RESUMO
Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of â¼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Escherichia coli/química , Sistemas de Secreção Tipo IV/antagonistas & inibidores , Sistemas de Secreção Tipo IV/química , Xanthomonas/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Relação Estrutura-Atividade , Sistemas de Secreção Tipo IV/genética , Xanthomonas/genéticaRESUMO
The main objective of this study was to clarify the topical mechanisms underlying diclofenac-induced gastric toxicity by considering for the first time both ionization states of this nonsteroidal anti-inflammatory drug. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes were the model system chosen to mimic the protective phospholipid layers of the gastric mucosa and to describe the interactions with diclofenac, considering the pH gradient found in the gastric mucosa (3 < pH < 7.4). Complementary experimental techniques were combined to evaluate the drug's affinity for DMPC bilayers, as well as to assess the drug's effects on the structural properties of the phospholipid bilayer. The diclofenac-DMPC interactions were clearly dependent on the drug's ionization state. Neutral diclofenac displayed greater affinity for DMPC bilayers than anionic diclofenac. Moreover, the protonated/neutral form of the drug induced more pronounced and/or distinct alterations in the structure of the DMPC bilayer than the deprotonated/ionized form, considering similar membrane concentrations. Therefore, neutral diclofenac-induced changes in the structural properties of the external phospholipid layers of the gastric mucosa may constitute an additional toxicity mechanism of this worldwide-used drug, which shall be considered for the development of safer therapeutic strategies. SIGNIFICANCE STATEMENT: Neutral or anionic diclofenac exerted distinct alterations in phosphatidylcholine bilayers, which are used in this work as models for the protective phospholipid layers of the gastric mucosa. Remarkable changes were induced by neutral diclofenac in the structural properties of the phospholipid bilayer, suggesting that both ionized and neutral states of nonsteroidal anti-inflammatory drugs must be considered to clarify their mechanisms of toxicity and to ultimately develop safer anti-inflammatory drugs.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diclofenaco/toxicidade , Dimiristoilfosfatidilcolina/química , Mucosa Gástrica/efeitos dos fármacos , Bicamadas Lipídicas/química , Mucosa Gástrica/química , Concentração de Íons de Hidrogênio , Lipossomos/química , Estrutura Molecular , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Specific ion effects in zwitterionic micelles, especially for anions, are evident in reaction kinetics, zeta potential, and critical micelle concentration measurements. However, anion adsorption to zwitterionic micelles does not produce significant changes in shape, aggregation number, or interfacial hydration. Here we used molecular dynamics simulation of systems containing sulfobetaine zwitterionic micelles of N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (DPS) and nine different salts to explore ion adsorption in terms of group dehydration. Our results, in line with those obtained for cationic micelles, showed that the adsorption degree of anions containing both hydrophobic and hydrophilic portions, i.e., hydrotropes, were correlated with the ion dehydration and were governed mainly by the hydrophobic portion dehydration upon adsorption.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Micelas , Simulação de Dinâmica Molecular , Compostos de Amônio Quaternário/química , Água/química , Adsorção , Conformação MolecularRESUMO
(1) Background: Membrane lipids have been disregarded in drug development throughout the years. Recently, they gained attention in drug design as targets, but they are still disregarded in the latter stages. Thus, this study aims to highlight the relevance of considering membrane lipids in the preclinical phase of drug development. (2) Methods: The interactions of a drug candidate for clinical use (licofelone) with a membrane model system made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were evaluated by combining Langmuir isotherms, Brewster angle microscopy (BAM), polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS), and grazing-incidence X-ray diffraction (GIXD) measurements. (3) Results: Licofelone caused the expansion of the DPPC isotherm without changing the lipid phase transition profile. Moreover, licofelone induced the reduction of DPPC packing density, while increasing the local order of the DPPC acyl chains. (4) Conclusions: The licofelone-induced alterations in the structural organization of phosphatidylcholine monolayers may be related to its pharmacological actions. Thus, the combination of studying drug-membrane interactions with the pharmacological characterization that occurs in the preclinical stage may gather additional information about the mechanisms of action and toxicity of drug candidates. Ultimately, the addition of this innovative step shall improve the success rate of drug development.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Pirróis/química , Desenvolvimento de Medicamentos , Lipídeos de Membrana/química , Microscopia , Estrutura Molecular , Análise Espectral , TemperaturaRESUMO
Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH2), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100alanylhexadecyl1amine (BP100-Ala-NH-C16H33) and cyclo(14)dCys1, Ile2, Leu3, Cys4-BP100 (Cyclo(14)cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C16H33 acquired α-helical conformation, while Cyclo(14)cILC-BP100) was partly α-helical and partly ß-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.
Assuntos
Anti-Infecciosos/síntese química , Oligopeptídeos/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Gastrointestinal (GI) toxicity is a major drawback of the chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs). The NSAIDs topical actions on the protective phospholipid layers of the GI mucosa seem to be a central toxicity mechanism of these pharmaceuticals. This work describes the interactions of acemetacin, a commercialized NSAID, with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at pH 3.0, 5.0, and 7.4. This pH range was chosen to mimic the pH gradient found in the gastric mucosa, and to ultimately gain insights into the mechanisms underlying the acemetacin-induced gastric toxicity. Various experimental techniques were combined to characterize the partitioning of acemetacin in DMPC bilayers, and its effects on the phase transition behavior, as well as the structure and dynamics of DMPC bilayers. The acemetacin-DMPC interactions were clearly pH-dependent. The neutral (protonated) form of acemetacin had more affinity for the DMPC bilayer than the negatively charged form. Due to the higher affinity of neutral acemetacin, the drug effects on the phase transition and the structure and dynamics of the DMPC bilayer were more pronounced at lower pH values. In general, acemetacin decreased the temperature and the cooperativity of the lipid phase transition and induced changes in the packing and dynamics of the DMPC bilayer. These results support the hypothesis that acemetacin-induced gastric toxicity may be related to its effects on the protective phospholipid layers of the mucosal barrier.
Assuntos
Anti-Inflamatórios não Esteroides/química , Indometacina/análogos & derivados , Fosfatidilcolinas/química , Concentração de Íons de Hidrogênio , Indometacina/química , Cinética , Bicamadas Lipídicas/química , Estrutura Molecular , Transição de Fase , Eletricidade Estática , TemperaturaRESUMO
The efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) against inflammation, pain, and fever has been supporting their worldwide use in the treatment of painful conditions and chronic inflammatory diseases until today. However, the long-term therapy with NSAIDs was soon associated with high incidences of adverse events in the gastrointestinal tract. Therefore, the search for novel drugs with improved safety has begun with COX-2 selective inhibitors (coxibs) being straightaway developed and commercialized. Nevertheless, the excitement has fast turned to disappointment when diverse coxibs were withdrawn from the market due to cardiovascular toxicity. Such events have once again triggered the emergence of different strategies to overcome NSAIDs toxicity. Here, an integrative review is provided to address the breakthroughs of two main approaches: (i) the association of NSAIDs with protective mediators and (ii) the design of novel compounds to target downstream and/or multiple enzymes of the arachidonic acid cascade. To date, just one phosphatidylcholine-associated NSAID has already been approved for commercialization. Nevertheless, the preclinical and clinical data obtained so far indicate that both strategies may improve the safety of nonsteroidal anti-inflammatory therapy.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Animais , Humanos , Relação Estrutura-AtividadeRESUMO
The properties of ionic micelles depend on the nature of the counterion, and these effects become more evident as the ion adsorption at the interface increases. Prediction of the relative extent of ion adsorption is required for rational design of ionic micellar aggregates. Unlike the well understood adsorption of monatomic ions, the adsorption of polyatomic ions is not easily predicted. We combined experimental and computational methods to evaluate the affinity of hydrotropic ions, i.e., ions with polar and apolar regions, to the surface of positively charged micelles. We analyzed cationic micelles of dodecyltrimethylammonium and six hydrotropic counterions: methanesulfonate, trifluoromethanesulfonate, benzenesulfonate, acetate, trifluoroacetate and benzoate. Our results demonstrated that the apolar region of hydrotropic ions had the largest influence on micellar properties. The dehydration of the apolar region of hydrotropic ions upon their adsorption at the micellar interface determined the ion adsorption extension, differently to what was expected based on Collins' law of matching affinities. These results may lead to more general models to describe the adsorption of ions, including polyatomic ions, at the micellar interface.
RESUMO
The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1(WT) and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp(32) residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp(32) residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1(WT) and hSOD1(G93A) mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp(32) residue in the process. The results showed that Trp(32) residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp(32) residue (bovine SOD1 and hSOD1(W32F) mutant). The results support a role for the oxidation products of the hSOD1-Trp(32) residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.
Assuntos
Superóxido Dismutase/química , Triptofano/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Bicarbonatos/química , Humanos , Dados de Sequência Molecular , Oxirredução , Agregação Patológica de Proteínas , Carbonilação Proteica , Multimerização Proteica , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Estrutura Secundária de Proteína , Eletricidade EstáticaRESUMO
Dodecyltrimethylammonium triflate (DTATf) micelles possess lower degree of counterion dissociation (α), lower hydration, and higher packing of monomers than other micelles of similar structure. Addition of sodium triflate ([NaTf] > 0.05 M) to DTATf solutions promotes phase separation. This phenomenon is commonly observed in oppositely charged surfactant mixtures, but it is rare for ionic surfactants and relatively simple counterions. While the properties of DTATf have already been reported, the driving forces for the observed phase separation with added salt remain unclear. Thus, we propose an interpretation for the observed phase separation in cationic surfactant solutions. Addition of up to 0.03 M NaTf to micellar DTATf solutions led to a limited increase of the aggregation number, to interface dehydration, and to a progressive decrease in α. The viscosity of DTATf solutions of higher concentration ([DTATf] ≥ 0.06 M) reached a maximum with increasing [NaTf], though the aggregation number slightly increased, and no shape change occurred. We hypothesize that this maximum results from a decrease in interaggregate repulsion, as a consequence of increased ion binding. This reduction in micellar repulsion without simultaneous infinite micellar growth is, probably, the major driving force for phase separation at higher [NaTf].
RESUMO
The dediazoniation of aryldiazonium salts in mixed solvents proceeds by a borderline SN1 and SN2 pathway, and product distribution should be proportional to the composition of the solvation shell of the carbon attached to the -N2 group (ipso carbon). The rates of dediazoniation of 2,4,6-trimethylbenzenediazonium in water, methanol, ethanol, propanol, and acetonitrile were similar, but measured product distributions were noticeably dependent on the nature of the water/cosolvent mixture. Here we demonstrated that solvent distribution in the first solvation shell of the ipso carbon, calculated from classical molecular dynamics simulations, is equal to the measured product distribution. Furthermore, we showed that regardless of the charge distribution of the initial state, i.e., whether the positive charge is smeared over the molecule or localized on phenyl moiety, the solvent distribution around the reaction center is nearly the same.
RESUMO
Micellar properties of dodecyltrimethylammonium triflate (DTA-triflate, DTATf) are very different from those of DTA-bromide (DTAB). DTATf aggregates show high aggregation numbers (Nagg), low degree of counterion dissociation (α), disk-like shape, high packing, ordering, and low hydration. These micellar properties and the low surface tension of NaTf aqueous solutions point to a high affinity of Tf(-) to the micellar and air/water interfaces. Although the micellar properties of DTATf are well defined, the source of the Tf(-) effect upon the DTA aggregates is unclear. Molecular dynamics (MD) simulations of Tf(-) (and Br(-)) at the air/water interface and as counterion of a DTA aggregate were performed to clarify the nature of Tf(-) preferences for these interfaces. The effect of NaTf or NaBr on surface tension calculated from MD simulations agreed with the reported experimental values. From the MD simulations a high affinity of Tf(-) toward the interface, which occurred in a specific orientation, was calculated. The micellar properties calculated from the MD simulations for DTATf and DTAB were consistent with experimental data: in MD simulations, the DTATf aggregate was more ordered, packed, and dehydrated than the DTAB aggregate. The Tf(-)/alkyltrimethylammonium interaction energies, calculated from the MD simulations, suggested ion pair formation at the micellar interface, stabilized by the preferential orientation of the adsorbed Tf(-) at the micellar interface.
RESUMO
Short-cationic alpha-helical antimicrobial peptides (SCHAMPs) are promising candidates to combat the growing global threat of antimicrobial resistance. They are short-sequenced, selective against bacteria, and have rapid action by destroying membranes. A full understanding of their mechanism of action will provide key information to design more potent and selective SCHAMPs. Molecular Dynamics (MD) simulations are invaluable tools that provide detailed insights into the peptide-membrane interaction at the atomic- and meso-scale level. We use atomistic and coarse-grained MD to look into the exact steps that four promising SCHAMPs-BP100, Decoralin, Neurokinin-1, and Temporin L-take when they interact with membranes. Following experimental set-ups, we explored the effects of SCHAMPs on anionic membranes and vesicles at multiple peptide concentrations. Our results showed all four peptides shared similar binding steps, initially binding to the membrane through electrostatic interactions and then flipping on their axes, dehydrating, and inserting their hydrophobic moieties into the membrane core. At higher concentrations, fully alpha-helical peptides induced membrane budding and protrusions. Our results suggest the carpet mode of action is fit for the description of SCHAMPs lysis activity and discuss the importance of large hydrophobic residues in SCHAMPs design and activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Simulação de Dinâmica Molecular , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Eletricidade EstáticaRESUMO
The properties of ionic micelles are affected by the nature of the counterion. Specific ion effects can be dramatic, inducing even shape and phase changes in micellar solutions, transitions apparently related to micellar hydration and counterion binding at the micellar interface. Thus, determining the hydration and dynamics of ions in micellar systems capable of undergoing such transitions is a crucial step in understanding shape and phase changes. For cationic micelles, such transitions are common with large organic anions as counterions. Interestingly, however, phase separation also occurs for dodecyltrimethylammonium triflate (DTATf) micelles in the presence of sodium triflate (NaTf). Specific ion effects for micellar solutions of dodecyltrimethylammonium chloride (DTAC), bromide (DTAB), methanesulfonate (DTAMs), and triflate (DTATf) were studied with dielectric relaxation spectroscopy (DRS), a technique capable of monitoring hydration and counterion dynamics of micellar aggregates. In comparison to DTAB, DTAC, and DTAMs, DTATf micelles were found to be considerably less hydrated and showed reduced counterion mobility at the micellar interface. The obtained DTATf and DTAMs data support the reported central role of the anion's -CF3 moiety with respect to the properties of DTATf micelles. The reduced hydration observed for DTATf micelles was rationalized in terms of the higher packing of this surfactant compared to that of other DTA-based systems. The decreased mobility of Tf(-) anions condensed at the DTATf interface strongly suggests the insertion of Tf(-) in the micellar interface, which is apparently driven by the strong hydrophobicity of -CF3.
Assuntos
Espectroscopia Dielétrica , Mesilatos/química , Compostos de Amônio Quaternário/química , Micelas , Estrutura Molecular , Água/químicaRESUMO
Specific ion effects in surfactant solutions affect the properties of micelles. Dodecyltrimethylammonium chloride (DTAC), bromide (DTAB), and methanesulfonate (DTAMs) micelles are typically spherical, but some organic anions can induce shape or phase transitions in DTA(+) micelles. Above a defined concentration, sodium triflate (NaTf) induces a phase separation in dodecyltrimethylammonium triflate (DTATf) micelles, a phenomenon rarely observed in cationic micelles. This unexpected behavior of the DTATf/NaTf system suggests that DTATf aggregates have unusual properties. The structural properties of DTATf micelles were analyzed by time-resolved fluorescence quenching, small-angle X-ray scattering, nuclear magnetic resonance, and electron paramagnetic resonance and compared with those of DTAC, DTAB, and DTAMs micelles. Compared to the other micelle types, the DTATf micelles had a higher average number of monomers per aggregate, an uncommon disk-like shape, smaller interfacial hydration, and restricted monomer chain mobility. Molecular dynamic simulations supported these observations. Even small water-soluble salts can profoundly affect micellar properties; our data demonstrate that the -CF3 group in Tf(-) was directly responsible for the observed shape changes by decreasing interfacial hydration and increasing the degree of order of the surfactant chains in the DTATf micelles.
Assuntos
Mesilatos/química , Micelas , Compostos de Amônio Quaternário/química , Cátions/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Tensoativos/químicaRESUMO
BP100, a short antimicrobial peptide, produces membrane perturbations that depend on lipid structure and charge, salts presence, and peptide/lipid molar ratios. As membrane perturbation mechanisms are not fully understood, the atomic scale nature of peptide/membrane interactions requires a close-up view analysis. Molecular Dynamics (MD) simulations are valuable tools for describing molecular interactions at the atomic level. Here, we use MD simulations to investigate alterations in membrane properties consequent to BP100 binding to zwitterionic and anionic model membranes. We focused on membrane property changes upon peptide binding, namely membrane thickness, order parameters, surface curvature, lipid lateral diffusion and membrane hydration. In agreement with experimental results, our simulations showed that, when buried into the membrane, BP100 causes a decrease in lipid lateral diffusion and lipid acyl-chain order parameters and sharp local membrane thinning. These effects were most pronounced on the closest lipids in direct contact with the membrane-bound peptide. In DPPG and anionic-aggregate-containing DPPC/DPPG membranes, peptide flip (rotation of its non-polar facet towards the membrane interior) induced marked negative membrane curvature and enhanced the water residence half-life time in the lipid hydrophobic core and transmembrane water transport in the direction of the peptide. These results further elucidate the consequences of the initial interaction of cationic alpha-helical antimicrobial peptides with membranes.
RESUMO
BP100 is a short antimicrobial peptide and can also act as a molecule-carrier into cells. Like with other antimicrobial peptides, the precise mechanism of membrane disruption is not fully understood. Here we use computer simulations to understand, at a molecular level, the initial interaction between BP100 and zwitterionic/negatively charged model membranes. In agreement with experimental results, our simulations showed BP100 folded into an alpha helix when in contact with negatively charged membranes. BP100 binding induced the aggregation of negatively charged lipids on mixed membranes composed of zwitterionic and anionic lipids. The peptide in alpha-helix conformation initially interacts with the membrane via electrostatic interactions between the negatively charged lipids and the positively charged residues of the peptide. At that point the peptide flips, burying the hydrophobic residues into the bilayer highlighting the importance of the hydrophobic effect contribution to the initial interaction of cationic antimicrobial peptides with membranes.
Assuntos
Anti-Infecciosos/farmacologia , Oligopeptídeos/farmacologia , 1,2-Dipalmitoilfosfatidilcolina/química , Anti-Infecciosos/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Fosfatidilgliceróis/química , Estrutura Secundária de Proteína , Água/químicaRESUMO
Violacein is a tryptophan-derived purple pigment produced by environmental bacteria, which displays multiple biological activities, including strong inhibition of Gram-positive pathogens. Here, we applied a combination of experimental approaches to identify the mechanism by which violacein kills Gram-positive bacteria. Fluorescence microscopy showed that violacein quickly and dramatically permeabilizes B. subtilis and S. aureus cells. Cell permeabilization was accompanied by the appearance of visible discontinuities or rips in the cytoplasmic membrane, but it did not affect the cell wall. Using in vitro experiments, we showed that violacein binds directly to liposomes made with commercial and bacterial phospholipids and perturbs their structure and permeability. Furthermore, molecular dynamics simulations were employed to reveal how violacein inserts itself into lipid bilayers. Thus, our combined results demonstrate that the cytoplasmic membrane is the primary target of violacein in bacteria. The implications of this finding for the development of violacein as a therapeutic agent are discussed.
Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Indóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Bacillus subtilis/química , Bacillus subtilis/crescimento & desenvolvimento , Membrana Celular/química , Indóis/química , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Staphylococcus aureus/química , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Lauric acid (LAH) strongly inhibits the growth of acne-causing bacteria. LAH is essentially water-insoluble and the solubility of laurate (LA) salts are medium and temperature dependent. Hence, LAH/LA preparations are difficult to formulate. Here we fully characterized phospholipid vesicles containing up to 50 mol% LAH. Vesicles of dipalmitoylphosphatidylcholine (DPPC) containing LAH, at pHs 7.4 and 5.0, were characterized measuring size, charge, bilayer phase transition temperature (Tm) and permeability of water-soluble probes. Small angle X-ray scattering and cryotransmission electron microscopy showed multilamellar vesicles at low LAH %. Increasing LAH % had a negligible effect on particle size. An internal aqueous compartment in all vesicle's preparations, even at equimolar DPPC: LAH fractions, was demonstrated using water-soluble probes. At pH 5.0, the interaction between DPPC and LAH increased the Tm and phase transition cooperativity showing a single lipid phase formed by hydrogen-bonded DPPC: LAH complexes. At pH 7.4, vesicles containing 50 mol% LAH exhibited distinct phases, ascribed to complex formation between LAH and LA or LAH and DPPC. LAH incorporated in the vesicles minimally permeated a skin preparation at both pHs, indicating that the primary sites of LAH solubilization were the skin layers. These results provide the foundations for developing processes and products containing DPPC: LAH.