Perturbation of peptide conformations induced in anisotropic environments

Reversed-phase high performance liquid chromatography (RP HPLC) has been found to be a convenient and powerful tool for the study of the secondary structure of peptides. Here, the ability of proline to perturb the secondary structures of peptides induced at aqueous-lipid interfaces and the induced conformation of polyproline peptides were investigated by means of RP HPLC. For these studies, four different complete sets of substitution analogues of model peptides expected to have specific induced conformations were used. In the first two studies, a single lysine was “walked” through two 18-residue polyproline sequences (one N-acetylated, the other not). In the remaining two studies, a proline was “walked” through two different sequences that had been found earlier to be induced into an α-helical conformation during RP HPLC (an 18-residue polyalanine sequence and the amphipathic 14-residue sequence Ac-LLKLLKKLLKKLKK-NH₂). Sixty-eight individual analogues were synthesized for this study and the effect of the respective substitutions on retention times was determined. The results are consistent with the concept that, upon interaction with the C-18 of the stationary phase during RP HPLC, polyproline is induced into a type II helical conformation, polyalanine into an α-helical conformation, and Ac-LLKLLKKLLKKLKK-NH₂ into an amphipathic α-helical array. In an extension of this study, the antimicrobial activities of Ac-LLKLLKKLLKKLKK-NH₂ and its 18 proline substitution analogues were found to be inversely correlated with their RP HPLC retention times.     

Probing the influence of sequence-dependent interactions upon α-helix stability in alanine-based linear peptides

The observation that short, linear alanine-based polypeptides form stable α-helices in aqueous solution has allowed the development of well-defined experimental systems with which to study the influence of amino acid sequence upon the stability of secondary structure. We have performed detailed conformational searches upon six alanine-based peptides in order to rationalize the observed variation in the α-helical stability in terms of side-chain-backbone and side-chain-side-chain interactions. Although a simple, gas-phase, potential model was used to obtain the conformational energies for these peptides, good agreement was obtained with experiment regarding their relative α-helical stabilities. Our calculations clearly indicate that valine, isoleucine, and phenylalanine residues should destabilize the α-helical conformation when included within alanine-based peptides because of energetically unfavorable side-chain-backbone interactions, which tend to result in the formation of regions of 3₁₀-helix. In the case of valine, the destabilization most probably arises from entropic effects as the isopropyl side chain can assume more orientations in the 3₁₀-helical form of the peptide. A detailed examination of very short-range interactions in these peptides has also indicated that an interaction, involving fewer than five consecutive residues, whose stabilizing effect reinforces that of the (i, i + 4) hydrogen bond may be the basis of the requirement for increased nucleation (σ) and propagation parameters (s) required by Zimm–Bragg theory to predict the α-helical content for compounds in this class of short peptides. Our calculations complement recent work using modified Zimm–Bragg and Lifson–Roig theories of the helix–coil transition, and are consistent with molecular dynamics simulations upon linear peptides in aqueous solution. © 1993 John Wiley & Sons, Inc.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: A consequence of the formation of “native-like conformation”

The influence of n-propanol on the overall α-helical conformation of β-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of α-helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of α-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of α-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The α-helical conformation induced into α- and β-globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the α-helical conformation of both α- and β-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced “α-helical conformation” of globins and the “α-helical conformation” of α- and β-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of β-globin with the corresponding increase in the α-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the α-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a “native-like structure.” The induction of α-helical conformation into these proteins in the presence of n-propanol and the consequent generation of “native-like conformation” is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an α-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume α-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high α-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of α-helical proteins. Consistent with this concept, the induction of α-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., α_1-30, which exists in an α-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Scrutiny of chain-length and N-terminal effects in α-helix folding: a molecular dynamics study on polyalanine peptides

Protein folding remains an unsolved problem as main-chain, side-chain, and solvent interactions remain entangled and have been hard to resolve. Polyalanines are promising models to analyze protein folding initiation and propagation structurally as well as energetically. In the present work, the effect of chain-length and N-terminal residue stereochemistry in polyalanine peptides are investigated for their role in the nucleation of α-helical conformation. The end-protected polyalanine peptides, tetra-alanine, Ac-^LAla₄-NHMe (Ia) and Ac-^DAla-^LAla₃-NHMe (Ib), hexa-alanine, Ac-^LAla₆-NHMe (IIa) and Ac-^DAla-^LAla₅-NHMe (IIb), and octa-alanine, Ac-^LAla₈-NHMe (IIIa) and Ac-^DAla-^LAla₇-NHMe (IIIb), are assessed as chain-length and stereochemical-structure perturbed models. The appreciable variations in the sampling of α-helical conformation, including a sampling of α-helix folds, due to the cooperative effect of chain-length and N-terminal residue stereochemistry have been noted. The electrostatics of α-helical conformation rather than the conformational entropy of the main-chain appear to be decisive in the initiation of α-helix folding. The results of the present work will enhance our understanding on the nucleation of α-helical conformation in short peptides and aid in the design of novel peptides with α-helical structure that can modulate disease-related protein–protein interactions.     

The binding constant for amyloid Abeta40 peptide interaction with human serum albumin

Human serum albumin (HSA) is the major carrier of Aβ peptides in blood plasma. 1:1 interaction stoichiometries were established in previous indirect antibody-based studies for both Aβ40 and Aβ42, but corresponding binding constants were not provided. In this study we applied direct titrations of HSA with Aβ40 monitored using circular dichroism spectroscopy and obtained a dissociation constant (K_d) of 5 ± 1 μM for a HSA complex with Aβ40. The interaction resulted in an increase of the α-helical contents in the complex, compared to its components, which is quantitatively consistent with the known ability of Aβ40 to adopt a partially α-helical conformation in a hydrophobic environment. The relevance of these findings for the role of HSA in Aβ physiology is discussed.     

Peptidic HIV integrase inhibitors derived from HIV gene products: Structure–activity relationship studies

Structure–activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an α-helical conformation, Glu-Lys pairs were introduced into the i and i + 4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an α-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.     

Carboxyl-terminal peptides from histone H1 variants: DNA binding characteristics and solution conformation

The carboxyl-terminal domains of the histone H1 proteins bind to DNA and are important in condensation of DNA. Little is known about the details of the interactions between H1 histones and DNA, and in particular, there is little known about differences among variant H1 histones in their interactions with DNA. Questions concerning H1 histone-DNA affinity and H1 conformation were investigated using peptide fragments from the carboxyl terminal domains of four nonallelic histone H1 variant proteins (mouse H1-1, H1-4 and H1^°, and rat H1T). Three of the four peptides showed a slight preference for binding to a GC-rich region of a 214-base-pair DNA fragment, rather than to an AT-rich region. The fourth peptide, H1t, appeared to bind preferentially to the AT-rich region of the 214-base-pair fragment. The results show that these small peptides bind preferentially to a subset of DNA sequences; such sequence preference might be exhibited by the intact H1 histones themselves. CD spectra of the peptides, which are from regions of the proteins that are not compactly folded, showed that the α-helical content of the peptides was minimal if the peptides were in 10 mM phosphate buffer, but increased if the peptides were in 1 M NaClO₄ and 50% trifluoroethanol, conditions that are postulated to approximate certain aspects of binding to DNA. H1-4 peptide, which was predicted to be 70% α-helix, but was not α-helical in 10 mM phosphate buffer, appeared from difference CD spectra to be more α-helical when it was bound to DNA. The regions of the proteins from which these peptides are derived, which are extended in solution, may fold, forming α-helices, upon binding to DNA. © 1996 John Wiley & Sons, Inc.     

Lecithin cholesterol acyltransferase (LCAT) activity in the presence of Apo-AI-derived peptides exposed to disorder–order conformational transitions

Although the association of Apo AI with HDLs has been proposed to activate LCAT activity, the detailed molecular mechanisms involved in the process are not known. Therefore, in this study we have investigated how conformational changes in several exposed regions of Apo-AI might cause LCAT activation and for this purpose, designed a strategy to investigate three Apo AI-derived peptides. Since these peptides present the ability to adopt several secondary structure conformations, they were used to determine whether LCAT activity could be modulated in the presence of a particular conformation. Circular dichroism experiments showed that Apo AI-derived peptides in PBS displayed a disordered arrangement, with a strong tendency to adopt β-sheet and random conformational structures as a function of concentration. However, in the presence of Lyso-C₁₂PC, maximal percentages of α-helical structures were observed. Performed in human plasma, time-course experiments of LCAT activity under control conditions reached the highest level of ³H-cholesteryl esters after 2.5 h incubation. In the presence of Apo AI-derived peptides, a significant increase in the production of ³H-cholesteryl esters was observed. The present study provides an important insight into the potential interactions between LCAT and lipoproteins and also suggests that peptides, initially present in a disordered conformation, are able to sense the lipid environment provided by lipoproteins of plasma and following a disorder-to-order transition, change their conformation to an ordered α-helix.     

A Hydrogen Bonded Chain in Bactereorhodopsin by Computer Modelling Approach

Abstract

The seven α-helical segments of Bacteriorhodopsin (BR) passing through the membrane are investigated for a continuous Hydrogen Bonded Chain (HBC). The study is carried out by computer modelling approach. It is assumed that the seven helices are placed as (AGFEDCB), which has been accepted as the best model by several groups. Helices A, D, E and G are considered to be present in right handed α-helical conformation. The inter-orientation of these helices are represented by Eulerian angles α, β and γ. For the helices B, C and F which contain Proline in the middle, several conformational possibilities were considered. In these cases apart from the Eulerian angles α, β and γ, the dihedral angles φ_{p_1} and ψ_{p_1} of the residues that are succeeded by Proline residue in the helical regions were also used in fixing the position of the helices with respect to each other. All these parameters were varied to fit with the top, middle and bottom distances reported by electron diffraction studies. Good fit was obtained for all right handed α-helical conformations and also for helices B, C and F with a left handed turn at the residue preceeding proline. Hence two structures were analysed for continuous HBC, Structure I which contained all the seven helices in right handed α-helical conformation and Structure II, which had the helices A, D, E and G in right handed conformation and the helices B, C and F in right handed α-helical conformation with a left handed turn at the residue preceeding proline. All possible staggered conformations were considered for the side chains and the inter atomic distances were analysed for Hydrogen bonds. It was possible to obtain a continuous chain in both the structures which includes most of the residues found to be important by the experiments. However Lys-216 has to be considered in two different conformations to connect the cytoplasmic side with the extra cellular side. The overall height spanned by HBC is about 25Å. The chains obtained by both the structures I and II are analysed in terms of the conformational parameters. It has also been possible to place the retinal in the region as predicted by the experiments. The Tryptophan residues which affect the spectral characterestics can be aligned on either side of the retinal.     

Proline-induced constraints in alpha-helices

The disrupting effect of a prolyl residue on an α-helix has been analyzed by means of conformational energy computations. In the preferred, nearly α-helical conformations of Ac-Ala₄-Pro-NHMe and of Ac-Ala₇-Pro-Ala₇-NHMe, only the residue preceding Pro is not α-helical, while all other residues can occur in the α-helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α-helix. Other low-energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near-α-helical A-conformational state (with up to 26° changes of their dihedral angles ? and ψ, and a 48° change in one ω from those of the ideal α-helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α-helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α-helices, 61 have a Pro at or next to their N-terminus, 21 have a Pro next to their C-terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α-helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.     

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic,alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7

Peptide XT-7 (GLLGP⁵LLKIA¹⁰AKVGS¹⁵NLL.NH₂) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC₅₀ = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC₅₀ > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d₃)-H₂O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu³ and Leu¹⁷ whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu⁶ and Leu¹⁷. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu³ and Leu¹⁷. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H₂O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly⁴ → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.     

Alzheimer's disease drug candidates stabilize A-β protein native structure by interacting with the hydrophobic core

Deposition of amyloid fibrils, consisting primarily of Aβ₄₀ and Aβ₄₂ peptides, in the extracellular space in the brain is a major characteristic of Alzheimer''s disease (AD). We recently developed new (to our knowledge) drug candidates for AD that inhibit the fibril formation of Aβ peptides and eliminate their neurotoxicity. We performed all-atom molecular-dynamics simulations on the Aβ₄₂ monomer at its α-helical conformation and a pentamer fibril fragment of Aβ₄₂ peptide with or without LRL and fluorene series compounds to investigate the mechanism of inhibition. The results show that the active drug candidates, LRL22 (EC₅₀ = 0.734 μM) and K162 (EC₅₀ = 0.080 μM), stabilize hydrophobic core I of Aβ₄₂ peptide (residues 17–21) to its α-helical conformation by interacting specifically in this region. The nonactive drug candidates, LRL27 (EC₅₀ > 10 μM) and K182 (EC₅₀ > 5 μM), have little to no similar effect. This explains the different behavior of the drug candidates in experiments. Of more importance, this phenomenon indicates that hydrophobic core I of the Aβ₄₂ peptide plays a major mechanistic role in the formation of amyloid fibrils, and paves the way for the development of new drugs against AD.     

Lactam bridge stabilization of α-helical peptides: Ring size,orientation and positional effects

A series of 14 residue amphipathic α-helical peptides, in which the sidechains of glutamic acid and lysine have been covalently joined, was synthesized in order to determine the effect of spacing, position and orientation of these lactam bridges. It was found that although an (i, i+3) spacing would position the lactam bridge on the same face of the helix, these lactams with 18-member rings were actually helix-destabilizing regardless of position or location. On the other hand, (i, i+4) lactams with 21-member rings were helix-stabilizing but this was dependent on orientation. Glutamic acid-lysine lactams increased the helical content of the peptide when compared with their linear homologue in benign conditions (50 mM KH₂PO₄, 100 mM KCl, pH 7). Two Glu-Lys (i, i+4) lactams located at the N- and C-termini gave rise to a peptide with greater than 99% helical content in benign conditions. Peptides with Lys-Glu oriented lactams were random structures in benign conditions but in the presence of 50% TFE could be induced into a helical conformation. The stability of the single-stranded α-helices, as measured by thermal denaturations in 25% TFE indicated that Glu-Lys oriented lactam bridges stabilized the helical conformation relative to the linear unbridged peptide. One Glu-Lys lactam in the middle of the peptide was more effective at stabilizing helical structure than two Glu-Lys lactams positioned one at each end of the molecule. The lactams with the Lys-Glu orientation were destabilizing relative to the unbridged peptide. This study demonstrates that correct orientation and position of a lactam bridge is critical in order to design peptides with high helical content in aqueous media.     

IQ-motif peptides as novel anti-microbial agents

The IQ-motif is an amphipathic, often positively charged, α-helical, calmodulin binding sequence found in a number of eukaryote signalling, transport and cytoskeletal proteins. They share common biophysical characteristics with established, cationic α-helical antimicrobial peptides, such as the human cathelicidin LL-37. Therefore, we tested eight peptides encoding the sequences of IQ-motifs derived from the human cytoskeletal scaffolding proteins IQGAP2 and IQGAP3. Some of these peptides were able to inhibit the growth of Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) comparable to LL-37. In addition some IQ-motifs had activity against the fungus Candida albicans. This antimicrobial activity is combined with low haemolytic activity (comparable to, or lower than, that of LL-37). Those IQ-motifs with anti-microbial activity tended to be able to bind to lipopolysaccharide. Some of these were also able to permeabilise the cell membranes of both Gram positive and Gram negative bacteria. These results demonstrate that IQ-motifs are viable lead sequences for the identification and optimisation of novel anti-microbial peptides. Thus, further investigation of the anti-microbial properties of this diverse group of sequences is merited.     

Effect of hydrocarbon stapling on the properties of α-helical antimicrobial peptides isolated from the venom of hymenoptera

The impact of inserting hydrocarbon staples into short α-helical antimicrobial peptides lasioglossin III and melectin (antimicrobial peptides of wild bee venom) on their biological and biophysical properties has been examined. The stapling was achieved by ring-closing olefin metathesis, either between two S-2-(4′-pentenyl) alanine residues (S ₅) incorporated at i and i + 4 positions or between R-2-(7′-octenyl) alanine (R ₈) and S ₅ incorporated at the i and i + 7 positions, respectively. We prepared several lasioglossin III and melectin analogs with a single staple inserted into different positions within the peptide chains as well as analogs with double staples. The stapled peptides exhibited a remarkable increase in hemolytic activity, while their antimicrobial activities decreased. Some single stapled peptides showed a higher resistance against proteolytic degradation than native ones, while the double stapled analogs were substantially more resistant. The CD spectra of the singly stapled peptides measured in water showed only a slightly better propensity to form α-helical structure when compared to native peptides, whereas the doubly stapled analogs exhibited dramatically enhanced α-helicity.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

Structural and biological characterization of mastoparans in the venom of Vespa species in Taiwan

Mastoparans, a family of small peptides, are isolated from the wasp venom. In this study, six mastoparans were identified in the venom of six Vespa species in Taiwan. The precursors of these mastoparans are composed of N-terminal signal sequence, prosequence, mature mastoparan, and appendix glycine at C-terminus. These mature mastoparans all have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bond. Therefore, these peptides could be predicted to adopt an amphipathic α-helical secondary structure. In fact, the CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 8 mM SDS or 40% 2,2,2-trifluoroethanol (TFE). All mastoparans exhibit mast cell degranulation activity, antimicrobial activity against both Gram-positive and -negative bacteria tested, various degree of hemolytic activity on chicken, human, and sheep erythrocytes as well as membrane permeabilization on Escherichia coli BL21. Our results also show that the hemolytic activity of mastoparans is correlated to mean hydrophobicity and mean hydrophobic moment.