Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Novel antimicrobial peptides identified from an endoparasitic wasp cDNA library

We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram‐negative and ‐positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1‐h incubation at a concentration of 62.5 µM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt‐dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13‐treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced α‐helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity

Currently, novel antibiotics are urgently required to combat the emergence of drug‐resistant bacteria. Antimicrobial peptides with membrane‐lytic mechanism of action have attracted considerable interest. Anoplin, a natural α‐helical amphiphilic antimicrobial peptide, is an ideal research template because of its short sequence. In this study, we designed and synthesized a group of analogues of anoplin. Among these analogues, anoplin‐4 composed of d ‐amino acids displayed the highest antimicrobial activity due to increased charge, hydrophobicity and amphiphilicity. Gratifyingly, anoplin‐4 showed low toxicity to host cells, indicating high bacterial selectivity. Furthermore, the mortality rate of mice infected with Escherichia coli was significantly reduced by anoplin‐4 treatment relative to anoplin. In conclusion, anoplin‐4 is a novel anoplin analogue with high antimicrobial activity and enzymatic stability, which may represent a potent agent for the treatment of infection. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Engineering of a linear inactive analog of human β‐defensin 4 to generate peptides with potent antimicrobial activity

Human β‐defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N‐terminus of HBD4. Our results show that l ‐arginine to d ‐arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l ‐arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d ‐arginine. Substitution of cysteine with the hydrophobic helix‐promoting amino acid α‐aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d ‐arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d ‐amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A novel defensin‐like peptide from salivary glands of the hard tick,Haemaphysalis longicornis

A novel defensin‐like antimicrobial peptide named longicornsin was isolated from the salivary glands of the hard tick, Haemaphysalis longicornis, using a 10‐kDa cut‐off Centriprep filter and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Its amino acid sequence was determined as DFGCGQGMIFMCQRRCMRLYPGSTGFCRGFRCMCDTHIPLRPPFMVG by Edman degradation. The cDNA encoding longicornsin was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 78 amino acids including a mature longicornsin. It showed similarity with defensin‐like peptides from other ticks by BLAST search. Different from most other tick defensin‐like peptides, longicornsin had a C‐terminal extension. Purified longicornsin exerted potent antimicrobial activities against bacteria and fungi. Interestingly, it even showed strong antimicrobial ability against drug‐resistant microorganisms and Helicobacter pylori. The results of this study indicated that longicornsin is a potential candidate for novel antimicrobial drug design.     

Two novel antimicrobial peptides from skin secretions of the frog,Rana nigrovittata

Two novel antimicrobial peptides with similarity to brevinin‐2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin‐2‐RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin‐2‐RN2), respectively, by Edman degradation. Different from brevinin‐2, which is composed of 33 amino acid residues (aa), both brevinin‐2‐RN1 and ‐RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465‐9) encoding precursors of brevinin‐2‐RN1 and ‐RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin‐2‐RN. Both brevinin‐2‐RN1 and ‐RN2 showed strong antimicrobial activities against gram‐positive and gram‐negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Modifications on amphiphilicity and cationicity of unnatural amino acid containing peptides for the improvement of antimicrobial activity against pathogenic bacteria

The widespread natural sources‐derived cationic peptides have been reported to reveal bacterial killing and/or growth‐inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug‐resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib‐Arg‐Aib‐Ala sequences, showed strong antibacterial activity against both Gram‐negative and Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug‐resistant Pseudomonas aeruginosa, known as proteases‐secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib‐derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A novel cysteine‐free venom peptide with strong antimicrobial activity against antibiotics‐resistant pathogens from the scorpion Opistophthalmus glabrifrons

Antibiotic‐resistant bacteria, such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococcus, pose serious threat to human health. The outbreak of antibiotic‐resistant pathogens in recent years emphasizes once again the urgent need for the development of new antimicrobial agents. Here, we discovered a novel antimicrobial peptide from the scorpion Opistophthalmus glabrifrons, which was referred to as Opisin. Opisin consists of 19 amino acid residues without disulfide bridges. It is a cationic, amphipathic, and α‐helical molecule. Protein sequence homology search revealed that Opisin shares 42.1–5.3% sequence identities to the 17/18‐mer antimicrobial peptides from scorpions. Antimicrobial assay showed that Opisin is able to potently inhibit the growth of the tested Gram‐positive bacteria with the minimal inhibitory concentration (MIC) values of 4.0–10.0 μM; in contrast, it possesses much lower activity against the tested Gram‐negative bacteria and a fungus. It is interesting to see that Opisin is able to strongly inhibit the growth of methicillin‐ and vancomycin‐resistant pathogens with the MICs ranging from 2.0 to 4.0 μM and from 4.0 to 6.0 μM, respectively. We found that at a concentration of 5 × MIC, Opisin completely killed all the cultured methicillin‐resistant Staphylococcus aureus. These results suggest that Opisin is a promising therapeutic candidate for the treatment of the antibiotic‐resistant bacterial infections. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Analogues of the frog skin peptide alyteserin‐2a with enhanced antimicrobial activities against Gram‐negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly¹¹→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC₅₀ = 24 µm ). Antimicrobial potency was increased further by the additional substitution Ser⁷→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC₅₀ = 38 µm ). However, the peptide containing d ‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm ) but appreciably lower haemolytic activity (LC₅₀ = 185 µm ) and cytotoxicity against A549 human alveolar basal epithelial cells (LC₅₀ = 65 µm ). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Bactrocerin‐1: A novel inducible antimicrobial peptide from pupae of oriental fruit fly Bactrocera dorsalis Hendel

A novel antimicrobial peptide, Bactrocerin‐1, was purified and characterized from an immunized dipteran insect, Bactrocera dorsalis. Bactrocerin‐1 has 20 amino acid residues with a mass of 2,325.95 Da. The amino acid sequence of Bactrocerin‐1 showed very high similarity to the active fragment (46V‐65S‐NH₂) of Coleoptericin A. The composition of amino acid residues revealed that Bactrocerin‐1 is a hydrophobic, positively charged, and Lys/Ile/Gly‐rich peptide. Minimal growth inhibition concentration (MIC) measurements for synthesized Bactrocerin‐1 showed a very broad spectrum of anti‐microbial activity against Gram‐positive bacteria, Gram‐negative bacteria, and fungi. Bactrocerin‐1 did not show hemolytic activity toward mouse red blood cells even at a concentration of 50 µM. Analysis of the Helical‐wheel projection and the CD spectrum suggested that Bactrocerin‐1 contains the amphipathic α‐helix. © 2009 Wiley Periodicals, Inc.     

The introduction of l-phenylalanine into antimicrobial peptide protonectin enhances the selective antibacterial activity of its derivative phe-Prt against Gram-positive bacteria

Protonectin was a typical amphiphilic antimicrobial peptide with potent antimicrobial activity against Gram-positive and Gram-negative bacteria. In the present study, when its eleventh amino acid in the sequence was substituted by phenylalanine, the analog named phe-Prt showed potent antimicrobial activity against Gram-positive bacteria, but no antimicrobial activity against Gram-negative bacteria, indicating a significant selectivity between Gram-positive bacteria and Gram-negative bacteria. However, when Gram-negative bacteria were incubated with EDTA, the bacteria were susceptible to phe-Prt. Next, the binding effect of phe-Prt with LPS was determined. Our result showed that LPS could hamper the bactericidal activity of phe-Prt against Gram-positive bacteria. The result of zeta potential assay further confirmed the binding effect of phe-Prt with LPS for it could neutralize the surface charge of E. coli and LPS. Then, the effect of phe-Prt on the integrity of outer membrane of Gram-negative bacteria was determined. Our results showed that phe-Prt had a much weaker disturbance to the outer membrane of Gram-negative bacteria than the parent peptide protonectin. In summary, the introduction of l-phenylalanine into the sequence of antimicrobial peptide protonectin made phe-Prt show significant selectivity against Gram-positive bacteria, which could partly be attributed to the delay effect of LPS for phe-Prt to access to cell membrane. Although further study is still needed to clarify the exact mechanism of selectivity, the present study provided a strategy to develop antimicrobial peptides with selectivity toward Gram-positive and Gram-negative bacteria.

     

Inhibitory effect of short cationic homopeptides against Gram‐positive bacteria

In the selection or design of antimicrobial peptides, the key role played by cationic amino acids and chain length on the inhibitory potency and specificity is not clear. A fundamental study was conducted using chemically synthesized homopeptides of l ‐Lys and l ‐Arg ranging from 7 to 14 residues. Their effect on growth inhibition was evaluated over a wide range of Gram‐positive bacteria at different levels of concentration. Interestingly, at lower concentrations (10 μM), Lys homopeptides with odd number of residues, especially with 11 residues, showed a broader inhibitory activity than those with even number of residues. At higher peptide concentrations (>20 μM), the inhibitory activity of Lys homopeptides was directly related to the number of residues in the chain. In contrast, Arg homopeptides, at lower concentrations, did not exhibit a defined pattern of bacterial inhibition related to the number of residues; however, at higher concentrations (>20 μM), the inhibitory effects were more pronounced. Lys homopeptides at concentrations up to 300 μM showed a remarkably lower toxicity against CHSE‐214 cells. Arg homopeptides exhibited negligible cytotoxicity up to chain length of 11 residues at concentrations lower than 100 μM, but an abrupt increase in toxicity resulted when the peptide chain length reached 12 amino acid residues and higher concentrations. All synthesized homopeptides displayed characteristic polyproline II helix conformation in both buffer and liposomes, as shown by CD spectroscopy. This result suggests that short Lys homopeptides with an odd number of residues (9 and 11) have a broad spectrum of activity against Gram‐positive bacterial cells compared with Arg homopeptides, which in turn showed a considerably higher selectivity toward those cells. By investigating the differences between Lys and Arg homopeptides, this study contributes to the understanding of their mechanism of growth inhibition and selectivity. Thus, it provides further guidelines for a rational design of short antimicrobial peptides. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Aminoarabinose is essential for lipopolysaccharide export and intrinsic antimicrobial peptide resistance in Burkholderia cenocepacia(†)

One common mechanism of resistance against antimicrobial peptides in Gram‐negative bacteria is the addition of 4‐amino‐4‐deoxy‐l ‐arabinose (l ‐Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires l ‐Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for l ‐Ara4N synthesis and transfer to the LPS. The absence of l ‐Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi‐protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that l ‐Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.     

Intramolecular cyclization of the antimicrobial peptide Polybia‐MPI with triazole stapling: influence on stability and bioactivity

Cationic antimicrobial peptides have attracted increasing attention as a novel class of antibiotics to treat infectious diseases caused by pathogenic bacteria. However, susceptibility to protease is a shortcoming in their development. Cyclization is one approach to increase the proteolytic resistance of peptides. Therefore, to improve the proteolytic resistance of Polybia‐MPI, we have synthesized the MPI cyclic analogs C‐MPI‐1 (i‐to‐i+4) and C‐MPI‐2 (i‐to‐i+6) by copper(I)‐catalyzed azide–alkyne cycloaddition. Compared with MPI, C‐MPI‐1 displayed sustained antimicrobial activity and had enhanced anti‐trypsin resistance, while C‐MPI‐2 displayed no antimicrobial activity. The relationship between peptide structure and bioactivity was further investigated by probing the secondary structure of the peptides by circular dichroism. This showed that C‐MPI‐1 adopted an α‐helical structure in aqueous solution and, interestingly, had increased α‐helical conformation in 30 mM sodium dodecyl sulfate and 50% trifluoroethyl alcohol compared with MPI. C‐MPI‐2 that was not α‐helical in structure, suggesting that the propensity for α‐helix conformation may play an important role in cyclic peptide design. In addition, scanning electron microscopy, propidium iodide uptake, and membrane permeabilization assays indicated that MPI and the optimized analog C‐MPI‐1 had membrane‐active action modes, indicating that the peptides would not be susceptible to conventional resistance mechanisms. Our study provides additional insight into the influence of intramolecular cyclization at various positions on peptide structure and biological activity. In conclusion, the design and synthesis of cyclic analogs via click chemistry offer a new strategy for the development of stable antimicrobial agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial activity of leucine‐substituted decoralin analogs with lower hemolytic activity

Linear cationic α‐helical antimicrobial peptides are promising chemotherapeutics. Most of them act by different mechanisms, making it difficult to microorganisms acquiring resistance. Decoralin is an example of antimicrobial peptide; it was described by Konno et al. and presented activity against microorganisms, but with pronounced hemolytic activity. We synthesized leucine‐substituted decoralin analogs designed based on important physicochemical properties, which depend on the maintenance of the amphiphilic α‐helical tendency of the native molecule. Peptides were synthesized, purified, and characterized, and the conformational studies were performed. The results indicated that the analogs presented both higher therapeutic indexes, but with antagonistic behavior. While [Leu]¹⁰‐Dec‐NH₂ analog showed similar activity against different microorganisms (c.a. 0.4–0.8 μmol L^?1), helical structuration, and some hemolytic activity, [Leu]⁸‐Dec‐NH₂ analog did not tend to helical structure and presented antimicrobial activities two orders higher than the other two peptides analyzed. On the other hand, this analog showed to be the less hemolytic (MHC value = 50.0 μmol L^?1). This approach provided insight for understanding the effects of the leucine substitution in the amphiphilic balance. They led to changes on the conformational tendency, which showed to be important for the mechanism of action and affecting antimicrobial and hemolytic activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial activity and stability to proteolysis of small linear cationic peptides with D-amino acid substitutions

Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D-amino acids were substituted for the L-amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L-peptide) rich in five arginine residues and consisting of an 11-amino acid peptide (residues 32-42) of human granulysin was synthesized. Subsequently, the L-amino acids of the 11-amino acid peptide were replaced partially (D-peptide) or wholly (AD-peptide) with D-amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D-amino acid substitutions were examined. Peptides with D-amino acid substitutions were found to lyse bacteria as efficiently as their all-L-amino acid parent, L-peptide. In addition, the peptide composed of L-amino acids was susceptible to trypsin, whereas peptides containing D-amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L-amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS-incubated peptide. Thus, D-amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D-amino acids are potential candidates for clinical therapeutic use.     

An amphipathic cyclic tetrapeptide scaffold containing halogenated β2,2‐amino acids with activity against multiresistant bacteria

The present study describes the synthesis and biological studies of a small series of head‐to‐tail cyclic tetrapeptides of the general structure c(Lys‐β^2,2‐Xaa‐Lys) containing one lipophilic β^2,2‐amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram‐positive and gram‐negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum β‐lactamase—carbapenemase (ESBL‐CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram‐positive clinical isolates with minimum inhibitory concentrations of 4–8 μg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic β^2,2‐amino acids form a valuable scaffold for designing novel antimicrobial agents.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.