Probing the Conformational States of Thimet Oligopeptidase in Solution.

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).

Revealing the Mode of Action of Halictine Antimicrobial Peptides: A Comprehensive Study with Model Membranes.

Antimicrobial peptides are innate host defense molecules with the ability to kill pathogens. They have been widely studied for their membrane lytic activity and their potential to overcome the ever-increasing threat of antimicrobial resistance against conventional antibiotics. Here, we focus on two halictines, antimicrobial peptides first obtained from the venom of the eusocial bee Halictus sexcinctus. The peptides, HAL-1 and HAL-2, are cationic (with +3 and +4 charges, respectively) and amphipathic, have 12 amino acid residues, and exhibit high biological activity. For this study, the mechanism of action of HAL-1 and HAL-2 was studied in detail using large and giant unilamellar vesicles composed of pure palmitoyl oleoyl phosphatidyl choline (POPC) and a mixture of POPC and the anionic lipid palmitoyl oleoyl phosphatidyl glycerol (POPG) as biomimetic models of the membranes of eukaryotes and microorganisms, respectively. A set of complementary techniques was put forward: carboxyfluorescein leakage assay, phase contrast optical microscopy, ζ-potential, static and dynamic light scattering, fluorescence and circular dichroism spectroscopies, and isothermal titration calorimetry. The results show that both halictines are able to interact strongly with anionic membranes: The interaction is exothermic and accompanied by structuring of the peptides as an α-helix and deep insertion into the membrane causing substantial membrane permeabilization at very low peptide/lipid molar ratios. Extensive vesicle aggregation was detected only at a high peptide concentration. On the other hand, the interaction of the halictines with POPC is significantly milder. Yet, the peptides were able to permeabilize the POPC membranes to some extent. Comparing both peptides, HAL-1 showed a somewhat stronger effect on model membranes. Fits to the data revealed apparent binding constants on the order of 103-104 M-1 for anionic membranes and 1 order of magnitude lower for zwitterionic bilayers. When lytic activity results were compared at the same bound peptide/lipid ratio, the halictines exhibited a higher activity toward zwitterionic membranes. As novel peptides, small and with powerful activity, these halictines are potential candidates for becoming antimicrobial agents.

Location of the Positive Charges in Cationic Amphiphiles Modulates Their Mechanism of Action against Model Membranes.

Synthetic cationic amphiphiles (CAms) with physicochemical properties similar to antimicrobial peptides are promising molecules in the search for alternative antibiotics to which pathogens cannot easily develop resistance. Here, we investigate two types of CAms based on tartaric acid and containing two hydrophobic chains (of 7 or 11 carbons) and two positive charges, located either at the end of the acyl chains (bola-like, B7 and B11) or at the tartaric acid backbone (gemini-like, G7 and G11). The interaction of the CAms with biomimetic membrane models (anionic and neutral liposomes) was studied with zeta potential and dynamic light scattering measurements, isothermal titration calorimetry, and a fluorescent-based leakage assay. We show that the type of molecule determines the mechanism of action of the CAms. Gemini-like molecules (G7 and G11) interact mainly via electrostatics (exothermic process) and reside in the external vesicle leaflet, altering substantially the vesicle surface potential but not causing significant membrane lysis. On the other hand, the interaction of bola-like CAms (B7 and B11) is endothermic and thus entropy-driven, and these molecules reach both membrane leaflets and cause substantial membrane permeabilization, likely after clustering of anionic lipids. The lytic ability is clearly higher against anionic membranes as compared with neutral membranes. Within each class of molecule, longer alkyl chains (i.e., B11 and G11) exhibit higher affinity and lytic ability. Overall, the molecule B11 exhibits a high potential as antimicrobial agent, since it has a high membrane affinity and causes substantial membrane permeabilization.

Antimicrobial Peptide K0-W6-Hya1 Induces Stable Structurally Modified Lipid Domains in Anionic Membranes.

Considering the known different mode of action of antimicrobial peptides in zwitterionic and anionic cell membranes, the present work compares the action of the antimicrobial peptide K0-W6-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with zwitterionic and negatively charged model membranes, namely, liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, and a mixture of the two. Differential scanning calorimetry (DSC), steady state fluorescence of the Trp residue, dynamic light scattering (DLS), and measurement of the leakage of an entrapped fluorescent dye (carboxyfluorescein, CF) were performed with large unilamellar vesicles (LUVs). All techniques evidenced the different action of the peptide in zwitterionic and anionic vesicles. Trp fluorescence spectroscopy shows that the differences are related not only to the partition of the cationic peptide in zwitterionic and anionic membranes, but also to the different penetration depth of the peptide into the lipid bilayers: Trp goes deeper into negatively charged membranes, both in the gel and fluid phases, than into zwitterionic ones. DSC shows that the peptide is strongly attached to anionic bilayers, giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disturbed, possibly a stable or transient polar pore, considering the leakage of CF. This contrasts with the homogeneous effect produced by the peptide in zwitterionic membranes, probably related to peptide-membrane diffusion. Moreover, in mixed bilayers (PC:PG), the peptide sequesters negatively charged lipids, creating peptide-rich anionic lipid regions, strongly disturbing the membrane. The distinct structural interaction displayed by the peptide in PC and PG membranes could be related to the different mechanisms of action of the peptide in anionic prokaryotic and zwitterionic eukaryotic cell membranes.

Metacaspase-binding peptide inhibits heat shock-induced death in Leishmania (L.) amazonensis.

Leishmania (Leishmania) amazonensis is an important agent of cutaneous leishmaniasis in Brazil. This parasite faces cell death in some situations during transmission to the vertebrate host, and this process seems to be dependent on the activity of metacaspase (MCA), an enzyme bearing trypsin-like activity present in protozoans, plants and fungi. In fact, the association between MCA expression and cell death induced by different stimuli has been demonstrated for several Leishmania species. Regulators and natural substrates of MCA are poorly known. To fulfill this gap, we have employed phage display over recombinant L. (L.) amazonensis MCA to identify peptides that could interact with the enzyme and modulate its activity. Four peptides were selected for their capacity to specifically bind to MCA and interfere with its activity. One of these peptides, similar to ecotin-like ISP3 of L. (L.) major, decreases trypsin-like activity of promastigotes under heat shock, and significantly decreases parasite heat shock-induced death. These findings indicate that peptide ligands identified by phage display affect trypsin-like activity and parasite death, and that an endogenous peptidase inhibitor is a possible natural regulator of the enzyme.

Membrane permeabilization induced by Triton X-100: The role of membrane phase state and edge tension.

Detergents are widely used to solubilize and separate biomembrane components. It is therefore relevant to study and understand the mechanistic details underlying detergent-lipid interactions using biomimetic systems. Here, we have investigated in detail the process of membrane permeabilization and the nature of pores induced by sub-solubilizing concentrations of the detergent Triton X-100 (TX-100) in bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), sphingomyelin (SM) and binary mixtures of these phospholipids with 30 mol% cholesterol (chol). A fluorescence quenching assay was used to evaluate the permeability of large unilamellar vesicles (LUVs) in the presence of increasing concentrations of TX-100. Confocal microscopy was employed to visualize and quantify the permeability of giant unilamellar vesicles (GUVs) to two fluorescent dyes of different sizes in the presence of TX-100. Both methods showed that POPC, POPC/chol and SM membranes become fully permeable at a specific TX-100 concentration, followed by complete (POPC and SM) and partial (POPC/chol) solubilization at a higher detergent concentration. The confocal microscopy experiments revealed that opening of pores occurs as a well-defined event and that for POPC and POPC/chol the pores were initially selective to the small probe and then grew and allowed passage of the larger dye as well. On the other hand, the insoluble SM/chol membranes exhibited only a mild TX-100-induced permeabilization. The membrane edge tension of the liquid phases was measured from the closure rate of macropores induced by electric pulses in GUVs. Membrane edge tension was shown to be sensitive to membrane composition and to decrease in the presence of TX-100. We propose that extensive permeabilization occurs below a critical membrane edge tension, which is eventually reached in the partially and fully soluble compositions, but not in the insoluble mixture.

Comparative study of the mechanism of action of the antimicrobial peptide gomesin and its linear analogue: The role of the ß-hairpin structure.

Gomesin (Gm) is an antimicrobial peptide first isolated from the hemolymph of a Brazilian spider. Its powerful antimicrobial activity is, however, accompanied by hemolysis. As an alternative to this issue, a linear analogue (named GmL) lacking the disulfide bonds was designed. Here, CD spectroscopy, a fluorescence-based leakage assay, isothermal titration calorimetry (ITC) and light scattering are used to study the interaction of both Gm and GmL with large unilamellar vesicles (LUVs) composed of POPC (palmitoyl oleoyl phosphatidylcholine) with 25 and 50 mol% POPG (palmitoyl oleoyl phosphatidylglycerol). The activities of Gm and GmL in respect to their binding affinity/enthalpy, ability to permeabilize membranes and to induce vesicle aggregation are correlated with peptide secondary structure. Whereas Gm displays a quite stable ß-hairpin motif irrespective of the environment, GmL assumes a random conformation in aqueous solution and in the presence of 25 mol% POPG but adopts a ß-like structure in the presence of 50 mol% POPG. Gm exhibited high lytic activity against both surface charge densities. Instead, the activity of GmL was found to be negligible in the presence of 25 mol% POPG LUVs, but comparable to that of the native peptide against 50 mol% POPG as a consequence of peptide structuring. We conclude that the activity of Gm and its linear analogue is intimately related to the formation of a ß-turn motif, in which the hydrophobic residues form a hydrophobic face able to insert into the membrane and disrupt it.

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies.

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.

Structure-activity relationship of the antimicrobial peptide gomesin: the role of peptide hydrophobicity in its interaction with model membranes.

Antimicrobial peptides are part of the innate immune system of animals and plants. Their lytic activity against microorganisms generally depends on their ability to disrupt and permeabilize membranes. Here we study the structure-activity relationship of the antimicrobial peptide gomesin (Gm), from the spider Acanthoscurria gomesiana, with large unilamellar vesicles (LUVs) composed of 3:7 palmitoyloleoyl phosphatidylglycerol: palmitoyloleoyl phosphatidylcholine. Several synthetic analogues of Gm were designed to alter the hydrophobicity/charge of the molecule, whereby selected amino acid residues were replaced by alanine. Isothermal titration calorimetry (ITC) was used to assess the thermodynamic parameters of peptide binding to LUVs and light scattering measurements were made to evaluated peptide-induced vesicle aggregation. The ability of the peptides to permeabilize vesicles was quantified through the leakage of an entrapped fluorescent probe. The activity of peptides could be quantified in terms of the leakage extent induced and their affinity to the membrane, which was largely dictated by the exothermic enthalpy change. The results show that analogues more hydrophobic than Gm display higher activity, whereas peptides more hydrophilic than Gm have their activity almost abolished. Vesicle aggregation, on the other hand, largely increases with peptide charge. We conclude that interaction of Gm with membranes depends on an interplay between surface electrostatic interactions, which drive anchoring to the membrane surface and vesicle aggregation, and insertion of the hydrophobic portion into the membrane core, responsible for causing membrane rupture/permeabilization.

Interaction of the antimicrobial peptide gomesin with model membranes: a calorimetric study.

Gomesin is a potent cationic antimicrobial peptide (z = +6) isolated from the Brazilian spider Acanthoscurria gomesiana . The interaction of gomesin with large unilamellar vesicles composed of a 1:1 mixture of zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and anionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) phospholipids is studied with isothermal titration calorimetry (ITC). In parallel, light scattering and optical microscopy are used to assess peptide-induced vesicle aggregation. The ability of gomesin to permeabilize the membrane is examined with fluorescence spectroscopy of the leakage of 5,6-carboxyfluorescein (CF). Vesicles coated with 3 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG) lipids are also investigated to assess the influence of peptide-induced vesicle aggregation in the activity of gomesin. The ITC and light scattering titrations are done in two ways: lipid into peptide and peptide into lipid injections. Although some differences arise between the two setups, the basic interaction of gomesin with anionic vesicles is preserved. A surface partition model combined with the Gouy-Chapman theory is put forward to fit the ITC results. The intrinsic binding constant of gomesin is found to be K ≈ 10(3) M(-1). The interaction of gomesin with anionic membranes is highly exothermic and enthalpy-driven. Binding of gomesin is virtually always accompanied by vesicle aggregation and changes in membrane permeability, leading to CF leakage. Addition of PE-PEG to the membrane strongly attenuates vesicle aggregation but does not significantly change the mode of action of gomesin. The results point to a strong interaction of gomesin with the membrane surface, causing membrane rupture without a deep penetration into the bilayer core.

Using multidimensional projection techniques for reaching a high distinguishing ability in biosensing.

Recent advances in the control of molecular engineering architectures have allowed unprecedented ability of molecular recognition in biosensing, with a promising impact for clinical diagnosis and environment control. The availability of large amounts of data from electrical, optical, or electrochemical measurements requires, however, sophisticated data treatment in order to optimize sensing performance. In this study, we show how an information visualization system based on projections, referred to as Projection Explorer (PEx), can be used to achieve high performance for biosensors made with nanostructured films containing immobilized antigens. As a proof of concept, various visualizations were obtained with impedance spectroscopy data from an array of sensors whose electrical response could be specific toward a given antibody (analyte) owing to molecular recognition processes. In addition to discussing the distinct methods for projection and normalization of the data, we demonstrate that an excellent distinction can be made between real samples tested positive for Chagas disease and Leishmaniasis, which could not be achieved with conventional statistical methods. Such high performance probably arose from the possibility of treating the data in the whole frequency range. Through a systematic analysis, it was inferred that Sammon's mapping with standardization to normalize the data gives the best results, where distinction could be made of blood serum samples containing 10(-7) mg/mL of the antibody. The method inherent in PEx and the procedures for analyzing the impedance data are entirely generic and can be extended to optimize any type of sensor or biosensor.