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João Ruggiero Neto
Department of Physics, IBILCE, UNESP-São Paulo State University, São José do Rio Preto, Brazil

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Original article
Published: 22 April 2021 in Amino Acids
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Antimicrobial peptides (AMPs) are part of the innate immune system of many species. AMPs are short sequences rich in charged and non-polar residues. They act on the lipid phase of the plasma membrane without requiring membrane receptors. Polybia-MP1 (MP1), extracted from a native wasp, is a broad-spectrum bactericide, an inhibitor of cancer cell proliferation being non-hemolytic and non-cytotoxic. MP1 mechanism of action and its adsorption mode is not yet completely known. Its adsorption to lipid bilayer and lytic activity is most likely dependent on the ionization state of its two acidic and three basic residues and consequently on the bulk pH. Here we investigated the effect of bulk acidic (pH 5.5) and neutral pH (7.4) solution on the adsorption, insertion, and lytic activity of MP1 and its analog H-MP1 to anionic (7POPC:3POPG) model membrane. H-MP1 is a synthetic analog of MP1 with lysines replaced by histidines. Bulk pH changes could modulate this peptide efficiency. The combination of different experimental techniques and molecular dynamics (MD) simulations showed that the adsorption, insertion, and lytic activity of H-MP1 are highly sensitive to bulk pH in opposition to MP1. The atomistic details, provided by MD simulations, showed peptides contact their N-termini to the bilayer before the insertion and then lay parallel to the bilayer. Their hydrophobic faces inserted into the acyl chain phase disturb the lipid-packing.

ACS Style

Ingrid Bernardes Santana Martins; Taisa Giordano Viegas; Dayane Dos Santos Alvares; Bibiana Monson de Souza; Mário Sérgio Palma; João Ruggiero Neto; Alexandre Suman de Araujo. The effect of acidic pH on the adsorption and lytic activity of the peptides Polybia-MP1 and its histidine-containing analog in anionic lipid membrane: a biophysical study by molecular dynamics and spectroscopy. Amino Acids 2021, 53, 753 -767.

AMA Style

Ingrid Bernardes Santana Martins, Taisa Giordano Viegas, Dayane Dos Santos Alvares, Bibiana Monson de Souza, Mário Sérgio Palma, João Ruggiero Neto, Alexandre Suman de Araujo. The effect of acidic pH on the adsorption and lytic activity of the peptides Polybia-MP1 and its histidine-containing analog in anionic lipid membrane: a biophysical study by molecular dynamics and spectroscopy. Amino Acids. 2021; 53 (5):753-767.

Chicago/Turabian Style

Ingrid Bernardes Santana Martins; Taisa Giordano Viegas; Dayane Dos Santos Alvares; Bibiana Monson de Souza; Mário Sérgio Palma; João Ruggiero Neto; Alexandre Suman de Araujo. 2021. "The effect of acidic pH on the adsorption and lytic activity of the peptides Polybia-MP1 and its histidine-containing analog in anionic lipid membrane: a biophysical study by molecular dynamics and spectroscopy." Amino Acids 53, no. 5: 753-767.

Journal article
Published: 22 April 2021 in Membranes
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Anionic lipid membrane electrostatic potential and solution pH can influence cationic peptide adsorption to these bilayers, especially those containing simultaneously acid and basic residues. Here, we investigate the effects of the pH solution on MP1 (IDWKKLLDAAKQIL-NH2) adsorption to anionic (7POPC:3POPG) lipid vesicles in comparison to its analog H-MP1, with histidines substituting lysines. We used the association of adsorption isotherms and constant pH molecular dynamic simulations (CpHMD) to explore the effects of membrane potential and pH on peptides’ adsorption on this lipid membrane. We analyzed the fluorescence and zeta potential adsorption isotherms using the Gouy–Chapman theory. In CpHMD simulations for the peptides in solution and adsorbed on the lipid bilayer, we used the conformations obtained by conventional MD simulations at a μs timescale. Non-equilibrium Monte Carlo simulations provided the protonation states of acidic and basic residues. CpHMD showed average pKa shifts of two to three units, resulting in a higher net charge for the analog than for MP1, strongly modulating the peptide adsorption. The fractions of the protonation of acidic and basic residues and the peptides’ net charges obtained from the analysis of the adsorption isotherms were in reasonable agreement with those from CpHMD. MP1 adsorption was almost insensitive to solution pH. H-MP1 was much more sensitive to partitioning, at acidic pH, with an affinity ten times higher than in neutral ones.

ACS Style

Dayane Alvares; Ingrid Martins; Taisa Viegas; Mario Palma; Alexandre de Araujo; Sidney de Carvalho; João Ruggiero Neto. Modulatory Effects of Acidic pH and Membrane Potential on the Adsorption of pH-Sensitive Peptides to Anionic Lipid Membrane. Membranes 2021, 11, 307 .

AMA Style

Dayane Alvares, Ingrid Martins, Taisa Viegas, Mario Palma, Alexandre de Araujo, Sidney de Carvalho, João Ruggiero Neto. Modulatory Effects of Acidic pH and Membrane Potential on the Adsorption of pH-Sensitive Peptides to Anionic Lipid Membrane. Membranes. 2021; 11 (5):307.

Chicago/Turabian Style

Dayane Alvares; Ingrid Martins; Taisa Viegas; Mario Palma; Alexandre de Araujo; Sidney de Carvalho; João Ruggiero Neto. 2021. "Modulatory Effects of Acidic pH and Membrane Potential on the Adsorption of pH-Sensitive Peptides to Anionic Lipid Membrane." Membranes 11, no. 5: 307.

Preprint content
Published: 11 December 2020
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Antimicrobial peptides (AMPs) are part of the innate immune system of many species and are compounds with potential application against the development of resistant bacterial strains promoted by conventional antibiotics. The AMPs are rich in cationic and hydrophobic residues and act directly on the lipidic phase of the cell membranes. The MP1 has a broad-spectrum bactericide activity in both Gram-negative and positive bacteria, not being hemolytic or cytotoxic. H-MP1 is a synthetic analog of MP1 with lysines replaced by histidines so that its net charge could be responsive to changes in solution pH. In the present work, we investigated the effect of the solution pH on the structural properties, in the adsorption and insertion, and on the lytic activity of these peptides in lipid bilayers mimicking the cell membrane of Gram-negative bacteria, using experimental and computational biophysical techniques. The results indicate that the lytic activity of H-MP1 is sensitive to pH, increasing to an acidic environment, matching that of MP1, which is not influenced by solution pH. Molecular Dynamic simulations indicated that the adsorption process of both peptides started by the interaction of the N-terminus with the bilayer, followed by the complete adsorption of the peptide laying parallel to the bilayer plane, inducing an increase in the peptide’s helical content enhancing peptides contact with the bilayer hydrophobic phase.

ACS Style

Ingrid Bernardes Santana Martins; Taisa Giordano Viegas; Bibiana Monson De Souza; Mário Sérgio Palma; João Ruggiero Neto; Alexandre Suman De Araujo. The effect of acidic pH on the interaction and lytic activity of MP1 and its H-MP1 analog in anionic lipid membrane: a biophysical study by Molecular Dynamics and Spectroscopy. 2020, 1 .

AMA Style

Ingrid Bernardes Santana Martins, Taisa Giordano Viegas, Bibiana Monson De Souza, Mário Sérgio Palma, João Ruggiero Neto, Alexandre Suman De Araujo. The effect of acidic pH on the interaction and lytic activity of MP1 and its H-MP1 analog in anionic lipid membrane: a biophysical study by Molecular Dynamics and Spectroscopy. . 2020; ():1.

Chicago/Turabian Style

Ingrid Bernardes Santana Martins; Taisa Giordano Viegas; Bibiana Monson De Souza; Mário Sérgio Palma; João Ruggiero Neto; Alexandre Suman De Araujo. 2020. "The effect of acidic pH on the interaction and lytic activity of MP1 and its H-MP1 analog in anionic lipid membrane: a biophysical study by Molecular Dynamics and Spectroscopy." , no. : 1.

Preprint content
Published: 23 June 2020 in bioRxiv
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It is now accepted that hopanoids act as sterol-surrogates in membranes of some sterol-lacking bacteria. Here we inquiry whether the hopanoid diplopterol (DP) could attenuate the activity of the antimicrobial peptide Polybia-MP1 (MP1) similarly to cholesterol (CHO). Survival of P. aeruginosa exposed to MP1 was lower for cells incubated with DP than those incubated with CHO, and the affinity and subsequent effect of the peptide on lipid bilayers were different in the presence of DP than in the presence of CHO. Membrane properties showed a non-monotonic behavior as the peptide adsorbed, penetrated, and translocated bilayers with DP suggesting a reorganization of MP1 during these processes. We conclude that MP1 selectivity is finely tuned by lipid composition, and propose the differential interaction and consequent effect promoted by the peptide in membranes with diplopterol as a promising starting point for targeting antimicrobial peptides to hopanoid-containing bacterial membranes.

ACS Style

Dayane S. Alvares; Mariela R. Monti; João Ruggiero Neto; Natalia Wilke. Regulation of the activity of an antimicrobial peptide by sterols or hopanoids: possible role in cell recognition. bioRxiv 2020, 1 .

AMA Style

Dayane S. Alvares, Mariela R. Monti, João Ruggiero Neto, Natalia Wilke. Regulation of the activity of an antimicrobial peptide by sterols or hopanoids: possible role in cell recognition. bioRxiv. 2020; ():1.

Chicago/Turabian Style

Dayane S. Alvares; Mariela R. Monti; João Ruggiero Neto; Natalia Wilke. 2020. "Regulation of the activity of an antimicrobial peptide by sterols or hopanoids: possible role in cell recognition." bioRxiv , no. : 1.

Review
Published: 24 September 2019 in Toxins
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Solitary wasps use their stinging venoms for paralyzing insect or spider prey and feeding them to their larvae. We have surveyed bioactive substances in solitary wasp venoms, and found antimicrobial peptides together with some other bioactive peptides. Eumenine mastoparan-AF (EMP-AF) was the first to be found from the venom of the solitary eumenine wasp Anterhynchium flavomarginatum micado, showing antimicrobial, histamine-releasing, and hemolytic activities, and adopting an α-helical secondary structure under appropriate conditions. Further survey of solitary wasp venom components revealed that eumenine wasp venoms contained such antimicrobial α-helical peptides as the major peptide component. This review summarizes the results obtained from the studies of these peptides in solitary wasp venoms and some analogs from the viewpoint of (1) chemical and biological characterization; (2) physicochemical properties and secondary structure; and (3) channel-like pore-forming properties.

ACS Style

Marcia Perez Dos Santos Cabrera; Marisa Rangel; João Ruggiero Neto; Katsuhiro Konno. Chemical and Biological Characteristics of Antimicrobial α-Helical Peptides Found in Solitary Wasp Venoms and Their Interactions with Model Membranes. Toxins 2019, 11, 559 .

AMA Style

Marcia Perez Dos Santos Cabrera, Marisa Rangel, João Ruggiero Neto, Katsuhiro Konno. Chemical and Biological Characteristics of Antimicrobial α-Helical Peptides Found in Solitary Wasp Venoms and Their Interactions with Model Membranes. Toxins. 2019; 11 (10):559.

Chicago/Turabian Style

Marcia Perez Dos Santos Cabrera; Marisa Rangel; João Ruggiero Neto; Katsuhiro Konno. 2019. "Chemical and Biological Characteristics of Antimicrobial α-Helical Peptides Found in Solitary Wasp Venoms and Their Interactions with Model Membranes." Toxins 11, no. 10: 559.

Journal article
Published: 22 September 2018 in Chemistry and Physics of Lipids
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Peptide sequences containing acidic and basic residues could potentially have their net charges modulated by bulk pH with a possible influence on their lytic activity in lipid vesicles. The present study reports on a biophysical investigation of these modulatory effects on the synthetic mastoparan-like peptide L1A (IDGLKAIWKKVADLLKNT-NH2). At pH 10.0 L1A was 6 times more efficient in lysing large anionic (1-palmitoyl-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) / (8:2)) unilamellar vesicles (LUVs) than at pH 4.0. Despite the reduction of 60% in the L1A net charge in basic pH its affinity for this vesicle was almost insensitive to pH. On the other hand, L1A insertion into monolayers was dramatically influenced by subphase condition, showing that, in the neutral and basic subphases, the peptide induced surface pressure changes that surpassed the membrane lateral pressure, being able to destabilize a bilayer structure. In addition, in the basic subphase, visualization of the compression isotherms of co-spread 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):POPG (8:2) + 4.8 mol% L1A showed that the peptide induced significant changes in solid lipid domains, indicating its capability in perturbing lipid-packing. An insight into L1A lytic activity was also obtained in giant unilamellar vesicles (GUVs) using phase contrast microscopy. The suppression of L1A lytic activity at acidic pH is in keeping with its lower insertion capability and ability to disturb the lipid monolayer. The lytic activity observed under neutral and basic conditions showed a quick and stochastic leakage following a lag-time. The permeability and the leakage-time averaged over at least 14 single GUVs were dependent on the bulk condition. At basic pH, permeability is higher and quicker than in a neutral medium in good accordance with the lipid-packing perturbation.

ACS Style

Dayane S. Alvares; Taisa Giordano Viegas; João Ruggiero Neto. The effect of pH on the lytic activity of a synthetic mastoparan-like peptide in anionic model membranes. Chemistry and Physics of Lipids 2018, 216, 54 -64.

AMA Style

Dayane S. Alvares, Taisa Giordano Viegas, João Ruggiero Neto. The effect of pH on the lytic activity of a synthetic mastoparan-like peptide in anionic model membranes. Chemistry and Physics of Lipids. 2018; 216 ():54-64.

Chicago/Turabian Style

Dayane S. Alvares; Taisa Giordano Viegas; João Ruggiero Neto. 2018. "The effect of pH on the lytic activity of a synthetic mastoparan-like peptide in anionic model membranes." Chemistry and Physics of Lipids 216, no. : 54-64.

Journal article
Published: 01 March 2018 in Biochimica et Biophysica Acta (BBA) - Biomembranes
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L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.

ACS Style

Dayane Dos Santos Alvares; Natalia Wilke; João Ruggiero Neto. Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide. Biochimica et Biophysica Acta (BBA) - Biomembranes 2018, 1860, 737 -748.

AMA Style

Dayane Dos Santos Alvares, Natalia Wilke, João Ruggiero Neto. Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2018; 1860 (3):737-748.

Chicago/Turabian Style

Dayane Dos Santos Alvares; Natalia Wilke; João Ruggiero Neto. 2018. "Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide." Biochimica et Biophysica Acta (BBA) - Biomembranes 1860, no. 3: 737-748.

Journal article
Published: 01 October 2017 in Chemistry and Physics of Lipids
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Polybia-MP1 or simply MP1 (IDWKKLLDAAKQIL-NH2) is a peptide with broad-spectrum bactericidal activity and a strong inhibitory effect against cancer cells. The aim of this work was to evaluate the effect of biophysical properties such as membrane texture and film thickness on MP1 interaction with neutral and anionic lipid membranes. For this purpose, we first explored the peptide's surface behavior. MP1 showed high surface activity, adsorbing onto bare air/aqueous interfaces up to higher surface pressures than the collapse pressure of MP1 Langmuir films. The MP1-lipid membrane interaction was studied using Langmuir phosphatidylcholine and phosphatidylserine (PS) monolayers as model membrane systems. PS was chosen since this negatively charged lipid was found predominantly on the outer leaflet of tumor cells, and it enhances MP1 activity for PS-containing membranes to a greater extent than for other negatively charged lipids. MP1 incorporated into anionic PS monolayers, which show a liquid-expanded (LE) phase or LE-liquid-condensed (LC) phase coexistence, up to lipid-packing densities higher than those of cell membranes. The mixed lipid/MP1 films were explored by Brewster angle microscopy and atomic force microscopy. MP1 partitioned preferentially into the LE phase state of PS films, and were thus excluded from the coexisting LC phase. This interaction had strong electrostatic bases: in pure water, the lipid-peptide interaction was strong enough to induce formation of reversible lipid-peptide 3D structures associated with the interface. MP1 incorporation into the LE phase was accompanied by a shift of the phase transition pressure to higher values and a thinning of the lipid film. These results showed a clear correlation between peptide penetration capacity and the presence or induction of the thin LE phase. This capacity to regulate membrane physical properties may be of relevance in the binding, incorporation and membrane selectivity of this promising antitumor peptide.

ACS Style

Dayane Dos Santos Alvares; Natalia Wilke; João Ruggiero Neto; Maria Laura Fanani. The insertion of Polybia-MP1 peptide into phospholipid monolayers is regulated by its anionic nature and phase state. Chemistry and Physics of Lipids 2017, 207, 38 -48.

AMA Style

Dayane Dos Santos Alvares, Natalia Wilke, João Ruggiero Neto, Maria Laura Fanani. The insertion of Polybia-MP1 peptide into phospholipid monolayers is regulated by its anionic nature and phase state. Chemistry and Physics of Lipids. 2017; 207 ():38-48.

Chicago/Turabian Style

Dayane Dos Santos Alvares; Natalia Wilke; João Ruggiero Neto; Maria Laura Fanani. 2017. "The insertion of Polybia-MP1 peptide into phospholipid monolayers is regulated by its anionic nature and phase state." Chemistry and Physics of Lipids 207, no. : 38-48.

Review
Published: 29 August 2017 in Biophysical Reviews
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The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.

ACS Style

Dayane Dos Santos Alvares; Taisa Giordano Viegas; João Ruggiero Neto. Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides. Biophysical Reviews 2017, 9, 669 -682.

AMA Style

Dayane Dos Santos Alvares, Taisa Giordano Viegas, João Ruggiero Neto. Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides. Biophysical Reviews. 2017; 9 (5):669-682.

Chicago/Turabian Style

Dayane Dos Santos Alvares; Taisa Giordano Viegas; João Ruggiero Neto. 2017. "Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides." Biophysical Reviews 9, no. 5: 669-682.

Journal article
Published: 01 June 2017 in Biochimica et Biophysica Acta (BBA) - Biomembranes
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Polybia-MP1 (IDWKKLLDAAKQIL-NH2) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1.

ACS Style

Dayane Dos Santos Alvares; João Ruggiero Neto; Ernesto E. Ambroggio. Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide. Biochimica et Biophysica Acta (BBA) - Biomembranes 2017, 1859, 1067 -1074.

AMA Style

Dayane Dos Santos Alvares, João Ruggiero Neto, Ernesto E. Ambroggio. Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2017; 1859 (6):1067-1074.

Chicago/Turabian Style

Dayane Dos Santos Alvares; João Ruggiero Neto; Ernesto E. Ambroggio. 2017. "Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide." Biochimica et Biophysica Acta (BBA) - Biomembranes 1859, no. 6: 1067-1074.

Journal article
Published: 27 February 2016 in Amino Acids
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We investigate the effect of the N-terminus modification of the L1A, a synthetic octadecapeptide, on its helical content, affinity and lytic action in model membranes and on its hemolytic and antibacterial activities. L1A and its acetylated analog displayed a selective antibacterial activity to Gram-negative bacteria without being hemolytic. The covalently linked 2-aminobezoic acid to the N-terminus impaired the antibacterial efficacy and increased hemolysis. Despite their lower net charge (+2), N-terminus modifications resulted in enhanced affinity and improved lytic efficiency in anionic vesicles. The analogs also showed higher helical content and consequently higher amphipathicity in these vesicles. The conformational analysis by molecular dynamics simulations in 30 % of TFE/water showed that the hydrophobic faces of the peptides are in close contact with CF3 groups of TFE while the hydrophilic faces with water molecules. Due to the loss of the amino charge, the N-termini of the analogs are buried in TFE molecules. The analysis of the pair distribution functions, obtained for the center of mass of the charged groups, has evidenced that the state of the N-terminus has influenced the possibility of different ion-pairing. The higher complexity of the bacterial cells compared with anionic vesicles hampers to establish correlations structure-function for the analogs.

ACS Style

Luciana Puia Moro Zanin; Alexandre Suman De Araujo; Maria Aparecida Juliano; Tiago Casella; Mara Correa Lelles Nogueira; João Ruggiero Neto. Effects of N-terminus modifications on the conformation and permeation activities of the synthetic peptide L1A. Amino Acids 2016, 48, 1433 -1444.

AMA Style

Luciana Puia Moro Zanin, Alexandre Suman De Araujo, Maria Aparecida Juliano, Tiago Casella, Mara Correa Lelles Nogueira, João Ruggiero Neto. Effects of N-terminus modifications on the conformation and permeation activities of the synthetic peptide L1A. Amino Acids. 2016; 48 (6):1433-1444.

Chicago/Turabian Style

Luciana Puia Moro Zanin; Alexandre Suman De Araujo; Maria Aparecida Juliano; Tiago Casella; Mara Correa Lelles Nogueira; João Ruggiero Neto. 2016. "Effects of N-terminus modifications on the conformation and permeation activities of the synthetic peptide L1A." Amino Acids 48, no. 6: 1433-1444.

Journal article
Published: 01 February 2016 in Biochimica et Biophysica Acta (BBA) - Biomembranes
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Polybia-MP1 (IDWKKLLDAAKQIL-NH2), extracted from the Brazilian wasp Polybia paulista, exhibits a broad-spectrum bactericidal activity without being hemolytic and cytotoxic. In the present study, we analyzed the surface properties of the peptide and its interaction with DPPC in Langmuir monolayers. Polybia-MP1 formed stable monolayers, with lateral areas and surface potential values suggesting a mostly α-helical structure oriented near perpendicular to the membrane plane. In DPPC–peptide mixed monolayers, MP1 co-crystallized with the lipid forming branched domains only when the subphase was pure water. On subphases with high salt concentrations or at acidic or basic conditions, the peptide formed less densely packed films and was excluded from the domains, indicating the presence of attractive electrostatic interactions between peptides, which allow them to get closer to each other and to interact with DPPC probably as a consequence of a particular peptide arrangement. The residues responsible of the peptide–peptide attraction are suggested to be the anionic aspartic acids and the cationic lysines, which form a salt bridge, leading to oriented interactions in the crystal and thereby to branched domains. For this peptide, the balance between total attractive and repulsive interactions may be finely tuned by the aqueous ionic strength and pH, and since this effect is related with lysines and aspartic acids, similar effects may also occur in other peptides containing these residues in their sequences.

ACS Style

Dayane Dos Santos Alvares; Maria Laura Fanani; João Ruggiero Neto; Natalia Wilke. The interfacial properties of the peptide Polybia-MP1 and its interaction with DPPC are modulated by lateral electrostatic attractions. Biochimica et Biophysica Acta (BBA) - Biomembranes 2016, 1858, 393 -402.

AMA Style

Dayane Dos Santos Alvares, Maria Laura Fanani, João Ruggiero Neto, Natalia Wilke. The interfacial properties of the peptide Polybia-MP1 and its interaction with DPPC are modulated by lateral electrostatic attractions. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2016; 1858 (2):393-402.

Chicago/Turabian Style

Dayane Dos Santos Alvares; Maria Laura Fanani; João Ruggiero Neto; Natalia Wilke. 2016. "The interfacial properties of the peptide Polybia-MP1 and its interaction with DPPC are modulated by lateral electrostatic attractions." Biochimica et Biophysica Acta (BBA) - Biomembranes 1858, no. 2: 393-402.

Journal article
Published: 01 September 2015 in Biophysical Journal
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Polybia-MP1 (MP1) is a bioactive host-defense peptide with known anticancer properties. Its activity is attributed to excess serine (phosphatidylserine (PS)) on the outer leaflet of cancer cells. Recently, higher quantities of phosphatidylethanolamine (PE) were also found at these cells’ surface. We investigate the interaction of MP1 with model membranes in the presence and absence of POPS (PS) and DOPE (PE) to understand the role of lipid composition in MP1’s anticancer characteristics. Indeed we find that PS lipids significantly enhance the bound concentration of peptide on the membrane by a factor of 7–8. However, through a combination of membrane permeability assays and imaging techniques we find that PE significantly increases the susceptibility of the membrane to disruption by these peptides and causes an order-of-magnitude increase in membrane permeability by facilitating the formation of larger transmembrane pores. Significantly, atomic-force microscopy imaging reveals differences in the pore formation mechanism with and without the presence of PE. Therefore, PS and PE lipids synergistically combine to enhance membrane poration by MP1, implying that the combined enrichment of both these lipids in the outer leaflet of cancer cells is highly significant for MP1’s anticancer action. These mechanistic insights could aid development of novel chemotherapeutics that target pathological changes in the lipid composition of cancerous cells

ACS Style

Natália Bueno Leite; Anders Aufderhorst-Roberts; Mario Sergio Palma; Simon D. Connell; João Ruggiero Neto; Paul A. Beales. PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties. Biophysical Journal 2015, 109, 936 -947.

AMA Style

Natália Bueno Leite, Anders Aufderhorst-Roberts, Mario Sergio Palma, Simon D. Connell, João Ruggiero Neto, Paul A. Beales. PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties. Biophysical Journal. 2015; 109 (5):936-947.

Chicago/Turabian Style

Natália Bueno Leite; Anders Aufderhorst-Roberts; Mario Sergio Palma; Simon D. Connell; João Ruggiero Neto; Paul A. Beales. 2015. "PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties." Biophysical Journal 109, no. 5: 936-947.

Journal article
Published: 01 October 2014 in Biochimica et Biophysica Acta (BBA) - Biomembranes
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Polycationic peptides may present their C-termini in either amidated or acidic form; however, the effects of these conformations on the mechanisms of interaction with the membranes in general were not properly investigated up to now. Protonectarina-MP mastoparan with an either amidated or acidic C-terminus was utilized to study their interactions with anionic and zwitterionic vesicles, using measurements of dye leakage and a combination of H/D exchange and mass spectrometry to monitor peptide–membrane interactions. Mast cell degranulation, hemolysis and antibiosis assays were also performed using these peptides, and the results were correlated with the structural properties of the peptides. The C-terminal amidation promotes the stabilization of the secondary structure of the peptide, with a relatively high content of helical conformations, permitting a deeper interaction with the phospholipid constituents of animal and bacterial cell membranes. The results suggested that at low concentrations Protonectarina-MP interacts with the membranes in a way that both terminal regions remain positioned outside the external surface of the membrane, while the α-carbon backbone becomes partially embedded in the membrane core and changing constantly the conformation, and causing membrane destabilization. The amidation of the C-terminal residue appears to be responsible for the stabilization of the peptide conformation in a secondary structure that is richer in α-helix content than its acidic congener. The helical, amphipathic conformation, in turn, allows a deeper peptide–membrane interaction, favoring both biological activities that depend on peptide structure recognition by the GPCRs (such as exocytosis) and those activities dependent on membrane perturbation (such as hemolysis and antibiosis)

ACS Style

Alessandra V.R. Da Silva; Bibiana M. De Souza; Marcia P. Dos Santos Cabrera; Nathalia B. Dias; Paulo C. Gomes; João Ruggiero Neto; Rodrigo Guerino Stabeli; Mario S. Palma. The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems. Biochimica et Biophysica Acta (BBA) - Biomembranes 2014, 1838, 2357 -2368.

AMA Style

Alessandra V.R. Da Silva, Bibiana M. De Souza, Marcia P. Dos Santos Cabrera, Nathalia B. Dias, Paulo C. Gomes, João Ruggiero Neto, Rodrigo Guerino Stabeli, Mario S. Palma. The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2014; 1838 (10):2357-2368.

Chicago/Turabian Style

Alessandra V.R. Da Silva; Bibiana M. De Souza; Marcia P. Dos Santos Cabrera; Nathalia B. Dias; Paulo C. Gomes; João Ruggiero Neto; Rodrigo Guerino Stabeli; Mario S. Palma. 2014. "The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems." Biochimica et Biophysica Acta (BBA) - Biomembranes 1838, no. 10: 2357-2368.

Journal article
Published: 11 July 2014 in Peptides
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Honeybee stings are a severe public health problem. Bee venom contains a series of active components, including enzymes, peptides, and biogenic amines. The local reactions observed after envenoming include a typical inflammatory response and pain. Honeybee venom contains some well-known polycationic peptides, such as Melittin, Apamin, MCD peptide, Cardiopep, and Tertiapin. Secapin in honeybee venom was described 38 years ago, yet almost nothing is known about its action. A novel, variant form of this peptide was isolated from the venom of Africanized honeybees (Apis mellifera). This novel peptide, named Secapin-2, is 25 amino acid residues long. Conformational analyses using circular dichroism and molecular dynamics simulations revealed a secondary structure rich in strands and turns, stabilized by an intramolecular disulfide bridge. Biological assays indicated that Secapin-2 did not induce hemolysis, mast cell degranulation or chemotactic activities. However, Secapin-2 caused potent dose-related hyperalgesic and edematogenic responses in experimental animals. To evaluate the roles of prostanoids and lipid mediators in the hyperalgesia and edema induced by this peptide, Indomethacin and Zileuton were used to inhibit the cyclooxygenase and lipoxygenase pathways, respectively. The results showed that Zileuton partially blocked the hyperalgesia induced by Secapin-2 and decreased the edematogenic response. In contrast, Indomethacin did not interfere with these phenomena. Zafirlukast, a leukotriene receptor antagonist, blocked the Secapin-2 induced hyperalgesia and edematogenic response. These results indicate that Secapin-2 induces inflammation and pain through the lipoxygenase pathway in both phenomena.

ACS Style

D. Mourelle; P. Brigatte; L.D.B. Bringanti; B.M. De Souza; H.A. Arcuri; P.C. Gomes; N.B. Baptista-Saidemberg; J. Ruggiero Neto; M.S. Palma. Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom. Peptides 2014, 59, 42 -52.

AMA Style

D. Mourelle, P. Brigatte, L.D.B. Bringanti, B.M. De Souza, H.A. Arcuri, P.C. Gomes, N.B. Baptista-Saidemberg, J. Ruggiero Neto, M.S. Palma. Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom. Peptides. 2014; 59 ():42-52.

Chicago/Turabian Style

D. Mourelle; P. Brigatte; L.D.B. Bringanti; B.M. De Souza; H.A. Arcuri; P.C. Gomes; N.B. Baptista-Saidemberg; J. Ruggiero Neto; M.S. Palma. 2014. "Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom." Peptides 59, no. : 42-52.

Journal article
Published: 05 March 2014 in European Biophysics Journal
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Polybia-MP1 (IDWKKLLDAAKQIL-NH2), a helical peptide extracted from the venom of a Brazilian wasp, has broad-spectrum antimicrobial activities without being hemolytic or cytotoxic. This peptide has also displayed anticancer activity against cancer cell cultures. Despite its high selectivity, MP1 has an unusual low net charge (Q = +2). The aspartic residue (D2) in the N-terminal region plays an important role in its affinity and selectivity; its substitution by asparagine (D2N mutant) led to a less selective peptide. Aiming to explore the importance of this residue for the peptides’ affinity, we compared the zwitterionic and anionic vesicle adsorption activity of Polybia-MP1 versus its D2N mutant and also mastoparan X (MPX). The adsorption, electrostatic, and conformational free energies were assessed by circular dichroism (CD) and fluorescence titrations using large unilamellar vesicles (LUVs) at the same conditions in association with measurement of the zeta potential of LUVs in the presence of the peptides. The adsorption free energies of the peptides, determined from the partition coefficients, indicated higher affinity of MP1 to anionic vesicles compared with the D2N mutant and MPX. The electrostatic and conformational free energies of MP1 in anionic vesicles are less favorable than those found for the D2N mutant and MPX. Therefore, the highest affinity of MP1 to anionic vesicles is likely due to other energetic contributions. The presence of D2 in MP1 makes these energetic components 1.2 and 1.5 kcal/mol more favorable compared with the D2N mutant and MPX, respectively.

ACS Style

Natália Bueno Leite; Dayane Dos Santos Alvares; Bibiana Monson De Souza; Mário Sérgio Palma; João Ruggiero Neto. Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles. European Biophysics Journal 2014, 43, 1 .

AMA Style

Natália Bueno Leite, Dayane Dos Santos Alvares, Bibiana Monson De Souza, Mário Sérgio Palma, João Ruggiero Neto. Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles. European Biophysics Journal. 2014; 43 (4):1.

Chicago/Turabian Style

Natália Bueno Leite; Dayane Dos Santos Alvares; Bibiana Monson De Souza; Mário Sérgio Palma; João Ruggiero Neto. 2014. "Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles." European Biophysics Journal 43, no. 4: 1.

Journal article
Published: 05 October 2013 in European Biophysics Journal
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Static and time-resolved fluorescence of tryptophan and ortho-aminobenzoic acid was used to investigate the interaction of the synthetic antimicrobial peptide L1A (IDGLKAIWKKVADLLKNT-NH2) with POPC and POPC:POPG. N-acetylated (Ac-L1A) and N-terminus covalently bonded ortho-aminobenzoic acid (Abz-L1A-W8V) were also used. Static fluorescence and quenching by acrylamide showed that the peptides adsorption to the lipid bilayers was accompanied by spectral blue shift and by a decrease in fluorescence quenching, indicating that the peptides moved to a less polar environment probably buried in the lipidic phase of the vesicles. These results also suggest that the loss of the N-terminus charge allowed deeper fluorophore insertion in the bilayer. Despite the local character of spectroscopic information, conclusions can be drawn about the peptides as a whole. The dynamic behaviors of the peptides are such that the mean intensity lifetimes, the long correlation time and the residual anisotropy at long times increased when the peptides adsorb in lipid vesicles, being larger in anionic vesicles. From the steady-state increase in fluorescence intensity and anisotropy, we observed that the partition coefficient of peptides L1A and its Abz analog in both types of vesicles are higher than the acetylated analog; moreover, the affinity to the anionic vesicle is higher than to the zwitterionic.

ACS Style

Luciana Moro Puia Zanin; Dayane Dos Santos Alvares; Maria Aparecida Juliano; Wallance Moreira Pazin; Amando Siuiti Ito; João Ruggiero Neto. Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy. European Biophysics Journal 2013, 42, 819 -831.

AMA Style

Luciana Moro Puia Zanin, Dayane Dos Santos Alvares, Maria Aparecida Juliano, Wallance Moreira Pazin, Amando Siuiti Ito, João Ruggiero Neto. Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy. European Biophysics Journal. 2013; 42 (11-12):819-831.

Chicago/Turabian Style

Luciana Moro Puia Zanin; Dayane Dos Santos Alvares; Maria Aparecida Juliano; Wallance Moreira Pazin; Amando Siuiti Ito; João Ruggiero Neto. 2013. "Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy." European Biophysics Journal 42, no. 11-12: 819-831.

Research article
Published: 06 June 2012 in Biochemistry
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This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Trp indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern–Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.

ACS Style

Marcia Perez Dos Santos Cabrera; Manoel Arcisio-Miranda; Renata Gorjão; Natália Bueno Leite; Bibiana Monson De Souza; Rui Curi; Joaquim Procopio; João Ruggiero Neto; Mario Palma. Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity. Biochemistry 2012, 51, 4898 -4908.

AMA Style

Marcia Perez Dos Santos Cabrera, Manoel Arcisio-Miranda, Renata Gorjão, Natália Bueno Leite, Bibiana Monson De Souza, Rui Curi, Joaquim Procopio, João Ruggiero Neto, Mario Palma. Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity. Biochemistry. 2012; 51 (24):4898-4908.

Chicago/Turabian Style

Marcia Perez Dos Santos Cabrera; Manoel Arcisio-Miranda; Renata Gorjão; Natália Bueno Leite; Bibiana Monson De Souza; Rui Curi; Joaquim Procopio; João Ruggiero Neto; Mario Palma. 2012. "Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity." Biochemistry 51, no. 24: 4898-4908.

Original article
Published: 01 March 2010 in Amino Acids
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Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.

ACS Style

Natália Bueno Leite; Laiana Cristina Da Costa; Dayane Dos Santos Alvares; Marcia P. Dos Santos Cabrera; Bibiana Monson De Souza; Mário Sérgio Palma; João Ruggiero Neto. The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy. Amino Acids 2010, 40, 91 -100.

AMA Style

Natália Bueno Leite, Laiana Cristina Da Costa, Dayane Dos Santos Alvares, Marcia P. Dos Santos Cabrera, Bibiana Monson De Souza, Mário Sérgio Palma, João Ruggiero Neto. The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy. Amino Acids. 2010; 40 (1):91-100.

Chicago/Turabian Style

Natália Bueno Leite; Laiana Cristina Da Costa; Dayane Dos Santos Alvares; Marcia P. Dos Santos Cabrera; Bibiana Monson De Souza; Mário Sérgio Palma; João Ruggiero Neto. 2010. "The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy." Amino Acids 40, no. 1: 91-100.

Journal article
Published: 28 January 2010 in Amino Acids
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In order to investigate the effect of the different positions of the positive charges generated by the ionization of the side-chain of lysine residues, on the structure-activity relationship of the mastoparans, the peptides Protonectarina-MP (INWKALLDAAKKVL-NH2), Parapolybia-MP (INWKKMAATALKMI-NH2) and Asn-2-Polybia-MP I (INWKKLLDAAKQIL-NH2) and MK-578 (INWLKAKKVAGMIL-NH2) were investigated as models. Thus, the four peptides had their secondary structure studied and were submitted to assays of mast cell degranulation, hemolysis, and antibiosis. The results of the bioassays made clear that those peptides bearing the positive charges positioned at the positions 4/5 and/or from 11 to 13 are the most active ones; meanwhile, the localization of the positive charges in the middle of peptide chain resulted in a poorly active peptide. Thus, Protonectarina-MP, Parapolybia-MP, and Asn-2-Polybia-MP I presented physiologically important hemolysis and antibiosis, while MK-578 presented only a reduced antibiotic activity. Circular dichroism analysis were carried-out in different environments revealing that the anionic environment of a mixture of phosphatidylcholine and phosphatidylglycerol (70:30) liposomes favored the higher helical content of the four peptides in this study in relation to the zwiterionic environment of 100% phosphatidylcholine liposomes. The positioning of the lysine residues at the strategic positions (4/5 and 11-13), flanking and maintaining stable α-helix which extends from the 4th to the 13th residue along the peptide chain, seems to contribute to maximal lytic efficiency of the mastoparans, which in turn results in a more homogeneous hydrophobic surface in the amphipathic structure.

ACS Style

Bibiana Monson De Souza; Marcia P. Dos Santos Cabrera; João Ruggiero Neto; Mário Sérgio Palma. Investigating the effect of different positioning of lysine residues along the peptide chain of mastoparans for their secondary structures and biological activities. Amino Acids 2010, 40, 77 -90.

AMA Style

Bibiana Monson De Souza, Marcia P. Dos Santos Cabrera, João Ruggiero Neto, Mário Sérgio Palma. Investigating the effect of different positioning of lysine residues along the peptide chain of mastoparans for their secondary structures and biological activities. Amino Acids. 2010; 40 (1):77-90.

Chicago/Turabian Style

Bibiana Monson De Souza; Marcia P. Dos Santos Cabrera; João Ruggiero Neto; Mário Sérgio Palma. 2010. "Investigating the effect of different positioning of lysine residues along the peptide chain of mastoparans for their secondary structures and biological activities." Amino Acids 40, no. 1: 77-90.