearly the full length of the peptide except for four N-terminal residues and six C-terminal residues. The electrostatic surface map shows that the N-terminal segment of the alpha-helix, residues 5-22, is an amphipathic alpha-helix with a clear separation of hydrophobic and hydrophilic faces, and that the C-terminal segment of the alpha-helix, residues 23-36, is a hydrophobic alpha-helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N-terminal segment of the alpha-helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.     

De novo design and structure-activity relationships of peptide emulsifiers and foaming agents

A series of eight amphipathic peptides (8, 11, 15, 2 x 18, 22, 26, 29 amino acids in length) were designed to investigate the effects of amino acid composition, peptide length and secondary structure on surface activity assessed as emulsification and foaming activity. The potential for alpha-helix formation at the hydrophobic/hydrophilic interface was maximized through the use of helix-forming amino acids, a relatively large hydrophobic surface of 200 degrees of arc and ion pairs between basic and acidic amino acids on the hydrophilic surface. Emulsification activity increased rapidly between 11 and 22 residues as alpha-helicity in aqueous solution increased. Despite their small size, the peptides produced exceptionally stable emulsions, compared with proteins. Foaming activity was enhanced by the presence of aromatic amino acids and the activity of the best peptide examined was superior to that of bovine serum albumin and beta-lactoglobulin.     

The expanding scope of antimicrobial peptide structures and their modes of action

Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protect a host from invading pathogenic bacteria. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as potential alternatives for antibiotics. Although extremely variable in length, amino acid composition and secondary structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent studies demonstrate that they achieve their antimicrobial activity by disrupting various key cellular processes. Some peptides can even use multiple mechanisms. Moreover, several intact proteins or protein fragments are now being shown to have inherent antimicrobial activity. A better understanding of the structure-activity relationships of AMPs is required to facilitate the rational design of novel antimicrobial agents.     

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.     

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

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

Design of model amphipathic peptides having potent antimicrobial activities.

Induced amphipathic alpha-helical conformations play an important role in the biological activity of peptides. By using reversed-phase high-performance liquid chromatography (RP-HPLC) as a means to study the secondary structure of peptides at aqueous/lipid interfaces, a sequence (Ac-LKLLKKLLKKLKKLLKKL-NH2) was found to readily adopt an amphipathic alpha-helical conformation upon interacting with the lipid groups of the stationary phase during RP-HPLC. This peptide exhibited potent antimicrobial activities against both Gram-positive and Gram-negative bacteria. We have prepared a complete set of omission, as well as of leucine and lysine substitution, analogs of this sequence. These analogs were used to investigate the effects of such alterations on the parent sequence's antimicrobial and hemolytic activities relative to each analog's behavior during RP-HPLC. The potential for the formation of ion channels through cell membranes by this amphipathic model peptide was also evaluated through preparation of analogs which varied in length from 8 to 22 residues, while maintaining their amphipathicity.     

Structure of the propeptide of prothrombin containing the gamma-carboxylation recognition site determined by two-dimensional NMR spectroscopy

The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a gamma-carboxylation recognition site that directs precursor forms of these proteins for posttranslational gamma-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing alpha-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues -13 to -3 form an amphipathic alpha-helix. NMR spectra indicate that a similar structure is present at 5 degrees C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the gamma-carboxylation recognition site, four residues (-18, -17, -16, and -15) are adjacent to the helical region and one residue (-10) is located within the helix. The potential role of the amphipathic alpha-helix in the gamma-carboxylation recognition site is discussed.     

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata.

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death.     

The consequence of sequence alteration of an amphipathic alpha-helical antimicrobial peptide and its diastereomers

The search for antibiotics with a new mode of action led to numerous studies on antibacterial peptides. Most of the studies were carried out with l-amino acid peptides possessing amphipathic alpha-helix or beta-sheet structures, which are known to be important for biological activities. Here we compared the effect of significantly altering the sequence of an amphipathic alpha-helical peptide (15 amino acids long) and its diastereomer (composed of both l- and d-amino acids) regarding their structure, function, and interaction with model membranes and intact bacteria. Interestingly, the effect of sequence alteration on biological function was similar for the l-amino acid peptides and the diastereomers, despite some differences in their structure in the membrane as revealed by attenuated total reflectance Fourier-transform infrared spectroscopy. However, whereas the all l-amino acid peptides were highly hemolytic, had low solubility, lost their activity in serum, and were fully cleaved by trypsin and proteinase K, the diastereomers were nonhemolytic and maintained full activity in serum. Furthermore, sequence alteration allowed making the diastereomers either fully, partially, or totally protected from degradation by the enzymes. Transmembrane potential depolarization experiments in model membranes and intact bacteria indicate that although the killing mechanism of the diastereomers is via membrane perturbation, it is also dependent on their ability to diffuse into the inner bacterial membrane. These data demonstrate the advantage of the diastereomers over their all l-amino acid counterparts as candidates for developing a repertoire of new target antibiotics with a potential for systemic use.     

Dephosphorylation-induced structural changes in beta-casein and its amphiphilic fragment in relation to emulsion properties

To promote the understanding of the relationship between emulsifying and molecular properties of proteins/peptides, intact beta-casein (betaCN) and its amphipathic fragment, i.e., betaCN (1-105/107) were dephosphorylated. Dephosphorylation was found not to change significantly their emulsifying properties. Since it is known that the structure of proteins can change upon adsorption onto an interface, the secondary structure of intact beta-casein, its amphipathic fragment, and their dephosphorylated forms, both in solution and after adsorption onto a hydrophobic teflon/water interface, were studied by far-UV circular dichroism spectroscopy. An increased content of secondary structure, especially alpha-helix, was found for all samples after adsorption onto teflon. Dephosphorylation increased the helix-forming propensity, especially for amphipathic fragment of beta-casein. No influence of the secondary structure properties on the emulsion-forming and -stabilizing properties was observed, but a relationship between the maximum surface load and the emulsion-stabilizing properties was found.     

High-level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain

Phosphatidylcholine transfer protein (PC-TP) is a 214-amino acid cytosolic protein that promotes intermembrane transfer of phosphatidylcholines, but no other phospholipid class. To probe mechanisms for membrane interactions and phosphatidylcholine binding, we expressed recombinant human PC-TP in Escherichia coli using a synthetic gene. Optimization of codon usage for bacterial protein translation increased expression of PC-TP from trace levels to >10% of the E. coli cytosolic protein mass. On the basis of secondary structure predictions of an amphipathic alpha-helix (residues 198-212) in proximity to a hydrophobic alpha-helix (residues 184-193), we explored whether the C-terminus might interact with membranes and promote binding of phosphatidylcholines. Consistent with this possibility, truncation of five residues from the C-terminus shortened the predicted amphipathic alpha-helix and decreased PC-TP activity by 50%, whereas removal of 10 residues eliminated the alpha-helix, abolished activity, and markedly decreased the level of membrane binding. Circular dichroic spectra of synthetic peptides containing one ((196-214)PC-TP) or both ((183-214)PC-TP) predicted C-terminal alpha-helices in aqueous buffer were most consistent with random coil structures. However, both peptides adopted alpha-helical configurations in the presence of trifluoroethanol or phosphatidylcholine/phosphatidylserine small unilamellar vesicles. The helical content of (196-214)PC-TP increased in proportion to vesicle phosphatidylserine content, consistent with stabilization of the alpha-helix at the membrane surface. In contrast, the helical content of (183-214)PC-TP was not influenced by vesicle composition, implying that the more hydrophobic of the alpha-helices penetrated into the membrane bilayer. These studies suggest that tandem alpha-helices located near the C-terminus of PC-TP facilitate membrane binding and extraction of phosphatidylcholines.     

Purification,structure-function analysis,and molecular characterization of novel linear peptides from scorpion Opisthacanthus madagascariensis

In the previous report [Biochem. Biophys. Res. Commun. 286 (2001) 820], we described a novel short linear peptide, IsCT, with cytolytic activity isolated from the venom of scorpion Opisthacanthus madagascariensis. From the same scorpion venom, we further purified and characterized three short linear peptides named IsCT2, IsCTf, and IsCT2f that shared high homology with IsCT, while with different C-terminal areas between IsCT/IsCT2 and IsCTf/IsCT2f. Structure-activity relationship was analyzed by performing vivo and vitro assays and circular dichroism spectroscopy. Like IsCT, IsCT2 showed broad activity spectra against microbes (Gram positive and negative bacteria as well as fungi) and relatively weak hemolytic activity against sheep red blood cells. It adopts an amphipathic alpha-helical structure in aqueous TFE and is able to disrupt the artificial membrane. However, the other two peptides IsCTf and IsCT2f showed no activity in antimicrobial or hemolytic assay. Furthermore, IsCTf and IsCT2f cannot form amphipathic alpha-helix while demonstrating random coil structure in aqueous TFE, which might result in their lost cytolytic activity. IsCTf and IsCT2f both exist in the crude venom and are proved to be enzymatic products from IsCT and IsCT2. Whether they have some other biological activity is still unclear. In addition, we got the cDNAs encoding the precursors of IsCT and IsCT2. Besides the signal peptide, they still contain an unusual acidic pro-peptide at the C-terminal that was quite different from other known precursors of scorpion venom peptides. The novel structure and biological activity of these peptides proposed them to be a new class in scorpion venom.     

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

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

N-terminal analogues of cecropin A: synthesis, antibacterial activity, and conformational properties

Six analogues of the 37-residue antibacterial peptide cecropin A were synthesized by the solid-phase method: cecropin A-(2-37), [Glu2]cecropin A, [Pro4]cecropin A, [Glu6]cecropin A, [Leu6]cecropin A, and [Pro8]cecropin A. Their antibacterial activities against four test organisms were determined and related to conformational changes observed in their CD spectra and were discussed on the basis of a previously proposed amphipathic alpha-helix model. An aromatic residue in position 2 was shown to be important for activity against all tested bacteria. The highly alpha-helical 1-11 region of cecropin A did not appear to play a significant role in its activity against Escherichia coli but was clearly involved in its interaction against Pseudomonas aeruginosa, Bacillus megaterium, and Micrococcus luteus.     

Alpha-helical antimicrobial peptides—Using a sequence template to guide structure-activity relationship studies

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.