Antibacterial Peptide Cecropin Research Articles

Overexpression of the Bivalent Antibacterial Peptide Genes in Pichia pastoris Delays Sour Rot in Citrus Fruit and Induces Geotrichum citri-aurantii Cell Apoptosis

ABSTRACTGenetic manipulation of antagonists shows tremendous potential for improving biocontrol activity of antagonists. Here, bivalent antibacterial peptide Cecropin A/PsdI gene was transformed and expressed in methylotrophic yeast, Pichia pastoris GS115. The bivalent yeast GS115/CEC-PSD showed a great ability in delaying sour rot in citrus fruit and based on observations of restrained growth of geminated Geotrichum citri-aurantii spores relative to non-transformed strain. Although there was no remarkable discrepancy between growth rates of strain GS115 and GS115/CEC-PSD in vivo and vitro, plenty of vacuoles were observed in the cellular space of G. citri-aurantii treated with the CEC-PSD via transmission electron microscopy. Meanwhile, a gel-retardation assay exhibited that the DNA of G. citri-aurantii was degraded by CEC-PSD. Furthermore, our research revealed apoptosis genes in G. citri-aurantii were expressed at different levels with the CEC-PSD levels, among these genes, the expression level of RLM1 and RLM1was decreased remarkably, and the expression level of others changed relatively a little. These findings suggest that bivalent antimicrobial peptides can inhibit G. citri-aurantii by harming cell membrane, disrupting the cell metabolism, inducing programmed cell death and can therefore heighten the inhibition effect of the biocontrol yeast against postharvest diseases and this provides a new approach for the biological control of postharvest diseases.

Effect of Musca Domestica Antimicrobial Peptide Cecropin on Cell Cycle of BEL- 7402 Cells and Its Mechanism

Objective To investigate the effect of Musca domestica antimicrobial peptide cecropin on cell cycle of human hepatocelluar carcinoma BEL-7402 cells and its underlying mechanism.Methods After BEL-7402 cell in vitro were treated with antimicrobial peptide cecropin of Musca domestica,the cell proliferation cycle was determined with flow cytometry analysis(FCM)with PI staining,and the expression of CHK1 and CDK2 were examined by FCM with indirect immunofluorescence technique.Results The optical microscope showed that the morphology of control BEL-7402 cells was regular,spindle-shaped adherent growth,but part of cecropin treated group cell was round,the adherent growth ability decreased and the cell number reduced.FCM showed antimicrobial peptide cecropin of Musca domestica made the cell cycle of BEL-7402 changed,decreased the ratio of G0/G1 and G2/M phase,but increased the proportion of cells in S phase,and increased the expression level of CHK1 and CDK2,compared with the control group significantly(P0.05).Conclusion Musca domestica antibacterial peptide cecropin can effect on BEL-7402 cell cycle by adjusting the CHK1 and CDK2 expression.

Expression, cDNA cloning, and characterization of the antibacterial peptide cecropin D from Agrius convolvuli

Cecropins are basic antibacterial peptides that have potent activities against microorganisms. We have cloned and characterized a cecropin-like peptide of the lepidopteran insect Agrius convolvuli and analyzed its expression in Escherichia coli. The full-length cDNA of A. convolvuli cecropin D3 (AcCec) was 318 bp, containing a 5′ untranslated region (UTR) of 47 bp, a 3′ UTR of 82 bp with a poly (A) tail, and an open reading frame (ORF) of 189 bp encoding a polypeptide of 63 amino acids, including a 24 amino acid signal sequence and a 38 amino acid mature peptide (GenBank accession no. GQ888768). The mature peptide is highly similar to D-type cecropin. To understand the effect of C-terminal amidation, while overcoming the disadvantage of its lack in the prokaryote system, we added a lysine residue to AcCec (AcCec-K) and compared its antibacterial activity to the purified AcCec. The recombinant AcCec (rAcCec) and AcCec-K were expressed, respectively, in E. coli Rosetta cells using a pGEX-4T-1 expression vector, which contained the glutathione S-transferase (GST) gene for fusion partner, and the fusion proteins were induced by isopropyl-β-d-thiogalactopyranoside (IPTG). The recombinant proteins were purified by fast protein liquid chromatography (FPLC) using GSTrap FF and Resource RPC column. The result of the inhibition zone suggests that C-terminal lysine residue could increase the activity due to activated phosphorylation.

Goat Mammary Gland Expression of Cecropin B to Inhibit Bacterial Pathogens Causing Mastitis

The antibacterial peptide Cecropin B (CB), isolated from the giant silk moth, has been shown to effectively eliminate bacteria. In this study, the effects of transgenic CB on dairy goat mammary epithelial cells (DGMECs) and dairy goat mammary gland were investigated. The DNA of CB from silkworm was amplified by reverse transcription PCR (RT-PCR) and then fused to the eukaryotic expression vector pECFP-C1. The recombinant plasmid pECFP-Cecropin B (pECFP-CB) was used for the transfection of DGMECs, and the expression of transgenic CB and the antibacterial activity of it were confirmed by western blot and agar diffusion reaction respectively. The stable DGMEC line transfected by pECFP-CB was obtained by screening with G418. In vivo experiment, pECFP-CB was injected into dairy goat mammary gland, and also the expression and antibacterial activity of transgenic CB were confirmed. Results of this study: transgenic CB can be expressed in DGMECs and dairy goat mammary gland, and inhibit the mastitis caused by Staphylococcus aureus.

CDNA cloning and characterization of the antibacterial peptide cecropin 1 from the diamondback moth, Plutella xylostella L

Cecropins are linear cationic antibacterial peptides that have potent activities against microorganisms. In the present study, a 480bp full-length cDNA encoding diamondback moth (Plutella xylostella) cecropin 1 (designated as Px-cec1) was obtained using RT-PCR. A Northern blot analysis showed that the Px-cec1 transcript was predominantly expressed in fat bodies, hemocytes, midgut and epidermis with the highest expression level in fat bodies. The expression of Px-cec1 mRNA in fat bodies was significantly increased 24h after microbial challenge, with the highest induced expression by Staphylococcus aureus. A circular dichroism (CD) analysis revealed that the recombinant Px-cec1 mainly contained α-helixes. Antimicrobial assays demonstrated that recombinant Px-cec1 exhibited a broad spectrum of anti-microbial properties against fungi, Gram-positive and Gram-negative bacteria, but it did not exhibit hemolytic activity against human erythrocytes. Furthermore, Px-cec1 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy and transmission electron microscopy. These results demonstrated that Px-cec1 exerts its antibacterial activity by acting on the cell membrane to disrupt bacterial cell structures.

Co-expression of apoptin (VP3) and antibacterial peptide cecropin B mutant (ABPS1) genes induce higher rate of apoptosis in HepG2 and A375 cell lines

The antibacterial peptide cecropin B mutant (ABPS1) gene has a broad range of antibacterial and antiproliferative properties. Apoptin (VP3), a chicken anaemia virus-encoded protein is known to induce apoptosis in human transformed cells. To explore drug combination in human tumor cells, apoptin and ABPS1 eukaryotic expression vector pIRES2-EGFP-apoptin and pIRES2-EGFP-ABPS1 were constructed and their expression effect individually and in combinations were studied in HepG2 and A375 cells. The vector pIRES2-EGFP-ABPS1 and pIRES2-EGFP-apoptin were transfected into tumor cells HepG2 and A375 by the lipofectamine-mediated DNA transfection procedure. At 48 h post transfection, the apoptotic rate obtained by flow cytometry and the morphological changes under light and scanning electron microscope of tumor cells were significant. In contrast, the microvilli on the surface of the control cells were disrupted, decreased and even disappeared. The cell membrane was injured and intracellular substances leaked out. Furthermore, our results indicate that the apoptotic rates of apoptin (27.32% in HepG2 and 9.34% in A375 cells), were higher than ABPS1 (23.79% in HepG2 and 8.33% in A375 cells). Moreover, the co-expression of Apoptin and ABPS1 showed higher apoptotic rates which were 27.66 and 10.33% in HepG2 and A375 cells respectively. However, the apoptotic rates obtained in HepG2 cells treated with apoptin and apoptin and ABPS1 together were closely similar, but, not in A375 cells. Therefore, the results of the present study showed that the combination of Apoptin and ABPS1 has synergistic effect in HepG2 and A375 cell lines. Keys words : Apoptin, ABPS1, apoptosis, co-expression, HepG2, A375.

Polymersome surface decoration by an EGFP fusion protein employing Cecropin A as peptide “anchor”

Polymer based nanocompartments have potential applications in synthetic biology, medicine (drug release) and industrial biotechnology (chiral nanoreactors, multistep syntheses, selective product recovery). A step towards the aforementioned goals is the polymer membrane functionalization through covalent bonding of chemical anchors or insertion of proteins/peptides, to obtain specific properties like recognition, catalytic activity and facilitated diffusion, mimicking the complexity of a biological membrane. The use of genetic engineering techniques widens the possible applications of peptides and proteins specifically designed for polymer membrane interactions. A fusion protein (CecEGFP) based on the antibacterial peptide Cecropin A and the EGFP (Enhanced Green Fluorescent Protein) was designed, expressed and biophysically characterized. CecEGFP interaction with the tri-block copolymer PIB-PEG-PIB (PIB=polyisobutylene, PEG=polyethylene glycol) based polymersome membrane was analyzed by circular dichroism as well as EGFP and Trp fluorescence measurements. Results proved that Cecropin A is usable as a "membrane surface anchor" for water soluble proteins, as it inserts into the polymer membrane. The aim and novelty of this study is within the design of fusion proteins specifically developed for polymer membrane interactions. The use of amphiphilic Cecropin A "anchoring" water soluble proteins to the polymersome surface, avoids chemical coupling between polymers and proteins.

Effect of nucleopolyhedrovirus infection on antibacterial activity and antibacterial peptide production in larvae of the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Effects of nucleopolyhedrovirus (NPV) on antibacterial peptide gene expression and peptide production in Bombyx mori larvae remain unclear. Antibacterial activity was first examined with the hemolymph from B. mori larvae infected with B. mori NPV (BmNPV) and injected with bacterial lipopolysaccharide (LPS) 72 h postinfection (p.i.). No antibacterial activity was detected. Expression of four antibacterial peptide genes were next analyzed in B. mori larvae infected with BmNPV. Although antibacterial peptide genes were activated upon injection of LPS in the larvae infected with BmNPV, none were triggered by BmNPV alone. Production of the antibacterial peptide cecropin B was next examined by Western blotting. Cecropin B was not detected from 72 to 96 h p.i. The absence of cecropin B was coincident with an increase in BmNPV cysteine proteinase (CP) activity. Cecropin B production was confirmed at 96 h p.i. in the larvae infected with CP-deficient BmNPV, indicating that the antibacterial peptides are degraded by BmNPV-CP at a very late stage of viral infection. These results suggest that BmNPV neither activates nor suppresses gene expression of antibacterial peptides and that absence of antibacterial activity is due to BmNPV-CP in B. mori larvae.

Diversity and community structure of culturable Bacillus spp. populations in the rhizospheres of transgenic potatoes expressing the lytic peptide cecropin B

A greenhouse experiment was carried out with transgenic potato plants expressing the antibacterial lytic peptide cecropin in order to determine whether non-target Bacillus communities in the rhizosphere are affected by such plants. Two cecropin expressing lines, a line containing only vector sequences and the parental variety Achirana Inta (AI) were included in the experiment. At the flowering and the tuber production stage a total of 621 Bacillus isolates was obtained from the potato rhizospheres, and strains were analysed by PCR–RFLP analysis of the 16S rRNA gene and the 16S–23S rDNA intergenic spacer. Representative isolates were further analyzed by partial 16S rDNA sequence analysis. At the flowering stage the cecropin expressing lines caused a transient, but significant effect on the diversity and community structure of culturable Bacillus spp. Populations associated with the plasmid-containing potato line showed diversity values comparable to the Bacillus communities found in the rhizospheres of wildtype plants, but community structures were highly different. At the tuber production stage the rhizosphere Bacillus populations showed only few differences. The observed effects can be partly explained by different sensitivities of the Bacillus community members towards cecropin. In addition, unintentionally altered plant characteristics seem to be responsible for the different population structures found in the rhizospheres of transgenic plants.

Cell-free production of biologically active polypeptides: application to the synthesis of antibacterial peptide cecropin.

An approach to preparative production of polypeptides, including uneasily testable, degradable, and toxic ones, is proposed on the basis of in vitro expression systems of last generation, such as continuous-exchange cell-free and continuous-flow cell-free transcription-translation systems. The approach implies that a polypeptide of interest is synthesized as a fusion protein with the polypeptide linked to green fluorescent protein (GFP) through a cleavable spacer. The GFP moiety provides direct visualization and quantitative monitoring of the polypeptide synthesis, as well as solubility and stability of the product. The synthesis of functionally active antibacterial polypeptide cecropin P1 (31 amino acid residues) has been demonstrated.

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding.

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.

Conformational study of a custom antibacterial peptide cecropin B1: implications of the lytic activity

Cecropin B1 (CB1) with two amphipathic α-helical segments is a derivative of the natural antibacterial peptide, cecropin B. The assays of cell lysis show that, compared with cecropin A (CA), CB1 has a similar ability to lyse bacteria with a higher potency (two- to six-fold higher) in killing cancer cells. The difference may be due to the fact that the peptides possess different structures and sequences. In this study, the solution structure of CB1 in 20% hexafluoroisopropanol was determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR resonances were assigned. A total of 350 inter-proton distances were used to calculate the solution structure of CB1. The final ensemble structures were well converged, showing the minimum root mean square deviation. The results indicate that CB1 has two stretches of helices spanning from residues 3 to 22 and from residues 26 to 33, which are connected by a hinge section formed by Gly-23 and Pro-24. Lys-25 is partially incorporated in the hinge region. The bent angle between two helical segments located in two planes was between 100 and 110°. With comparisons of the known NMR structure of CA and its activities on bacteria and cancer cells, the structure–function relationship of the peptides is discussed.

The Dependence of Membrane Permeability by the Antibacterial Peptide Cecropin B and Its Analogs, CB-1 and CB-3, on Liposomes of Different Composition

A natural antibacterial peptide, cecropin B (CB), and designed analogs, CB-1 and CB-3, were synthesized. The three peptides have different structural characteristics, with CB having one hydrophobic and one amphipathic alpha-helix, CB-1 having two amphipathic alpha-helices, and CB-3 having two hydrophobic alpha-helices. These differences were used as the rationale for a study of their efficacy in breaking liposomes with different combinations of phosphatidic acid and phosphatidylcholine. Biosensor binding measurements and encapsulating dye leakage studies showed that the higher binding affinity of CB and CB-1 to the polar heads of lipids is not necessary for the peptides to be more effective at lysing lipid bilayers, especially when liposomes have a higher phosphatidic acid content. Kinetic studies, by intrinsic and extrinsic fluorescence stopped-flow measurements, revealed two transitional steps in liposome breakage by CB and CB-1, although only one kinetic step was found for CB-3. Circular dichroism stopped-flow measurements, monitoring the formation of secondary structure in the peptides, found one kinetic step for the interaction of all of the peptides with the liposomes. Also, the alpha-helical motif of the peptides was maintained after interacting with the liposomes. Based on these results, the mechanisms of liposome lysis by CB, CB-1, and CB-3 are discussed.

Direct expression of PCR products in a cell-free transcription/translation system: synthesis of antibacterial peptide cecropin

A simple and effective methodology is proposed for direct expression of PCR-generated linear DNAs in cell-free transcription/translation systems without cloning DNA fragments in plasmids. This methodology is realized for the synthesis of the active antibacterial peptide cecropin using the synthetic coding sequence. Possible scientific applications and perspectives of the proposed approach are discussed.

Effects of the anti-bacterial peptide cecropin B and its analogs, cecropins B-1 and B-2, on liposomes, bacteria, and cancer cells

Custom designed analogs of the natural anti-bacterial peptide cecropin B (CB) have been synthesized; cecropin B-1 (CB-1) was constructed by replacing the C-terminal segment (residues 26 to 35) with the N-terminal sequence of CB (positions 1 to 10 which include five lysine residues). The second analog, CB-2, is identical to CB-1 except for the insertion of a Gly-Pro residue pair between Pro-24 and Ala-25. These peptides were used to investigate their anti-liposome, anti-bacterial and anti-cancer activities. The strength of anti-liposome activity is reduced two- to three-fold when the analogs are used instead of natural CB based on DL 50 analysis. Similarly, the potency of these analogs on certain bacteria is about two- to four-fold lower than those of CB based on LC measurements. In contrast, on leukemia cancer cells, the potency of CB-1 and CB-2 is about two- to three-fold greater than that of natural CB based on IC 50 measurements. All CB, CB-1 and CB-2 peptides have comparable helix contents according to CD measurements. These results indicate that the designed cationic lytic peptides, having extra cationic residues, are less effective in breaking liposomes and killing bacteria but more effective in lysing cancer cells. The possible interpretations for these observations are discussed. © 1997 Elsevier Science B.V.

Structure and Orientation of the Mammalian Antibacterial Peptide Cecropin P1 within Phospholipid Membranes

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly α-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospho lipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to a cispositive transmembrane voltage difference. Taken together, the results further support a “carpet-like” mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.

Interaction of the Mammalian Antibacterial Peptide Cecropin P1 with Phospholipid Vesicles

Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect cecropins), P22-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P22-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Gram-positive/Gram-negative antibacterial specificity.

Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A

A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy.

The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study.

The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.

Synthesis of the antibacterial peptide cecropin A (1-33).

Cecropin A(1-33) was synthesized by an improved stepwise solid-phase method. The synthesis was designed to give high coupling yields and minimal amounts of byproducts. All coupling steps were monitored for completion by a new ninhydrin procedure, and the fully protected peptide-resin was analyzed for deletion peptides by the solid-phase Edman preview technique. Both methods indicated that the average coupling yield was greater than 99.8%. The unpurified peptide mixture resulting from HF cleavage and extraction into 10% acetic acid was analyzed by reverse-phase high-pressure liquid chromatography, and 93% of the total product was shown to be the desired [Trp(For)2]cecropin A(1-33), indicating an average yield per synthetic cycle of 99.8%. Removal of the formyl group at pH 9, followed by ion-exchange chromatography, gave the purified product. Cecropin A(1-33) showed antibacterial activity against both Gram-positive and Gram-negative bacteria. Against Escherichia coli, the activity was only slightly lower than that of the natural 37-residue cecropin A when tested over a 100-fold concentration range; the minimum inhibitory concentration was approximately 1 microM. The formyl derivative was somewhat less effective in killing E. coli than the free 1-33 peptide. The antibacterial activity was discussed in terms of an amphipathic alpha-helix structure and the binding of the peptide to bacterial membranes.