Macromolecular properties of cepacian in water and in dimethylsulfoxide

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.     

Hydrophobic interactions modulate antimicrobial peptoid selectivity towards anionic lipid membranes

Hydrophobic interactions govern specificity for natural antimicrobial peptides. No such relationship has been established for synthetic peptoids that mimic antimicrobial peptides. Peptoid macrocycles synthesized with five different aromatic groups are investigated by minimum inhibitory and hemolytic concentration assays, epifluorescence microscopy, atomic force microscopy, and X-ray reflectivity. Peptoid hydrophobicity is determined using high performance liquid chromatography. Disruption of bacterial but not eukaryotic lipid membranes is demonstrated on the solid supported lipid bilayers and Langmuir monolayers. X-ray reflectivity studies demonstrate that intercalation of peptoids with zwitterionic or negatively charged lipid membranes is found to be regulated by hydrophobicity. Critical levels of peptoid selectivity are demonstrated and found to be modulated by their hydrophobic groups. It is suggested that peptoids may follow different optimization schemes as compared to their natural analogues.     

Mechanical force analysis of peptide interactions using atomic force microscopy

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.     

Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens

The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an alpha-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide-polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide-peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic Acid

Amylose-conjugated linoleic acid (CLA) complexes were produced by water/dimethyl sulfoxide (DMSO) and KOH/HCl complexation methods. The formation of amylose V form was confirmed by X-ray diffraction (XRD), and complexes formed at 30, 60, and 90 degrees C exhibit melting temperatures exceeding 88 degrees C. Atomic force microscopy (AFM) images showed distinct difference in complex organization, with complexes formed in water/DMSO showing spherical shape with typical diameter of 150 nm. Complexes formed by KOH/HCl showed elongated structure with typical width of 43-160 nm. Water/DMSO complexes exhibit superior protection to CLA against oxidation. All complexes showed high retention of CLA in simulated stomach conditions, and the digestion of complexes by amylases results in high hydrolysis and CLA release by pancreatin and alpha-amylase. Only moderate release was detected following hydrolysis by amyloglucosidase and beta-amylase. It is therefore suggested that amylose-CLA complexes can serve as molecular nanocapsules for protection and delivery of CLA.     

A radish seed antifungal peptide with a high amyloid fibril-forming propensity

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.     

A Flexible Nanoarray Approach for the Assembly and Probing of Molecular Complexes

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers’ rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.     

Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees (Apis mellifera)

Four antimicrobial peptides were purified from Royal Jelly of honeybees, by using reverse phase-HPLC and sequenced by using Q-Tof-MS/MS: PFKLSLHL-NH(2) (Jelleine-I), TPFKLSLHL-NH(2) (Jelleine-II), EPFKLSLHL-NH(2) (Jelleine-III), and TPFKLSLH-NH(2) (Jelleine-IV). The peptides were synthesized on-solid phase, purified and submitted to different biological assays: antimicrobial activity, mast cell degranulating activity and hemolysis. The Jelleines-I-III presented exclusively antimicrobial activities against yeast, Gram+ and Gram- bacteria; meanwhile, Jelleine-IV was not active in none of the assays performed. These peptides do not present any similarity with the other antimicrobial peptides from the honeybees; they are produced constitutively by the workers and secreted into Royal Jelly.     

Single molecule recognition between cytochrome C 551 and gold-immobilized azurin by force spectroscopy

Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of approximately 95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be approximately 14 s(-1), much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.     

Membrane and lipopolysaccharide interactions of C-terminal peptides from S1 peptidases

The mechanisms underlying antimicrobial and anti-endotoxic effects were investigated for a series of structurally related peptides derived from the C-terminal region of S1 peptidases. For this purpose, results on bacterial killing were compared to those on peptide-induced liposome leakage, and to ellipsometry and dual polarization interferometry results on peptide binding to, and disordering of, supported lipid bilayers. Furthermore, the ability of these peptides to block endotoxic effects caused by bacterial lipopolysaccharide (LPS), monitored through NO production in macrophages, was compared to the binding of these peptides to LPS, and to secondary structure formation in the peptide/LPS complex. Bacteria killing, occurring through peptide-induced membrane lysis, was found to correlate with liposome rupture, and with the extent of peptide binding to the lipid membrane, no adsorption threshold for peptide insertion being observed. Membrane and LPS binding was found to depend on peptide net charge, illustrated by LPS binding increasing with increasing peptide charge, and peptides with net negative charge being unable to lyse membranes, kill bacteria, and block LPS-induced endotoxic effect. These effects were, however, also influenced by peptide hydrophobicity. LPS binding was furthermore demonstrated to be necessary, but not sufficient, for anti-endotoxic effect of these peptides. Circular dichroism spectroscopy showed that pronounced helix formation occurs in peptide/LPS complexes for all peptides displaying anti-endotoxic effect, hence potentially linked to this functionality. Similarly, ordered secondary structure formation was correlated to membrane binding, lysis, and antimicrobial activity of these peptides. Finally, preferential binding of these peptides to LPS over the lipid membrane was demonstrated.     

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.     

H1 helix of colicin U causes phospholipid membrane permeation

In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.     

Detection of immune complexes using atomic force microscopy

Complex formation between immunoglobulins and ligands immobilized on mica was studied by atomic force microscopy in two different systems. In the first system, 60-kDa ligands possessing only one site for antibody recognition were used. In the other system, a more complex interaction of human immunoglobulin with immobilized polyclonal antibodies was studied. In both systems, specific complexes with proper ligand appeared, and unspecific interaction was not detected. The method of revealing immunocomplexes by image atomic force microscopy can be used in the development of modern diagnostic systems.     

Molecular evolution of animal antimicrobial peptides: widespread moderate positive selection

An increasing number of studies in both vertebrates and invertebrates show that the evolution of antimicrobial peptides is driven by positive selection. Because these diverse molecules show potential for therapeutic applications, they are currently the targets of much structural and functional research, providing extensive background data for evolutionary studies. In this paper, patterns of molecular evolution in antimicrobial peptide genes are reviewed. Evidence for positive selection on antimicrobial peptides includes an excess of nonsynonymous nucleotide substitutions, an excess of charge-changing amino acid substitutions, nonneutral patterns of allelic variation, and functional assays in vivo and in vitro that show improved antimicrobial effects for derived sequence variants. Positive selection on antimicrobial peptides may be as common as, but perhaps weaker than, selection on the best-known example of adaptively evolving immunity genes, the major histocompatibility complex. Thus, antimicrobial peptides present a useful and underutilized model for the study of adaptive molecular evolution.     

Anti-antimicrobial Peptides: FOLDING-MEDIATED HOST DEFENSE ANTAGONISTS*

Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.     

Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream

Chrysophsin-1 is an amphipathic alpha-helical antimicrobial peptide produced in the gill cells of red sea bream. The peptide has broad range activity against both Gram-positive and Gram-negative bacteria but is more hemolytic than other antimicrobial peptides such as magainin. Here we explore the membrane interaction of chrysophsin-1 and determine its toxicity, in vitro, for human lung fibroblasts to obtain a mechanism for its antimicrobial activity and to understand the role of the unusual C-terminal RRRH sequence. At intermediate peptide concentrations, solid-state NMR methods reveal that chrysophsin-1 is aligned parallel to the membrane surface and the lipid acyl chains in mixed model membranes are destabilized, thereby being in agreement with models where permeabilization is an effect of transient membrane disruption. The C-terminal RRRH sequence was shown to have a large effect on the insertion of the peptide into membranes with differing lipid compositions and was found to be crucial for pore formation and toxicity of the peptide to fibroblasts. The combination of biophysical data and cell-based assays suggests likely mechanisms involved in both the antibiotic and toxic activity of chrysophsins.     

Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation

The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.     

An insight into the gene delivery mechanism of the arginine peptide system: role of the peptide/DNA complex size

Cationic peptides have been used successfully to transfer macromolecules into living cells. Previously, we have reported a short arginine peptide-based gene delivery system. However, the mechanisms that allow arginine peptides to promote gene delivery yet remain unknown. In the present study, we investigated the effect of the arginine peptide/DNA complex size on the transfection efficiency. After combining peptides with DNA, a 400 nm complex was observed. As the incubation time was increased, the complex grew larger, reaching 6 microm after 1 h of incubation. Transfection and cellular uptake efficiency were likewise investigated for the effects of the different sizes of complexes. Large complexes were found to be advantageous for transfection. However, better internalization efficiency was found with small complexes, indicating that the amount of peptide/DNA complexes taken up by cells is not the rate-limiting step in the final transfection efficiency. The intracellular path of the peptide/DNA complex was studied using fluorescent labeling and confocal microscopy. In the early stages of transfection, complexes were observed only on the cell surface, and these complexes migrated into cytoplasm however, after 6 h, the presence of complexes in the perinuclear region was noted. We were able to detect colocalization of green and red fluorescence in both the cytoplasm and the nucleus. These results suggest that peptide/DNA complexes reach the nucleus as associated complexes.     

Investigating the binding behaviour of two avidin‐based testosterone binders using molecular recognition force spectroscopy

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand–receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin‐based proteins called sbAvd‐1 and sbAvd‐2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone‐binding protein was immobilized on the surface. Repeated formation and rupture of the ligand–receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex‐rupturing force. In this way, we obtained the molecular dissociation rate (k_off) and energy landscape distances (x_β) of the four possible complexes: sbAvd‐1‐biotin, sbAvd‐1‐testosterone, sbAvd‐2‐biotin and sbAvd‐2‐testosterone. It was found that the kinetic off‐rates for both proteins and both ligands are similar. In contrast, the x_β values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone‐binding proteins, implying a decreased cross‐reactivity of sbAvd‐2. Unravelling the binding behaviour of the investigated testosterone‐binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd.