De Novo Hydrocarbon-Stapling Design of Single-Turn α-Helical Antimicrobial Peptides

International Journal of Peptide Research and Therapeutics - Most existing antimicrobial peptides (AMPs) are α-helical and cationic that exhibit typical amphipathic feature to facilitate...     

Brazilian Kefir-Fermented Sheep’s Milk,a Source of Antimicrobial and Antioxidant Peptides

Fermented milks are a source of bioactive peptides and may be considered as functional foods. Among these, sheep’s milk fermented with kefir has not been widely studied and its most relevant properties need to be more thoroughly characterized. This research study is set out to investigate and evaluate the antioxidant and antimicrobial properties of peptides from fermented sheep’s milk in Brazil when produced by using kefir. For this, the chemical and microbiological composition of the sheep’s milk before and after the fermentation was evaluated. The changes in the fermented milk and the peptides extracted before the fermentation and in the fermented milk during its shelf life were verified. The antimicrobial and antioxidant activities of the peptides from the fermented milk were evaluated and identified according to the literature. The physicochemical properties and mineral profile of the fermented milk were like those of fresh milk. The peptide extract presented antimicrobial activity and it was detected that 13 of the 46 peptides were able to inhibit the growth of pathogenic microorganisms. A high antioxidant activity was observed in the peptides extracted from fermented milk (3.125 mg/mL) on the 28th day of storage. Two fractions displayed efficient radical scavenging properties by DPPH and ABTS methods. At least 11 peptides distributed in the different fractions were identified by tandem mass spectrometry. This sheep’s milk fermented by Brazilian kefir grains, which has antioxidant and antimicrobial activities and probiotic microorganisms, is a good candidate for further investigation as a source for bioactive peptides. The fermentation process was thus a means by which to produce potential bioactive peptides.     

Design, Recombinant Expression, and Antibacterial Activity of the Cecropins–Melittin Hybrid Antimicrobial Peptides

In order to evaluate their antibacterial activities and toxicities, the cecropins–melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp’s 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect.     

Synergistic Insertion of Antimicrobial Magainin-Family Peptides in?Membranes Depends on the Lipid Spontaneous Curvature

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state ¹⁵N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.The two antimicrobial peptides PGLa and magainin 2 (MAG2) from the African frog Xenopus laevis, which are active against Gram-positive and Gram-negative bacteria, show intriguing synergistic effects that are not yet well understood (1). Structural insights into this synergy may help in the development of a new antibiotic-combination therapy. Both peptides are known to form α-helices when bound to lipid bilayers (2–4). The orientation of such α-helices in a membrane can be readily determined from the ¹⁵N-NMR chemical shift in oriented lipid bilayers that are aligned with the sample normal parallel to the external magnetic field (5). If the ¹⁵N chemical shift is ∼90 ppm, the peptide lies flat on the membrane surface (in the so-called S-state). On the other hand, when the ¹⁵N chemical shift is ∼200 ppm, the peptide is fully inserted (the I-state) in a transmembrane alignment. For intermediate orientations, where the peptide is tilted (the T-state) with an angle of typically 30°–60° relative to the membrane normal, intermediate chemical shifts are expected, but the exact tilt angle cannot be determined from a single label in such cases (6). Using several selectively ²H- or ¹⁹F-labeled peptide analogs, more exact orientations can be obtained, since both the tilt and the azimuthal angles can be measured with high accuracy, and valuable information about dynamics also can be deduced (3,7–10).The orientation of PGLa and MAG2 in membranes has been extensively studied with solid-state NMR, and some clues about the synergistic mechanism have been observed. Notably, in DMPC/DMPG membranes, it has been shown that the peptides on their own are in the S-state or T-state, but when the peptides are mixed in a 1:1 molar ratio, PGLa changes to the I-state, whereas MAG2 stays on the membrane surface (1,11,12). Thus, in the mixed system, transmembrane pores, which would not be spontaneously formed by each peptide on its own, appear to be stable, which could be the basis for synergy. On the other hand, it was also reported that in POPC/POPG there is no change in orientation when PGLa and MAG2 are mixed, as both peptides stay always in the S-state (12). This observation was attributed to the greater hydrophobic thickness of the POPC/POPG bilayer, compared to the DMPC/DMPG bilayer, suggesting that the PGLa helix is so short that it can only insert into thin DMPC/DMPG membranes. However, we have recently shown for MSI-103, a designer-made antimicrobial peptide based on the PGLa sequence, that the orientation determined by ²H-NMR depends not on the bilayer thickness but rather on the intrinsic spontaneous curvature of the lipids (13). Accordingly, an insertion of PGLa and MAG2 into POPC/POPG should be prevented by the pronounced negative spontaneous curvature induced by the unsaturated acyl chains. However, a simple comparison of only two lipid systems does not yield an answer as to which of these two hypotheses is correct. Therefore, we have now collected data over a wide range of lipid systems, with systematic variations of acyl chain lengths (to address bilayer thickness) as well as chain saturations (to address lipid curvature) (see Table S1 in the Supporting Material). In this way, we found unambiguously that lipid curvature is also the decisive factor in the insertion of PGLa/MAG2.Here, ¹⁵N-NMR spectra of singly labeled peptides were recorded, and for each liquid-crystalline lipid system we prepared four oriented samples: ¹⁵N-MAG2 alone, ¹⁵N-MAG2 with PGLa, ¹⁵N-PGLa with MAG2, and ¹⁵N-PGLa alone. The total peptide/lipid molar ratio (P/L) was 1:50. ¹⁵N-NMR spectra are shown in Fig. 1, and the chemical shifts are listed in Fig. 2. The quality of each oriented sample was checked with ³¹P-NMR (see Fig. S1). Our previous detailed ²H- and ¹⁹F-NMR analysis of PGLa in DMPC and DMPC/DMPG showed that the helix realigns depending on the peptide concentration. Namely, at low concentration, PGLa is in an S-state with a tilt angle of ∼98° (3), but above a threshold concentration around P/L = 1:100, it flips into a tilted T-state with a tilt angle of ∼125° (9,14). In the presence of MAG2, it was found that PGLa inserts almost upright in an I-state with a tilt angle of ∼158° (11). The present ¹⁵N-NMR study confirms that in DMPC/DMPG (3:1), PGLa is in the I-state when mixed with MAG2, as indicated by the ¹⁵N chemical shift of 205 ppm. PGLa alone at P/L = 1:50 has a chemical shift of 116 ppm, which corresponds to a tilted orientation, as expected. MAG2 alone is found to be in the S-state (91 ppm), but when it is mixed with PGLa its signal moves to 105 ppm, indicating a small change in the alignment. MAG2 is, however, clearly not inserted like PGLa.Open in a separate windowFigure 1¹⁵N-NMR spectra of ¹⁵N-labeled PGLa or MAG2, alone or in a synergistic 1:1 mixture with the other peptide, in differently oriented lipids. Powder spectra are shown in the top row. Red, green, and blue lines indicate chemical shifts associated with the S-, T-, and I-state orientations, respectively.Open in a separate windowFigure 2Schematic overview of the orientation of PGLa (red), MAG2 (green), and MSI-103 (orange (13)) in different lipids. The corresponding ¹⁵N-NMR chemical shifts (in ppm) of the spectra in Fig. 1 are indicated beneath each peptide.In thin DLPC bilayers (12 carbon atoms in the chains) we see a behavior similar to that in DMPC (14 carbons), even though the exact chemical shifts are slightly different. PGLa alone is in the T-state, but in combination with MAG2, it flips into the I-state. MAG2, on the other hand, stays in the S-state with and without PGLa. In DPPC bilayers (16 carbons) also, the behavior is similar. PGLa alone is in the T-state but flips into the I-state in the presence of MAG2. MAG2 is slightly more tilted than in DMPC, but it never reaches the I-state.In contrast, in unsaturated lipids, both peptides are always in the S-state, both alone and in the presence of the synergistic partner. In POPC/POPG (9:1), the ¹⁵N chemical shifts of both PGLa and MAG2, alone and in the 1:1 mixture, are between 84 and 89 ppm, clearly indicating a flat alignment on the bilayer surface. As there are no changes in chemical shift with or without the other peptide, this could indicate that there are no interactions between them, in contrast to the situation in saturated lipids. Also, in thin DMoPC bilayers (with 14 carbon atoms and a double bond), the chemical shifts of all samples show that both peptides remain always in the S-state, whether alone or mixed.These results clearly demonstrate that the hydrophobic membrane thickness is not a critical factor for the insertion of PGLa in the presence of MAG2. In DMoPC (thinner than DMPC), there is no insertion, whereas in DPPC (thicker than POPC) insertion occurs. On the other hand, the results fully support the lipid-curvature hypothesis, which states that peptides remain on the surface in membranes composed of lipids with a negative spontaneous curvature, but are more easily tilted or inserted when the lipids have a positive spontaneous curvature (13).In a special lipid mixture, POPE/POPG/TOCL (72:23:5), often used to mimic the composition of the inner membrane of Escherichia coli (15), the result is practically the same as in POPC/POPG (9:1). Also here, chemical shifts of ∼84 ppm indicate that PGLa and MAG2 are always in the S-state, both alone and as a mixture. This behavior is in accordance with the curvature hypothesis, since PE and CL both have a strong negative curvature. On the other hand, when lyso-MPC is added to DMPC to increase the positive curvature, the chemical shift of MAG2 increases to 117 ppm, indicating a more tilted orientation in the membrane with enhanced curvature compared to DMPC or DMPC/DMPG, both with and without PGLa. PGLa alone gives a somewhat larger chemical shift but stays in the T-state, whereas PGLa together with MAG2 flips again into the I-state.We can now compare the results presented here with those from our previous study on the related peptide MSI-103 (13) to find strong correlations. Fig. 2 gives an overview of all results, illustrating the peptide orientations in the different lipid systems. PGLa on its own behaves just like MSI-103 and assumes the same S-state or T-state in the same systems, in full accordance with the lipid-curvature hypothesis. MAG2 alone behaves similarly but seems to have a higher concentration threshold to flip from the S-state to the T-state. In DMPC and DMPC/DMPG, where PGLa is already in the T-state, MAG2 is still in the S-state at P/L = 1:50. However, at P/L = 1:10 (Fig. S2), MAG2 has also reached the T-state. Since MAG2 is charged at both termini, whereas PGLa and MSI-103 are amidated and thus uncharged on the C terminus, it is indeed expected that MAG2 should not start to tilt as easily as PGLa or MSI-103. The polar sector of MAG2 is also larger (Fig. S3).When PGLa and MAG2 are mixed 1:1, their behavior correlates well with that of the individual peptides. In systems where PGLa and MSI-103 are in the S-state, the mixture of PGLa and MAG2 also remains in the S-state. Only when PGLa alone prefers the T-state does it get fully pushed into the I-state by the presence of MAG2. Thus, the model of MAG2-assisted insertion of PGLa proposed previously (12), which suggested that MAG2 would facilitate a thinning of the membrane such that PGLa would be able to insert into it, cannot be correct. We can instead conclude that only lipid systems that encourage peptide insertion per se show the MAG2-induced I-state of PGLa. The relationship between lipid shape and the tendency of peptides to insert into the membrane, as previously discussed (13), is illustrated in Fig. S4. Interestingly, common bacterial lipids like PE and CL have a negative spontaneous curvature and should thus not support peptide insertion and stable pores. However, pores could still be transient in native membranes, or other components like membrane proteins could influence the overall spontaneous curvature.In conclusion, we propose several criteria that encourage a peptide to insert from the surface-bound S-state more deeply into the membrane (i.e., into a T-state or I-state): 1), positive lipid spontaneous curvature, which is enhanced by large headgroups and ordered lipid chains (due to saturation, but also found at low temperatures close to the gel-to-liquid-crystalline phase transition); 2), a narrow polar sector and uncharged termini of the peptide; and 3), the presence of another peptide. The other peptide might have an indirect effect by changing the membrane properties via crowding. However, for PGLa/MAG2, a distinct synergistic activity has been demonstrated, indicating more specific interactions between these two peptides. The present ¹⁵N-NMR analysis shows that the two partner peptides are not aligned side-by-side as a dimer. Further solid-state NMR distance measurements will be required to clarify their detailed mode of assembly.     

Determining the Mode of Action Involved in the Antimicrobial Activity of?Synthetic Peptides: A Solid-State NMR and FTIR Study

We have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular β-sheet peptides on both types of bilayers. ³¹P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. ²H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core.     

Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides: STRUCTURES AND ACTIVITIES IN LIPOPOLYSACCHARIDE*?

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.LPS² or endotoxin, a major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is critically involved in health and diseases of humans (1, 2). LPS is essential for bacterial survival through establishing an efficient permeability barrier against a variety of antimicrobial compounds including hydrophobic antibiotics, detergents, host-defense proteins, and antimicrobial peptides (3, 4). Several studies have demonstrated that LPS catalyzes folding of outer membrane proteins as a chaperone (5–7).LPS, a potent inducer of innate immune systems, hence called endotoxin, is primarily responsible for lethality in sepsis and septic shock syndromes associated with serious Gram-negative infections (8–10). Circulating LPS in bloodstream is intercepted by the phagocytic cells of the innate immune system. Once induced by LPS, these phagocytes produce proinflammatory cytokines, e.g. tumor necrosis factor-α, interleukin-6, and interleukin-1β, through the activation of a Toll-like pattern recognition receptor (11, 12). The release of cytokines in response to microbial invasion is a natural function of the innate immunity. However, an uncontrolled and overwhelming production of these cytokines may cause “endotoxic shock” or septic shock, typified by endothelial tissue damage, loss of vascular tone, coagulopathy, and multiple organ failure, often resulting in death (9, 10). Sepsis is the major cause of mortality in the intensive care unit, accounting for 200,000 deaths every year in the United States alone (13). It was demonstrated that release of LPS from antibiotic-treated Gram-negative bacteria can indeed enhance sepsis (14). Therefore, an effective antibiotic should not only exert antibacterial activities but also have the ability to sequester LPS and ameliorate its toxicity. Therefore, an amalgamated property of LPS-neutralizing and antimicrobial activity would be highly desirable for antimicrobial agents. Polymyxin B is a prototypical antimicrobial and antiendotoxic antibiotic; however, its neurotoxicity and nephrotoxicity limit its application to topical use (15). The increasing emergence of bacterial strains that are resistant to conventional antibiotics has initiated vital structure/function studies of membrane-perturbing cationic antimicrobial peptides (16–20). More recent studies have been conducted to understand interactions between antimicrobial peptides with LPS to gain insights into the mechanism of outer membrane perturbation, antibacterial activities, and LPS neutralization (21–26). These studies have delineated the role of amino acid sequence properties, LPS-peptide interactions by biophysical methods, and global structural parameters, obtained by CD and FTIR.Designing synthetic peptides and elucidation of three-dimensional structures in complex with LPS would be useful for the purpose of rational development of non-toxic antisepsis and antimicrobial therapeutics. Such studies will also be potentially instructive in establishing rules by which folded structures can be stabilized on the LPS surface. Extensive work in the field of peptide design primarily focuses on mimicking secondary structures and tertiary folds of proteins. Usually, short linear peptides are often structurally flexible; however, the functions of these peptides are highly dependent on their ability to adopt folded structures upon complex formation with their cognate receptors. In this regard, designed peptides that would yield high resolution structures in complex with LPS have not been well pursued. LPS, being a negatively charged amphiphilic molecule, interacts with naturally occurring peptides or protein fragments containing basic/polar and hydrophobic amino acids, although there are considerable variations in lengths, sequences, and amino acid compositions among these peptides (27, 28).Here, we have determined the three-dimensional structures of a series of 12-residue peptides in the context of LPS. To the best of our knowledge, these results show, for the first time, that atomic resolution structures of designed peptides obtained in LPS could be correlated with their antiendotoxic activities. Furthermore, the LPS-induced structures of active, inactive, and short peptide motif, presented here, may provide building blocks for the designing novel proteins for the outer membrane.     

Antimicrobial mechanism of β-glycyrrhetinic acid isolated from licorice, Glycyrrhiza glabra

-Glycyrrhetinic acid isolated from Glycyrrhiza glabra had an antibacterial activity of 7.6 and 12.5 g ml^–1 against Bacillus subtilis and Staphylococcus epidermidis without causing hemolysis of human erythrocytes, whereas it was not inhibitory against Escherichia coli, Proteus vulgaris and various fungi. Confocal microscopy showed that -glycyrrhetinic acid was located within the bacteria but had not caused membrane disruption. It then inhibited synthesis of DNA, RNA and protein.     

Staphylococcus epidermidis Antimicrobial δ-Toxin (Phenol-Soluble Modulin-γ) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus

Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis δ-toxin (PSMγ) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic δ-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), δ-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of δ-toxin exerted a bacteriostatic effect on GAS, and in NETs, δ-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of δ-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with δ-toxin. Thus, these data suggest that S. epidermidis–derived δ-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.     

Structural Analysis of Peptides of PSⅡ Light-harvesting Complexes in Siphonous Green Algae,Codium fragile

Peptide composition and arrangement of 4 major light harvesting complexes LHCP1-3 and LHCP3′isolated from siphonous green algae （Codium fragile (Sur.) Hariot.） were investigated. LHCP1 showed five main peptides, 34.4, 31.5, 29.5, 28.2 and 26.5 kD in SDS PAGE, the 34.4 and 31.5 kD peptides were never found in higher plants. LHCP3 contained the other four kinds of LHCP1 peptides except 34.4 kD, while LHCP3′consisted of only 28.2 and 26.5 kD peptides. We found that 34.4, 28.2 and 26.5 kD peptides were easy to decompose from LHCP 1 when subjected to SDS PAGE without pretreatment. They might be located at the exterior of LHCP1, while the 31.5 and 29.5 kD peptides were at the central part. The 28.2 and 26.5 kD peptides often occurred in CPa, the center complex of PSⅡ. They are possibly the LHCⅡ peptides tightly associated with CCⅡ. According to the results described above, a peptide map of LHCP1 was sketched.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Several Fmoc-,-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satisfactory.     

Regulation of Aggregation of Self-Associated Peptides,Including N-Terminal Fragments of the Alzheimer’s β-Amyloid Peptide,by Nitro Derivatives of Azoloazine

Russian Journal of Bioorganic Chemistry - The potential of the nitro compounds of the azoloazine class as regulators of aggregation of natural self-associating peptides was demonstrated by the...     

β-Hairpin-Mediated Formation of Structurally Distinct Multimers of Neurotoxic Prion Peptides

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A₁₁₇V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies.     

Antimicrobial actions of α-mangostin against oral streptococci

The increasing prevalence of dental caries is making it more of a major world health problem. Caries is the direct result of acid production by cariogenic oral bacteria, especially Streptococcus mutans. New and better antimicrobial agents active against cariogenic bacteria are badly needed, especially natural agents derived directly from plants. We have evaluated the inhibitory actions of α-mangostin, a xanthone purified from ethanolic extracts of the tropical plant Garcinia mangostana L., by repeated silica gel chromatography. α-Mangostin was found to be a potent inhibitor of acid production by S. mutans UA159, active against membrane enzymes, including the F(H+)-ATPase and the phosphoenolpyruvate - sugar phosphotransferase system. α-Mangostin also inhibited the glycolytic enzymes aldolase, glyceraldehyde-3-phosphate dehydrogenase, and lactic dehydrogenase. Glycolysis by intact cells in suspensions or biofilms was inhibited by α-mangostin at concentrations of 12 and 120 μmol·L?1, respectively, in a pH-dependent manner, with greater potency at lower pH values. Other targets for inhibition by α-mangostin included (i) malolactic fermentation, involved in alkali production from malate, and (ii) NADH oxidase, the major respiratory enzyme for S. mutans. The overall conclusion is that α-mangostin is a multitarget inhibitor of mutans streptococci and may be useful as an anticaries agent.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Summary Several Fmoc-α,α-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satistactory.     

Antimicrobial efficacy of some N,N’-Dialkyl-N,N’-dimethyl-1, 6-hexanediamine dioxides

A total of 17 N,N'-dialkyl-N,N'-dimethyl-1,6-hexanediamine dioxides were tested for activity against three microorganisms. A relationship was found between the length of the alkyl substituent and antimicrobial activity.     

Copper and Zinc Mediated Oligomerisation of Aβ Peptides

The accumulation of senile plaques composed primarily of aggregated amyloid β-peptide (Aβ), is the major characteristic of Alzheimer’s disease. Many studies correlate plaque accumulation and the presence of metal ions, particularly copper and zinc. The metal binding sites of the amyloid Aβ peptide of Alzheimer’s disease are located in the N-terminal region of the full-length peptide. In this work, the interactions with metals of a model peptide comprising the first 16 amino acid residues of the amyloid Aβ peptide, Aβ(1–16), were studied. The effect of Cu²⁺ and Zn²⁺ binding to Aβ(1–16) on peptide structure and oligomerisation are reported. The results of ESI-MS, gel filtration chromatography and NMR spectroscopy demonstrated formation of oligomeric complexes of the peptide in the presence of the metal ions and revealed the stoichiometry of Cu²⁺ and Zn²⁺ binding to Aβ(1–16), with Cu²⁺ showing a higher affinity for binding the peptide than Zn²⁺.     

Synthesis of Modified Peptides with C-Terminal α-Amino Aldehydes

Various synthetic approaches to modified peptides with the C-terminal aldehyde group, capable of inhibiting a number of proteolytic enzymes belonging to the classes of thiol, serine, and aspartyl proteases, are considered. Both chemical methods, including solid phase peptide synthesis now widely used, and biocatalytic synthetic methods for obtaining these substances are discussed in detail.