Proline Hinge Research Articles

A histone H2A amino terminal derived peptide Etroplusin from Pearl spot, Etroplus suratensis (Bloch, 1790): Phylogenetic and functional characterization

Antimicrobial peptides (AMPs) derived from histone precursors form a significant class of therapeutic molecules. The study explores the comprehensive molecular characterization of core histone H2A-derived antimicrobial peptide, Etroplusin from Pearl spot, Etroplus suratensis. Etroplusin exhibited characteristic features of AMP such as net +11 charge and amphipathic alpha-helical structure. Multiple sequence alignment showed the homologous nature of etroplusin with previously reported H2A peptides. Phylogenetic analysis revealed that etroplusin is similar to Carassius auratus H2A-derived peptide and comes under the fish H2A peptide clade. Structure modelling with bioinformatics tools predicted alpha-helical structure and random coils with a proline hinge. The functional characterization of etroplusin revealed its antibacterial, antifungal, and anticancer activities. Significant upregulation of etroplusin was observed in the systemic tissues of E. suratensis on a challenge with the bacterial fish pathogen, Streptococcus agalactiae. The present study will shed light on fish innate immune responses and the importance of etroplusin, the H2A-derived peptide as a defense molecule.

Identification and in silico structural and functional analysis of a histone H2A-derived antimicrobial peptide from teleost fishes

BackgroundAntimicrobial peptides (AMPs) are small peptides that form the first line of defence in all living organisms. In addition to conventional AMPs, there exist molecules that are often not regarded as part of the innate defences, but possess antimicrobial activity. Histone H2A-derived peptides are a group of unconventional AMPs that represent very ancient defence factors, re-used in new ways during evolution. The present study attempts to compare histone H2A-derived AMPs from four commercially important teleost fishes inhabiting various habitats, viz. Asian sea bass (Lates calcarifer), tank goby (Glossogobius giuris), grey mullet (Mugil cephalus), and Malabar trevally (Carangoides malabaricus). Methods and resultsRT-PCR amplification of the mRNAs extracted from these fishes yielded H2A-derived AMP genes with slight differences in the nucleotide sequences, nevertheless with similar translated amino acid sequences. The amino acid sequence was found to possess marked similarity at the N-terminal region and showed antimicrobial motif sequence characteristic of histone H2A-derived AMPs. Phylogenetic analysis showed an evolutionary relationship with invertebrate ones suggesting a common ancestral origin for the vertebrate and invertebrate histone H2A. The secondary structure of histone H2A-derived AMP showed the presence of α-helices and intermittent coils having a proline hinge connecting the N- and C-terminus. The peptide was also predicted to be a cell-penetrating one. ConclusionIn silico structural and functional characterization of histone H2A-derived AMP revealed its potential antibacterial property and point towards the possibility of being an efficient AMP with the potential of being made use of in therapeutics.

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52PPQ54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52PPQ54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.

Members of the histone-derived antimicrobial peptide family from the pearl oyster Pinctada fucata martensii: Inhibition of bacterial growth

Because it is difficult to isolate standard antimicrobial peptides (AMPs) using traditional biochemical approaches, we designed, synthesized, and evaluated a series of structurally altered histone-derived AMPs (HDAPs) from the pearl oyster Pinctada fucata martensii using molecular cloning approaches. Four histone-homolog genes (PmH2A, PmH2B, PmH3, and PmH4-1) were identified, of which PmH2A and PmH2B had yet to be described. PmH2A and PmH2B were therefore cloned using Rapid Amplification of cDNA Ends (RACE) and characterized. Constitutive PmH2A and PmH2B mRNA expression was detected in all six pearl oyster tissues tested, with comparatively greater transcript abundance in the gonads. Because α-helical content, hydrophilicity index, and the presence of a proline hinge may be the three important factors influencing the antimicrobial efficacy of HDAPs, we synthesized a series of eight N- and C-terminally truncated or amino acid-substituted synthetic candidate HDAP analogs derived from PmH2A, PmH2B, PmH3, and PmH4-1. Only the PmH2A- and PmH4-derived AMPs inhibited bacterial growth. The PmH2A-derived AMPs were α-helical proteins, while the PmH4-derived AMPs were extended strand/random coil proteins. Our results suggested that having an α-helical structure was particularly important for the antibacterial efficacy of the PmH2A-derived peptides; amphipathic structures (hydrophilic index, 0.3 to −0.3) may enhance the antimicrobial function of both the PmH2A- and PmH4-derived peptides. The high antibacterial efficacy of one of the HDAP analogs studied, PmH2A-AMP (5–13) [KLLK]3, indicated that this protein may represent a promising candidate for the treatment of bacterial infections in aquaculture mollusk species. This first study of HDAPs from the pearl oyster P. f. martensii provides new insights into the design and function of highly effective antimicrobial peptides.

The N-Terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-Encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

The N-Terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-Encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

A histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himanturawalga: Molecular and functional characterisation.

Antimicrobial peptides (AMPs) are biologically dynamic molecules produced by all type of organisms as a fundamental component of their innate immune system. The present study deals with the identification of a histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himanturawalga. A 243 base pair fragment encoding 81 amino acid residues amplified from complementary DNA was identified as Hipposin and termed as Hw-Hip. Homologous analysis showed that Hw-Hip belongs to the Histone H2A superfamily and shares sequence identity with other histone-derived AMPs from fishes. Phylogenetic analysis of Hw-Hip displayed clustering with the fish H2A histones. Secondary structure analysis showed the presence of three α-helices and four random coils with a prominent proline hinge. The physicochemical properties of Hw-Hip are in agreement with the properties of antimicrobial peptides. A 39-mer active peptide sequence was released by proteolytic cleavage in silico. Functional characterisation of active peptide in silico revealed antibacterial, anticancer and antibiofilm activities making Hw-Hip a promising candidate for further exploration.

A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin

Despite high-resolution crystal structures of both inactive and active G protein-coupled receptors (GPCRs), it is still not known how ligands trigger the large structural change on the intracellular side of the receptor since the conformational changes that occur within the extracellular ligand-binding region upon activation are subtle. Here, we use solid-state NMR and Fourier transform infrared spectroscopy on rhodopsin to show that Trp2656.48 within the CWxP motif on transmembrane helix H6 constrains a proline hinge in the inactive state, suggesting that activation results in unraveling of the H6 backbone within this motif, a local change in dynamics that allows helix H6 to swing outward. Notably, Tyr3017.48 within activation switch 2 appears to mimic the negative allosteric sodium ion found in other family A GPCRs, a finding that is broadly relevant to the mechanism of receptor activation.

Molecular Simulations of Melittin-Induced Membrane Pores.

Membrane-active peptides (MAPs) are able to induce pores in cell membranes via molecular mechanisms, which are still subject to ongoing research. In this work, we present molecular dynamics simulations that suggest a precursor membrane defect plays an important role in the pore-inducing activity of the prototypical antimicrobial peptide melittin. The simulations reveal that the hydrophobic N-terminus of melittin is able to recognize and insert into the membrane defect in the lipid bilayer and that this leads to a cascading transfer of adsorbed peptides to the membrane defect, leading to peptide aggregation in the pore. We show that this mechanism also acts in the case of a melittin mutant without the flexible central proline hinge, thus indicating the latter is not crucial to the activity of melittin, which is consistent with experiments.

Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity.

Antimicrobial peptides (AMPs) are important components of the host innate immune system. Papiliocin is a 37-residue AMP purified from larvae of the swallowtail butterfly Papilio xuthus. Magainin 2 is a 23-residue AMP purified from the skin of the African clawed frog Xenopus laevis. We designed an 18-residue hybrid peptide (PapMA) incorporating N-terminal residues 1-8 of papiliocin and N-terminal residues 4-12 of magainin 2, joined by a proline (Pro) hinge. PapMA showed high antimicrobial activity but was cytotoxic to mammalian cells. To decrease PapMA cytotoxicity, we designed a lysine (Lys) peptoid analogue, PapMA-k, which retained high antimicrobial activity but displayed cytotoxicity lower than that of PapMA. Fluorescent dye leakage experiments and confocal microscopy showed that PapMA targeted bacterial cell membranes whereas PapMA-k penetrated bacterial cell membranes. Nuclear magnetic resonance experiments revealed that PapMA contained an N-terminal α-helix from Lys(3) to Lys(7) and a C-terminal α-helix from Lys(10) to Lys(17), with a Pro(9) hinge between them. PapMA-k also had two α-helical structures in the same region connected with a flexible hinge residue at Nlys(9), which existed in a dynamic equilibrium of cis and trans conformers. Using lipopolysaccharide-stimulated RAW264.7 macrophages, the anti-inflammatory activity of PapMA and PapMA-k was confirmed by inhibition of nitric oxide and inflammatory cytokine production. In addition, treatment with PapMA and PapMA-k decreased the level of ultraviolet irradiation-induced expression of genes encoding matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in human keratinocyte HaCaT cells. Thus, PapMA and PapMA-k are potent peptide antibiotics with antimicrobial and anti-inflammatory activity, with PapMA-k displaying enhanced bacterial selectivity.

Novel antibacterial peptides from the skin secretion of the Indian bicoloured frog Clinotarsus curtipes

HPLC elution profile and MALDI TOF MS analysis of electro-stimulated skin secretion of the Indian Ranid frog Clinotarsus curtipes of the Western Ghats confirmed the presence of multiple peptides. Peptides eluted out of the C18 column at higher hydrophobic solvent region showed antibacterial activity against diverse bacterial strains, including the clinical isolates of V. cholerae and methicillin resistant Staphylococcus aureus (MRSA). Peptidomic analysis of the most potent chromatographic effluent fraction identified five novel peptide amides having sequence homology with brevinin family. These peptides are named as brevinin1CTcu1 (B1CTcu1) to brevinin1CTcu5 (B1CTcu5). Peptide B1CTcu1 is non-haemolytic while the others are haemolytic in nature but all elicited potential antibacterial activity. B1CTcu5 is a twenty-one residue peptide amide having proline hinge region in the middle and the typical C-terminal intramolecular disulfide-bridged hepta peptide domain (Rana box) that is present in most of the brevinin peptides. Analysis of their killing kinetics with E. coli and S. aureus and the ability to induce membrane depolarization proved that these are two independent events. These novel multifunctional peptides play an important role to protect C. curtipes from invading pathogenic microorganisms present in the environment.

Activation and Inactivation Steps Altered by Proline Hinge Mutations in Kv1.4

Several voltage-gated channels share a Proline-Valine-Proline (“proline hinge”) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine (P558G) or alanine (P558A), and studied these mutations in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N and C-type inactivation.Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that the transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement plays a significant role in inactivation and recovery.To analyze the effect of these mutations in changing channel gating kinetics, we modified our previously published model of Kv1.4 by adding a primed but non-conducting state connected to the open state by a voltage insensitive step. In order to reproduce the experimental observation that N-type inactivation occurs at more positive potentials than C-type, the C-type inactivated state must be coupled to earlier voltage-dependent steps. Analyses of this model suggest that both P558G and P558A mutations not only modified early voltage dependent steps, but also made the voltage insensitive transition from the primed state to the open state very slow relative to activation.

Interaction of the S6 Proline Hinge with N-Type and C-Type Inactivation in Kv1.4 Channels

Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F6] and [V8]) in the proline hinge region with other hydrophobic residues ([W6] and [I8]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR]3 and KLLKQWPIG[KLLK]3, respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides’ mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties.

The Importance of the Proline Hinge in the Action of Histone-Derived Antimicrobial Peptides

Antimicrobial peptides (AMPs) are short, polycationic proteins capable of killing a wide variety of bacterial species through a number of different mechanisms. The ability to effectively engineer potent, cell-penetrating AMPs would maximize the full potential of these molecules. Buforin II (BF2), a cell-penetrating histone-derived antimicrobial peptide (HDAP), served as a model for the design of three novel histone-derived peptides, DesHDAPs1-3. BF2 is has a C-terminal α-helix that is broken by a proline hinge. Because this proline hinge determines, in part, BF2's ability to translocate into and kill bacterial cells, DesHDAPs1-3 were designed to also contain a helix-breaking proline residue. To determine whether this structural feature plays the same role in the designed peptides, the activity of proline to alanine mutants of each designed peptide were tested against a variety of bacterial species. As expected, circular dichroism measurements indicate that the proline to alanine mutation increases the α-helical character of BF2 and all designed peptides. For both BF2 and DesHDAP1, proline to alanine mutants show decreased antimicrobial activity against all species tested. In contrast, proline to alanine mutations in both DesHDAP2 and DesHDAP3 either do not affect or slightly increase the observed antimicrobial activity. This suggests that α-helicity is a poor predictor of antimicrobial activity for this family of HDAPs, and that the proline residue may play a different role in DesHDAP2 and DesHDAP3 than it does in BF2 and DesHDAP1. In order to explain these trends, we have further characterized the translocation and membrane permeabilization properties of the proline to alanine mutations. As well, we have used molecular dynamics simulations to explore the structure of the membrane bound peptides.

Inactivation In Kv1.4 Channels Involves Significant Intracellular Structural Rearrangements Mediated By A Proline Hinge

Several voltage gated channel families share a common structural motif in the intracellular side of their S6 segment: a Proline-Valine-Proline sequence known as a proline hinge. We studied the proline hinge in Kv1.4 channels which activate and then inactivate via two distinct inactivation mechanisms, N- and C-type. We made several point mutations to the two prolines in the P-V-P hinge of Kv1.4 channels, most of which did not result in a functional channel. Two mutations did result in a functional channel: a glycine or alanine for the second proline.

Conformational Dynamics of Minimal Elastin‐Like Polypeptides: The Role of Proline Revealed by Molecular Dynamics and Nuclear Magnetic Resonance

Previous molecular dynamics studies of the elastin-like peptide (ELP) GVG(VPGVG) predict that this ELP undergoes a conformational transition from an open to a more compact closed state upon an increase in temperature. These structural changes occurring in this minimal elastin model at the so-called inverse temperature transition (ITT), which takes place when elastin is heated to temperatures of about 20-40 (omicron)C, are investigated further in this work by means of a combined theoretical and experimental approach. To do this, additional extensive classical molecular dynamics (MD) simulations of the capped octapeptide are carried out, analyzed, and compared to data obtained from homonuclear magnetic resonance (NMR) spectroscopy of the same octapeptide. Moreover, in the previous simulations, the proline residue in the ELP is found to act as a hinge, thereby allowing for the large-amplitude opening and closing conformational motion of the ITT. To explore the role of proline in such elastin repeating units, a point mutant (P5I), which replaces the proline residue with an isoleucine residue, is also investigated using the aforementioned theoretical and experimental techniques. The results show that the site-directed mutation completely alters the properties of this ELP, thus confirming the importance of the highly conserved proline residue in the ITT. Furthermore, a correlation between the two different methods employed is seen. Both methods predict the mutant ELP to be present in an unstructured form and the wild type ELP to have a beta-turn-like structure. Finally, the role of the peptidyl cis to trans isomerization of the proline hinge is assessed in detail.

A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.

Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II.

Buforin II is a 21-aa potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 1-4), an extended helical region (residues 5-10), a hinge (residue 11), and a C-terminal regular alpha-helical region (residues 12-21). To elucidate the structural features of buforin II that are required for its potent antimicrobial activity, we synthesized a series of N- and C-terminally truncated or amino acid-substituted synthetic buforin II analogs and examined their antimicrobial activity and mechanism of action. Deletion of the N-terminal random coil region increased the antibacterial activity approximately 2-fold, but further N-terminal truncation yielded peptide analogs with progressively decreasing activity. Removal of four amino acids from the C-terminal end of buforin II resulted in a complete loss of antimicrobial activity. The substitution of leucine for the proline hinge decreased significantly the antimicrobial activity. Confocal fluorescence microscopic studies showed that buforin II analogs with a proline hinge penetrated the cell membrane without permeabilization and accumulated in the cytoplasm. However, removal of the proline hinge abrogated the ability of the peptide to enter cells, and buforin II analogs without a proline hinge localized on the cell surface, permeabilizing the cell membrane. In addition, the cell-penetrating efficiency of buforin II and its truncated analogs, which depended on the alpha-helical content of the peptides, correlated linearly with their antimicrobial potency. Our results demonstrate clearly that the proline hinge is responsible for the cell-penetrating ability of buforin II, and the cell-penetrating efficiency determines the antimicrobial potency of the peptide.

Structure-activity analysis of brevinin 1E amide, an antimicrobial peptide from Rana esculenta

Brevinin 1E, consisting of 24 amino acid residues, from Rana esculenta has potent antimicrobial and hemolytic activity. From a structural point of view, this peptide has a N-terminal hydrophobic region, a proline hinge region in the middle and a C-terminal loop region delineated by an intra-disulfide bridge, which is a common structural feature of antimicrobial peptides from Rana species. To investigate the structural features for antimicrobial and hemolytic activity, truncated and linearized brevinin 1E amides were synthesized and characterized. A deletion of three amino acids from the N-terminal region did not greatly affect antimicrobial activity but dramatically reduced hemolytic activity. The contribution of the intra-disulfide bridge to antimicrobial and hemolytic activity was somewhat different between brevinin 1E amide and truncated fragments. In brevinin 1E amide, the elimination of the intra-disulfide bridge did not greatly affect antimicrobial and hemolytic activity whereas the elimination of the intra-disulfide bridge in the truncated fragments did not decrease antimicrobial activity but did decrease hemolytic activity. Circular dichroism spectra and the retention time on the C 18 reverse phase column revealed that the intra-disulfide bridge ( i, i+6) formed an amphipathic loop which increased hydrophobicity and helped to induce the α-helical structure in the membrane-mimetic environment. Even though the intra-disulfide bridge and the N-terminal region were responsible for the α-helical structure and hydrophobicity, these two structural features were not essential for antimicrobial activity. The hemolytic activity of brevinin 1E amide and its analogs also correlated well with the retention time rather than the α-helicity.