Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.     

Increased potency of a novel d-β-naphthylalanine-substituted antimicrobial peptide against fluconazole-resistant fungal pathogens

The antifungal activities of the known antimicrobial peptide, P-113, as well as a new type of Trp-rich peptide, Ac-KWRRWVRWI-NH₂, Pac-525, and its modified peptide, d -Nal-Pac-525, were determined using the broth microdilution method in three different media. All peptides had similar activities against yeast pathogens in low-salt LYM media. However, only d -Nal-Pac-525 retained its antifungal activity in the media containing high concentrations of salt. Hence, d -Nal-Pac-525 has the potential of becoming a promising antifungal agent, especially for fungal pathogens with intrinsic resistance to fluconazole.     

Expression, purification and structural studies of a short antimicrobial peptide

We have produced a small antimicrobial peptide PFWRIRIRR in bacteria utilizing production in the form of insoluble fusion protein with ketosteroid isomerase. The recombinant peptide was rapidly and efficiently isolated by acidic cleavage of the fusion protein based on the acid labile Asp-Pro bond at the N-terminus of the peptide. The peptide has antibacterial activity and neutralizes macrophage activation by LPS. The selectivity of the peptide against bacteria correlates with preferential binding to acidic phospholipid vesicles. Solution structure of the peptide in SDS and DPC micelles was determined by NMR. The peptide adopts a well-defined structure, comprising a short helical segment. Cationic and hydrophobic clusters are segregated along the molecular axis of the short helix, which is positioned perpendicular to the membrane plane. The position of the helix is shifted in two micellar types and more nonpolar surface is exposed in anionic micelles. Overall structure explains the advantageous role of the N-terminal proline residue, which forms an integral part of the hydrophobic cluster.     

Model membrane interaction and DNA-binding of antimicrobial peptide Lasioglossin II derived from bee venom

Lasioglossins, a new family of antimicrobial peptide, have been shown to have strong antimicrobial activity with low haemo-lytic and mast cell degranulation activity, and exhibit cytotoxic activity against various cancer cells in vitro. In order to understand the active conformation of these pentadecapeptides in membranes, we have studied the interaction of Lasioglossin II (LL-II), one of the members of Lasioglossins family with membrane mimetic micelle Dodecylphosphocholine (DPC) by fluorescence, Circular Dichroism (CD) and two dimensional (2D) ¹H NMR spectroscopy. Fluorescence experiments provide evidence of interaction of the N-terminal tryptophan residue of LL-II with the hydrophobic core of DPC micelle. CD results show an extended chain conformation of LL-II in water which is converted to a partial helical conformation in the presence of DPC micelle. Moreover we have determined the first three-dimensional NMR structure of LL-II bound to DPC micelle with rmsd of 0.36 Å. The solution structure of LL-II shows hydrophobic and hydrophilic core formation in peptide pointing towards different direction in the presence of DPC. This amphipathic structure may allow this peptide to penetrate deeply into the interfacial region of negatively charged membranes and leading to local membrane destabilization. Further we have elucidated the DNA binding ability of LL-II by agarose gel retardation and fluorescence quenching experiments.     

The interaction of an antimicrobial decapeptide with phospholipid vesicles

Previously, by using combinatorial peptide libraries, we have identified activity-optimized decapeptide (KSL, KKVVFKVKFK-NH(2)), which exhibited a broad spectrum of the activity against bacteria and fungi without hemolytic activity. In order to examine lipid requirements and to understand the mode of KSL action, we investigated interactions of the peptide with vesicles consisting of various lipid compositions. KSL increased the permeability of negatively charged but not zwitterionic phospholipid membranes, and the leakage was independent on the size of encapsulated molecules (calcein, 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS)/N,N'-p-xylene bis(pyridinium) bromide (DPX), and fluorescein isothiocyanate (FITC)-dextran with different molecular weight), indicating that the peptide did not form pores or channels in this leakage process. KSL ability to permeabilize vesicles with negatively charged surface was dramatically reduced upon the addition of zwitterionic phospholipid rather than cholesterol, which revealed that the surface charge of lipid membranes played a major role in the activity and selectivity of KSL. Moreover, KSL diastereomer did not increase the permeability of negatively charged vesicles, indicating that the secondary structure of KSL was also required for membrane perturbation activity. Interestingly, KSL had an ability to cause aggregation and subsequent fusion of the acidic vesicles, which seemed to be related to the biological action. Structural studies performed by circular dichroism (CD) spectroscopy indicated that in the presence of acidic vesicles, the beta sheet structure of KSL must be required for the ability to (1) induce a leakage of dye from the acidic vesicles (2) to fuse the acidic vesicles.     

Interactions of the antimicrobial peptide Ac-FRWWHR-NH(2) with model membrane systems and bacterial cells.

The acetylated and amidated hexapeptide FRWWHR (combi-2), previously identified by combinatorial chemistry methods, shows strong antimicrobial activity. The binding of the peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-[(phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles was performed to determine changes in the lipid phase behaviour upon binding the peptide. Two-dimensional proton nuclear magnetic resonance (NMR) spectroscopy, to solve the bound peptide structure, was performed in the presence of dodecylphosphatidylcholine (DPC) and sodium dodecyl sulphate (SDS) micelles. The fluorescence, ITC and DSC studies indicate that the peptide interacts preferentially with lipid vesicles containing negatively charged head groups. Conformational information determined using NMR indicate that the combi-2 peptide adopts a coiled amphipathic conformation when bound to SDS and DPC micelles. Leakage assays indicate that the peptide is not very efficient at causing leakage from calcein-filled large unilamellar vesicles comprised of POPG/POPC (1 : 1). The rapid passage of either the fluorescent-tagged peptides combi-2 or the previously studied peptide Ac-RRWWRF-NH(2) (combi-1) into Escherichia coli and Staphylococcus aureus suggests that instead of membrane disruption, the main bactericidal site of action of these peptides might be located inside bacteria.     

Novel short antibacterial and antifungal peptides with low cytotoxicity: Efficacy and action mechanisms

Short antimicrobial peptides with nine and eleven residues were developed against several clinically important bacterial and fungal pathogens (specifically Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Fusarium solani). Twelve analogues of previously reported peptides BP76 (KKLFKKILKFL) and Pac-525 (KWRRWVRWI) were designed, synthesized, and tested for their antimicrobial activities. Two of our eleven amino acid peptides, P11-5 (GKLFKKILKIL) and P11-6 (KKLIKKILKIL), have very low MICs of 3.1-12.5 μg ml⁻¹ against all five pathogens. The MICs of these two peptides against S. aureus, C. albicans and F. solani are four to ten times lower than the corresponding MICs of the reference peptide BP76. P9-4 (KWRRWIRWL), our newly designed nine-amino acid analogue, also has particularly low MICs of 3.1-6.2 μg ml⁻¹ against four of the tested pathogens; these MICs are two to eight times lower than those reported for Pac-525 (6.2-50 μg ml⁻¹).These new peptides (P11-5, P11-6 and P9-4) also exhibit improved stability in the presence of salts, and have low cytotoxicity as shown by the hemolysis and MTT assays. From the results of field-emission scanning electron microscopy, membrane depolarization and dye-leakage assays, we propose that these peptides exert their action by disrupting membrane lipids. Molecular dynamics simulation studies confirm that P11-6 peptide maintains relatively stable helical structure and exerts more perturbation action on the order of acyl tail of lipid bilayer.     

Solution structure of a designed amphipathic antimicrobial synthetic peptide, PGAa

A designed peptide, PGAa showed an excellent antifungal activity as well as an efficient bactericidal activity toward gram-positive, especially in the pathogenic yeast Candida albicans 28838. The solution structures of PGAa have been determined both in 40% TFE/water solution and DPC micelle by CD and NMR spectroscopy. Based on NOEs, vicinal coupling constants, backbone amide exchange rates, and chemical shift indices, PGAa formed a long amphipathic alpha-helical conformation in both TFE and DPC micelle environments, spanning the residues Ile(2)-Ala(19) in TFE and Lys(5)-Ala(19) in DPC micelle, respectively. Solution structures suggested that the hydrophobic residues would interact with the fatty acyl chains of the lipid bilayer, while the positively charged side-chains exposed to aqueous environments. Therefore, we conclude that the alpha-helical structure as well as the highly amphiphatic nature of PGAa peptide may play a critical role in its antimicrobial activity as well as selectivities in different species.     

Molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers

The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two β-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the β-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.     

Structural determinants of the specificity of a membrane binding domain of the scaffold protein Ste5 of budding yeast: Implications in signaling by the scaffold protein in MAPK pathway

In the mitogen activated protein kinase (MAPK) cascades of budding yeast, the scaffold protein Ste5 is recruited to the plasma membrane to transmit pheromone induced signal. A region or domain of Ste5 i.e. residues P44-R67, referred here as Ste5PM24, has been known to be involved in direct interactions with the membrane. In order to gain structural insights into membrane interactions of Ste5, here, we have investigated structures and interactions of two synthetic peptide fragments of Ste5, Ste5PM24, and a hyperactive mutant, Ste5PM24LM, by NMR, ITC, and fluorescence spectroscopy, with lipid membranes. We observed that Ste5PM24 predominantly interacted only with the anionic lipid vesicles. By contrast, Ste5PM24LM exhibited binding with negatively charged as well as zwitterionic or mixed lipid vesicles. Binding of Ste5 peptides with the negatively charged lipid vesicles were primarily driven by hydrophobic interactions. NMR studies revealed that Ste5PM24 assumes dynamic or transient conformations in zwitterionic dodecylphosphocholine (DPC) micelles. By contrast, NMR structure, obtained in anionic sodium dodecyl sulphate (SDS), demonstrated amphipathic helical conformations for the central segment of Ste5PM24. The hydrophobic surface of the helix was found to be buried inside the micelles. Taken together, these results provide important insights toward the structure and specificity determinants of the scaffold protein interactions with the plasma membrane.     

High-resolution solution structure of a designed peptide bound to lipopolysaccharide: transferred nuclear Overhauser effects, micelle selectivity, and anti-endotoxic activity

Designing peptides that would interact with lipopolysaccharides (LPS) and acquire a specific folded conformation can generate useful structural insights toward the development of anti-sepsis agents. In this work, we have constructed a 12-residue linear peptide, YW12, rich in aromatic and aliphatic amino acid residues with a centrally located stretch of four consecutive positively charged (KRKR) residues. In absence of LPS, YW12 is predominantly unstructured in aqueous solution. Using transferred nuclear Overhauser effect (Tr-NOE) spectroscopy, we demonstrate that YW12 adopts a well-folded structure as a complex with LPS. Structure calculations reveal that YW12 assumes an extended conformation at the N-terminus followed by two consecutive beta-turns at its C-terminus. A hydrophobic core is formed by extensive packing between number of aromatic and nonpolar residues, whereas the positively charged residues are segregated out to a separate region essentially stabilizing an amphipathic structure. In an in vitro LPS neutralization assay using NF-kappaB induction as the readout, YW12 shows moderate activity with an IC50 value of approximately 10 microM. As would be expected, tryptophan fluorescence studies demonstrate that YW12 shows selective interactions only with the negatively charged lipid micelles including sodium dodecyl sulfate (SDS), 1-palmitoyl-2-oleoylphosphatidyl-dl-glycerol (POPG), and LPS, and no significant interactions are detected with zwitterionic lipid micelles such as dodecyl-phosphocholine (DPC). Far-UV CD studies indicate the presence of beta-turns or beta-sheet-like conformations of the peptide in negatively charged micelles, whereas no structural transitions are apparent in DPC micelles. These results suggest that structural features of YW12 could be utilized to develop nontoxic antisepsis compounds.     

Transient vesicle leakage initiated by a synthetic apoptotic peptide derived from the death domain of neurotrophin receptor, p75NTR.

Peptides that induce apoptosis have potential as anticancer therapeutics. The design of safe, effective cancer therapeutic peptides requires characterization of the physical and chemical properties that influence activation of cell death in neoplastic cells. NTR365 is a synthetic pro-apoptotic peptide with an amino acid sequence derived from the death domain of p75(NTR). These studies were initiated to identify a potential mechanism for the apoptotic activity of NTR365 identified by Rabizadeh et al. We examined the interactions of this synthetic pro-apoptotic peptide with phospholipid vesicles. Fluorescence experiments demonstrate that the peptide induces leakage from large unilamellar vesicles. Leakage activity is transient and dependent on the presence of anionic lipid in the vesicles. Circular dichroism studies show that the NTR365 adopts a different conformation and may have altered vesicle affinity under conditions conducive to leakage. The active conformation of NTR365 differs from that of the NMR derived conformation. A related peptide with a single substitution is not apoptotically active, does not form a helical structure in the presence of vesicles and does not induce appreciable vesicle leakage under the same conditions as NTR365. These studies suggest that the demonstrated apoptotic activity of a closely related NTR364 peptide is linked to disruption of a membrane barrier and to the ability of the peptide to form a helical structure.     

The interactions of antimicrobial peptides derived from lysozyme with model membrane systems

Two peptides, RAWVAWR-NH2 and IVSDGNGMNAWVAWR-NH2, derived from human and chicken lysozyme, respectively, exhibit antimicrobial activity. A comparison between the L-RAWVAWR, D-RAWVAWR, and the longer peptide has been carried out in membrane mimetic conditions to better understand how their interaction with lipid and detergent systems relates to the reported higher activity for the all L-peptide. Using CD and 2D 1H NMR spectroscopy, the structures were studied with DPC and SDS micelles. Fluorescence spectroscopy was used to study peptide interactions with POPC and POPG vesicles and DOPC, DOPE, and DOPG mixed vesicle systems. Membrane-peptide interactions were also probed by ITC and DSC. The ability of fluorescein-labeled RAWVAWR to rapidly enter both E. coli and Staphylococcus aureus was visualized using confocal microscopy. Reflecting the bactericidal activity, the long peptide interacted very weakly with the lipids. The RAWVAWR-NH2 peptides preferred lipids with negatively charged headgroups and interacted predominantly in the solvent-lipid interface, causing significant perturbation of membrane mimetics containing PG headgroups. Peptide structures determined by 1H NMR indicated a well-ordered coiled structure for the short peptides and the C-terminus of the longer peptide. Using each technique, the two enantiomers of RAWVAWR-NH2 interacted in an identical fashion with the lipids, indicating that any difference in activity in vivo is limited to interactions not involving the membrane lipids.     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

Phospholipid interactions of synthetic peptides representing the N-terminus of HIV gp41

Peptides representing the N-terminal 23 residues of the surface protein gp41 of LAV1a and LAVmal strains of the human immunodeficiency virus were synthesized and their interactions with phospholipid vesicles studied. The peptides are surface-active and penetrate lipid monolayers composed of negatively charged but not neutral lipids. Similarly, the peptides induce lipid mixing and solute (6-carboxyfluorescein) leakage of negatively charged, but not neutral, vesicles. Circular dichroism and infrared spectroscopy show that at low peptide:lipid ratios (approximately 1:200), the peptides bind to negatively charged vesicles as alpha-helices. At higher peptide:lipid ratios (1:30), a beta conformation is observed for the LAV1a peptide, accompanied by a large increase in light scattering. The LAVmal peptide showed less beta-structure and induced less light scattering. With neutral vesicles, only the beta conformation and a peptide:lipid ratio-dependent increase in vesicle suspension light scattering were observed for both peptides. We hypothesize that the inserted alpha-helical form causes vesicle membrane disruption whereas the surface-bound beta form induces aggregation.     

Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles

Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusually rich in tryptophan and proline. Its antimicrobial action involves the bacterial cytoplasmic membrane. Fluorescence and circular dichroism spectra demonstrated the structural similarity of indolicidin in complexes with large unilamellar phospolipid vesicles and with detergent micelles. The structure of indolicidin bound to zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) micelles was determined using NMR methods and shown to represent a unique membrane-associated peptide structure. The backbone structure in DPC, well defined between residues 3 and 11, was extended, with two half-turns at residues Lys-5 and Trp-8. The backbone structure in SDS, well defined between residues 5 and 11, was also extended, but lacked the bend in the C-terminal half. Indolicidin in complexes with DPC had a central hydrophobic core composed of proline and tryptophan, which was bracketed by positively charged regions near the peptide termini. The tryptophan side chains, with one exception, folded flat against the peptide backbone, thus giving the molecule a wedge shape. Indolicidin in complexes with SDS had an arrangement of hydrophobic and cationic regions similar to that found in the presence of DPC. The tryptophan side chains were less well defined than for indolicidin in DPC and extended away from the peptide backbone. The preferred location of indolicidin in DPC micelles and lipid bilayers, analyzed using spin-label probes, was at the membrane interface.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences, model membrane binding and biological activities.

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt beta-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Revisiting peptide amphiphilicity for membrane pore formation

It has previously been shown that an amphipathic de novo designed peptide made of 10 leucines and four phenylalanines substituted with crown ethers induces vesicle leakage without selectivity. To gain selectivity against negatively charged dimyristoylphosphatidylglycerol (DMPG) bilayers, one or two leucines of the peptide were substituted with positively charged residues at each position. All peptides induce significant calcein leakage of DMPG vesicles. However, some peptides do not induce significant leakage of zwitterionic dimyristoylphosphatidylcholine vesicles and are thus active against only bacterial model membranes. The intravesicular leakage is induced by pore formation instead of membrane micellization. Nonselective peptides are mostly helical, while selective peptides mainly adopt an intermolecular β-sheet structure. This study therefore demonstrates that the position of the lysine residues significantly influences the secondary structure and bilayer selectivity of an amphipathic 14-mer peptide, with β-sheet peptides being more selective than helical peptides.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Conformation of a bactericidal domain of puroindoline a: structure and mechanism of action of a 13-residue antimicrobial peptide

Puroindoline a, a wheat endosperm-specific protein containing a tryptophan-rich domain, was reported to have antimicrobial activities. We found that a 13-residue fragment of puroindoline a (FPVTWRWWKWWKG-NH(2)) (puroA) exhibits activity against both gram-positive and gram-negative bacteria. This suggests that puroA may be a bactericidal domain of puroindoline a. PuroA interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the microbicidal effect of puroA may be due to interactions with bacterial membranes. A variety of biophysical and biochemical methods, including fluorescence spectroscopy and microcalorimetry, were used to examine the mode of action of puroA. These studies showed that puroA is located at the membrane interface, probably due to its high content of Trp residues that have a high propensity to partition into the membrane interface. The penetration of these Trp residues in negatively charged phospholipid vesicles resembling bacterial membranes was more extensive than the penetration in neutral vesicles mimicking eukaryotic membranes. Peptide binding had a significant influence on the phase behavior of the former vesicles. The three-dimensional structure of micelle-bound puroA determined by two-dimensional nuclear magnetic resonance spectroscopy indicated that all the positively charged residues are oriented close to the face of Trp indole rings, forming energetically favorable cation-pi interactions. This characteristic, along with its well-defined amphipathic structure upon binding to membrane mimetic systems, allows puroA to insert more deeply into bacterial membranes and disrupt the regular membrane bilayer structure.