Characterization of the structure and membrane interaction of the antimicrobial peptides aurein 2.2 and 2.3 from Australian southern bell frogs

The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH(2)), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH(2)), both from Litoria aurea, and a carboxy C-terminal analog of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied to determine which features of this class of peptides are key to activity. Circular dichroism and solution-state NMR data indicate that all three peptides adopt an alpha-helical structure in the presence of trifluoroethanol or lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide/lipid molar ratios (P/L) = 1:15-1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface adsorbed over a range of low peptide concentrations but insert into the bilayers at high peptide concentrations. This finding is corroborated by (31)P-solid-state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. Oriented circular dichroism data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, show that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15-1:120), whereas the aurein 2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied interact with phosphatidylcholine membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in phosphatidylcholine/phosphatidylglycerol (PC/PG) membranes, whereas aurein 2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations of the three peptides against Staphylococcus aureus (strain C622, ATCC 25923) and Staphylococcus epidermidis (strain C621--clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.     

Effect of amidation on the antimicrobial peptide aurein 2.5 from Australian southern bell frogs

Aurein 2.5 is a naturally C-terminally amidated amphibian antimicrobial peptide. C-terminal amidation can increase efficacy and hence a comparison was made between aurein 2.5-CONH2 and its nonamidated analogue. Amidation of the C-terminal carboxyl of aurein 2.5 enhanced antimicrobial activity 2.5- fold against Klebsiella pneumonia. Our results demonstrate that both peptide analogues had high surface activities (23 mN m-1for aurein 2.5-COOH and 26 mN m-1 aurein 2.5-CONH2). Circular dichroism measurements suggest that the helical content of the amidated form, in the presence of trifluoroethanol, was significantly enhanced (33.66 % for aurein 2.5-COOH and 60.89 % aurein 2.5-CONH2). The interaction of aurein 2.5 with bacterial cell membrane mimics was investigated using Langmuir monolayers. Aurein 2.5-CONH2 induced stable surface pressure changes in monolayers formed from K. pneumonia (circa 4.7 mN m-1), however, lower surface pressure changes were observed for aurein 2.5- COOH (circa 3.8 mN m-1). The data shows that in the case of aurein 2.5, amidation is able to enhance antibacterial activity and it is proposed that the increase in effectiveness is due to stabilization of the α-helical structure at the membrane interface.     

The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and 31P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.     

Spatial structure and activity mechanism of a novel spider antimicrobial peptide

Latarcins (Ltc), linear peptides (ca. 25 amino acid long) isolated from the venom of the Lachesana tarabaevi spider, exhibit a broad-spectrum antibacterial activity, most likely acting on the bacterial plasmatic membrane. We study the structure-activity relationships in the series of these compounds. At the first stage, we investigated the spatial structure of one of the peptides, Ltc2a, and its mode of membrane perturbation. This was done by a combination of experimental and theoretical methods. The approach includes (i) structural study of the peptide by CD spectroscopy in phospholipid liposomes and by (1)H NMR in detergent micelles, (ii) determination of the effect on the liposomes by a dye leakage fluorescent assay and (31)P NMR spectroscopy, (iii) refinement of the NMR-derived spatial structure via Monte Carlo simulations in an implicit water-octanol slab, and (iv) calculation of the molecular hydrophobicity potential. The molecule of Ltc2a was found to consist of two helical regions (residues 3-9 and 13-21) connected via a poorly ordered fragment. The effect of the peptide on the liposomes suggests the carpet mechanism of the membrane deterioration. This is also supported by the analysis of hydrophobic/hydrophilic characteristics of Ltc2a and homologous antimicrobial peptides. These peptides exhibiting a helix-hinge-helix structural motif are characterized by a distinct and feebly marked amphiphilicity of their N- and C-terminal helices, respectively, and by a hydrophobicity gradient along the peptide chain. The approach we suggested may be useful in studying not only other latarcins but also a wider class of membrane-active peptides.     

Designing the antimicrobial peptide with centrosymmetric and amphipathic characterizations for improving antimicrobial activity

Antibiotic-resistant bacterial infections are becoming a serious health issue and will cause 10 million deaths per year by 2050. As a result, the development of new antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs) are found in the innate immune systems of various organisms to effectively fend off invading pathogens. In this study, we designed a series of AMPs (THL-2-1 to THL-2-9) with centrosymmetric and amphipathic properties, through substituting different amino acids on the hydrophobic side and at the centrosymmetric position to improve their antimicrobial activity. The results showed that leucine as a residue on the hydrophobic side of the peptide could enhance its antimicrobial activity and that glutamic acid as a centrosymmetric residue could increase the salt resistance of the peptide. Thus, the THL-2-3 peptide (KRLLRELKRLL-NH₂) showed the greatest antimicrobial activity (MIC₉₀ of 16 μM) against Gram-negative bacteria and had the highest salt resistance and cell selectivity among all the designed peptides. In summary, the results of this study provide useful references for the design of AMPs to enhance antimicrobial activity.     

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

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

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers

Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction.     

Importance of each residue within secretin for receptor binding and biological activity

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.     

β-发卡抗菌肽的全新设计及其生物学活性

通过缬氨酸和精氨酸的交替连接形成β-发卡结构的两条侧链,D-脯氨酸和甘氨酸形成β-转角单元以及侧链末端的两个半胱氨酸连接形成一个二硫键,来设计得到全新的由16残基构成的β-发卡抗菌肽VR。对设计得到的抗菌肽VR的生物学活性进行了检测,主要测定了新型β-发卡抗菌肽VR的最小杀菌浓度、对红细胞的溶血活性、杀菌动力学和盐敏感性。结果发现,VR和蜂毒素具有相似的杀菌活性,而溶血活性远低于蜂毒素,这表明VR比蜂毒素具有更高的细胞选择性。在NaCl的浓度低于100 mmol/L时,VR的杀菌活性没有受到影响;在NaCl的浓度为100 mmol/L时,VR具有50%的杀菌活性。综上可见,VR具有较优异的生物学活性,拥有成为抗生素替代物的发展潜力。     

Structural and functional implications of a proline residue in the antimicrobial peptide gaegurin.

Although it is commonly known as a helix breaker, proline residues have been found in the alpha-helical regions of many peptides and proteins. The antimicrobial peptide gaegurin displays alpha-helical structure and has a central proline residue (P14). The structure and activity of gaegurin and its alanine derivative (P14A) were determined by various spectroscopic methods, restrained molecular dynamics, and biological assays. Both P14 and P14A exhibited cooperative helix formation in solution, but the helical stability of P14 was reduced substantially when compared to that of P14A. Chemical-shift analysis indicated that both of the peptides formed curved helices and that P14 showed diminished stability in the region around the central proline. However, hydrogen-exchange data revealed remarkable differences in the location of stable amide protons. P14 showed a stable region in the concave side of the curved helix, while P14A exhibited a stable region in the central turn of the helix. The model structure of P14 exhibited a pronounced kink, in contrast to the uniform helix of P14A. Both peptides showed comparable binding affinities for negatively charged lipids, while P14 had a considerably reduced affinity for a neutral lipid. With its destabilized alpha-helix, P14 exhibited greater antibacterial activity than did P14A. Hence, electrostatic interaction between helical peptides and lipid membranes is believed to be the dominant factor for antibacterial activity. Moreover, helical stability can modulate peptide binding to membranes that is driven by electrostatic interactions. The observation that P14 is a more potent antibacterial agent than P14A implies that the helical kink of P14 plays an important role in the disruption of bacterial membranes.     

Kinetic microplate assay for determining immobilized antimicrobial peptide activity

Antimicrobial peptide immobilization onto surfaces is of great interest, although characterization of activity can be problematic. The kinetic microplate method described here determines the minimum bactericidal concentration (MBC) of immobilized antimicrobial peptides through a combination and modification of traditional solution assays, overcoming the difficulties of working with a solid substrate. The technique enables rapid, accurate evaluation of immobilized peptide lytic behavior, elucidating both dose- and time-dependent activity at multiple concentrations. Furthermore, the method yields information regarding sublethal concentrations not realized in the traditional assays.     

The antimicrobial activity of the appetite peptide hormone ghrelin

The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.     

A Leu-Lys-rich antimicrobial peptide: activity and mechanism

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1-8)-magainin 2 (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu⁵]PF4, [Ala²]PF4, [Gly²]PF4, [Ala²,Leu⁵]PF4, and [Gly²,Leu⁵]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu⁵]PF4 [Al²]PF4 = [Ala²,Leu⁵]PF4 [Gly²]PF4 = [Gly²,Leu⁵]PF4. Leu⁵ for Ile⁵ substitutions in PF4 did not alter the activity of this peptide; however, Gly²/Ala² for Pro² substitutions reduced, bud did not abolish, peptide activity. Peptide stability studies revealed that [Gly²]PF4(2–7) and -(3–7) and [Ala²]PF4(2–7), -(3–7), and -(4–7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro² in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases.     

Importance of the C-terminal alpha-helical structure for glucagon's biological activity

The synthetic glucagon analogues [Glu21]glucagon, 2, and [Lys17,18,Glu21]glucagon, 3, were designed using Chou-Fasman calculations for the purpose of enhancing the probability for the formation of a C-terminal amphipathic alpha-helical conformation. Circular dichroism indicates increased alpha-helical content for these analogues in solution relative to glucagon. Analogues 2 and 3 also exhibit a 3-fold and 5-fold increase in receptor binding potency, respectively. The adenylate cyclase stimulating potencies of 2 and 3 relative to glucagon are 2.1 and 7 times greater, respectively. Attempts were made at further alpha-helical enhancement by further substitutions in the 10-13 region of glucagon, as represented by the glucagon analogues [Phe13,Lys17,18 Glu21]glucagon, 4, and [Phe10,13,Lys17,18,Glu21]glucagon, 5. These latter substitutions resulted in lowered receptor binding and adenylate cyclase potencies for 4 and 5 relative to 3 despite increased alpha-helical content in solution as observed by circular dichroism spectroscopy.     

The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective alpha-helical IsCT-derived antimicrobial peptide

IsCT-P (ILKKIWKPIKKLF-NH2) is a novel alpha-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of L- or D-Pro kink on the structure and the mode of action of a short alpha-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT-p, in which D-Pro is substituted for L-Pro8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical alpha-helical structure in various membrane-mimicking conditions, whereas IsCT-p showed a random structure. This result indicated that D-Pro in the central position of a short alpha-helical peptide provides more remarkable structural flexibility than L-Pro. Despite its higher antibacterial activity, IsCT-p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT-p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT-p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central D-Pro kink in alpha-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.     

Influence of the N- and C-terminal regions of leu-lys rich antimicrobial peptide on antimicrobial activity

P5 (KWKKLLKKPLLKKLLKKL-NH(2)) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K(3)K(4)L(5)L(6)-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.     

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis the solution structure of aurein 1.2.

Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1-3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic alpha-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10-5-10-4 M range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5. 1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.     

Molecular dynamics investigation of the influence of anionic and zwitterionic interfaces on antimicrobial peptides' structure: implications for peptide toxicity and activity

Molecular dynamics simulations of three related helical antimicrobial peptides have been carried out in zwitterionic diphosphocholine (DPC) micelles and anionic sodiumdodecylsulfate (SDS) micelles. These systems can be considered as model mammalian and bacterial membrane interfaces, respectively. The goal of this study is to dissect the differences in peptide composition which make the mutant peptides (novispirin-G10 and novispirin-T7) less toxic than the parent peptide ovispirin (OVIS), although all three peptides have highly antibacterial properties. Compared to G10 and T7, OVIS inserts deepest into the DPC micelle. This correlates well with the lesser toxicity of G10 and T7. There is strong evidence which suggests that synergistic binding of hydrophobic residues drives binding of OVIS to the micelle. The helical content of G10 and T7 is reduced in the presence of DPC, and this leads to less amphipathic peptide structures, which bind weakly to the micelle. Simulations in SDS were carried out to compare the influence of membrane electrostatics on peptide structure. All three peptides bound strongly to SDS, and retained helical form. This corresponds well with their equally potent antibacterial properties. Based on the simulations, we argue that secondary structure stability often leads to toxic properties. We also propose that G10 and T7 operate by the carpet mechanism of cell lysis. Toxicity of peptides operating by the carpet mechanism can be attenuated by reducing the peptide helical content. The simulations successfully capture experimental binding states, and the different depths of binding of the three peptides to the two micelles correlate with their antibacterial and toxic properties.