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1.
Chemistry ; 28(50): e202201477, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-35695822

RESUMO

HBDI-like chromophores represent a novel set of biomimetic switches mimicking the fluorophore of the green fluorescent protein that are currently studied with the hope to expand the molecular switch/motor toolbox. However, until now members capable of absorbing visible light in their neutral (i. e. non-anionic) form have not been reported. In this contribution we report the preparation of an HBDI-like chromophore based on a 3-phenylbenzofulvene scaffold capable of absorbing blue light and photoisomerizing on the picosecond timescale. More specifically, we show that double-bond photoisomerization occurs in both the E-to-Z and Z-to-E directions and that these can be controlled by irradiating with blue and UV light, respectively. Finally, as a preliminary applicative result, we report the incorporation of the chromophore in an amphiphilic molecule and demonstrate the formation of a visible-light-sensitive nanoaggregated state in water.


Assuntos
Luz , Proteínas de Fluorescência Verde/química
2.
J Phys Chem B ; 125(36): 10213-10223, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34464136

RESUMO

Calorimetric and incoherent neutron scattering methods were employed to investigate the action of magainin 2 and PGLa peptides on the phase behavior and molecular dynamics of lipids mimicking cytoplasmic membranes of Gram-negative bacteria. The impact of the peptides, tested individually and cooperatively by differential scanning calorimetry, presented a broadened peak, sometimes with a second shoulder, depicting the phase transition temperature around 21 °C. Neutron scattering revealed a small but significant variation of the membrane dynamics due to the peptides in both in-plane and out-of-plane directions. Although we did not find a clear hint for synergy in the interplay of the two peptides, the calorimetric and neutron data give compatible results in terms of a decrease of the enthalpy due to the presence of the peptides, which destabilize the membrane. The dynamics in the two directions was differentiated when the individual peptides were added to the membranes, but the impact was smaller when both peptides were added together.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bactérias Gram-Negativas , Bicamadas Lipídicas , Magaininas/química , Varredura Diferencial de Calorimetria , Membrana Celular , Simulação de Dinâmica Molecular , Termodinâmica
3.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201610

RESUMO

The accumulation of aggregated protein is a typical hallmark of many human neurodegenerative disorders, including polyglutamine-related diseases such as chorea Huntington. Misfolding of the amyloidogenic proteins gives rise to self-assembled complexes and fibres. The huntingtin protein is characterised by a segment of consecutive glutamines which, when exceeding ~ 37 residues, results in the occurrence of the disease. Furthermore, it has also been demonstrated that the 17-residue amino-terminal domain of the protein (htt17), located upstream of this polyglutamine tract, strongly correlates with aggregate formation and pathology. Here, we demonstrate that membrane interactions strongly accelerate the oligomerisation and ß-amyloid fibril formation of htt17-polyglutamine segments. By using a combination of biophysical approaches, the kinetics of fibre formation is investigated and found to be strongly dependent on the presence of lipids, the length of the polyQ expansion, and the polypeptide-to-lipid ratio. Finally, the implications for therapeutic approaches are discussed.


Assuntos
Membrana Celular/metabolismo , Proteína Huntingtina/metabolismo , Peptídeos/metabolismo , Benzotiazóis/química , Dicroísmo Circular , Difusão Dinâmica da Luz , Éxons , Fluorescência , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Bicamadas Lipídicas , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/química
4.
Biochim Biophys Acta Biomembr ; 1862(8): 183212, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057757

RESUMO

The LAH4 family of amphipathic peptides exhibits pronounced antimicrobial, cell penetrating and nucleic acid transfection activities. Furthermore, variants were designed with potent lentiviral transduction enhancement. When viewed along a helical wheel the four histidines are arranged to form an amphipathic structure. In order to optimize some of these biological activities the number of leucine and alanine residues exposed to the hydrophilic surface was systematically varied which resulted in the design of vectofusin a peptide with strong lentiviral transduction enhancement activities. Here the series of peptides with varying numbers of alanine or leucine residues, respectively, framed by the histidines was tested for their calcein release activity. Interestingly, the membrane pore formation and DNA transfection activities show a clear correlation with the hydrophilic angle. In contrast the membrane partitioning and the propensity to adopt helical conformations was hardly affected as long as the hydrophilic angle did not exceed a limiting value of 150°.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , DNA/genética , Histidina/genética , Membranas/efeitos dos fármacos , Alanina/genética , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , DNA/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lentivirus/genética , Leucina/genética , Membranas/metabolismo , Porosidade , Transfecção
5.
Biochim Biophys Acta Biomembr ; 1862(2): 183149, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816324

RESUMO

Viral protein R (Vpr) is a small accessory protein of 96 amino acids that is present in Human and simian immunodeficiency viruses. Among the very different properties that Vpr possesses we can find cell penetrating capabilities. Based on this and on its capacity to interact with nucleic acids we previously investigated the DNA transfection properties of Vpr and subfragments thereof. We found that fragments of the C-terminal helical domain of Vpr are able to deliver efficiently plasmid DNA into different cell lines. As the amphipathic helix may play a role in the interactions with membranes, we investigated whether insertion of a proline residue in the α-helix modifies the transfection properties of Vpr. Unexpectedly, we found that the resulting Vpr55-82 Pro70 peptide was even more efficient than wild type Vpr55-82 in the gene delivery assays. Using circular dichroism, light scattering and solid-state NMR techniques, we characterized the secondary structure and interactions of Vpr and several mutants with model membranes. A model is proposed where the proline shifts the dissociation equilibrium of the peptide-cargo complex and thereby its endosomal release.


Assuntos
Peptídeos Penetradores de Células/química , Técnicas de Transferência de Genes , Bicamadas Lipídicas/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Substituição de Aminoácidos , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Células HEK293 , HIV-1/química , Humanos , Isoleucina/química , Isoleucina/genética , Prolina/química , Prolina/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo
7.
Adv Exp Med Biol ; 1117: 33-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980352

RESUMO

Even 30 years after the discovery of magainins, biophysical and structural investigations on how these peptides interact with membranes can still bear surprises and add new interesting detail to how these peptides exert their antimicrobial action. Early on, using oriented solid-state NMR spectroscopy, it was found that the amphipathic helices formed by magainins are active when being oriented parallel to the membrane surface. More recent investigations indicate that this in-planar alignment is also found when PGLa and magainin in combination exert synergistic pore-forming activities, where studies on the mechanism of synergistic interaction are ongoing. In a related manner, the investigation of dimeric antimicrobial peptide sequences has become an interesting topic of research which bears promise to refine our views how antimicrobial action occurs. The molecular shape concept has been introduced to explain the effects of lipids and peptides on membrane morphology, locally and globally, and in particular of cationic amphipathic helices that partition into the membrane interface. This concept has been extended in this review to include more recent ideas on soft membranes that can adapt to external stimuli including membrane-disruptive molecules. In this manner, the lipids can change their shape in the presence of low peptide concentrations, thereby maintaining the bilayer properties. At higher peptide concentrations, phase transitions occur which lead to the formation of pores and membrane lytic processes. In the context of the molecular shape concept, the properties of lipopeptides, including surfactins, are shortly presented, and comparisons with the hydrophobic alamethicin sequence are made.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Biofísica , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas , Magaininas/química , Espectroscopia de Ressonância Magnética
8.
Methods Mol Biol ; 1943: 39-59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838608

RESUMO

Amphipathic, pH-responsive, membrane-active peptides such as LAH4 and derivatives thereof have the ability to effectively deliver genes and small interfering RNA (siRNA) into mammalian cells. Their ability to bind and protect nucleic acids and then disrupt membranes when activated at low pH enables them to harness the endocytic machinery to deliver cargo efficiently and with low associated toxicity. This chapter describes protocols for the chemical synthesis of transfection peptides of the LAH4 family, complex formation with nucleic acids, and their use for the in vitro delivery of either plasmid DNA or siRNA into mammalian cell lines.


Assuntos
Peptídeos Penetradores de Células/química , Transfecção/métodos , Cátions/química , Linhagem Celular , Membrana Celular/metabolismo , DNA/administração & dosagem , DNA/genética , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Plasmídeos/genética , RNA Interferente Pequeno/genética
9.
Front Cell Neurosci ; 12: 464, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30559651

RESUMO

Morphine is an analgesic alkaloid used to relieve severe pain, and irreversible binding of morphine to specific unknown proteins has been previously observed. In the brain, changes in the expression of energy metabolism enzymes contribute to behavioral abnormalities during chronic morphine treatment. Creatine kinase B (CK-B) is a key enzyme involved in brain energy metabolism. CK-B also corresponds to the imidazoline-binding protein I2 which binds dopamine (a precursor of morphine biosynthesis) irreversibly. Using biochemical approaches, we show that recombinant mouse CK-B possesses a µM affinity for morphine and binds to morphine in vitro. The complex formed by CK-B and morphine is resistant to detergents, reducing agents, heat treatment and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). CK-B-derived peptides CK-B1-75 and CK-B184-258 were identified as two specific morphine binding-peptides. In vitro, morphine (1-100 µM) significantly reduces recombinant CK-B enzymatic activity. Accordingly, in vivo morphine administration (7.5 mg/kg, i.p.) to mice significantly decreased brain extract CK-B activity compared to saline-treated animals. Together, these results show that morphine strongly binds CK-B and inhibits its activity in vitro and in vivo.

10.
Biomolecules ; 8(2)2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29670065

RESUMO

Biophysical and structural investigations are presented with a focus on the membrane lipid interactions of cationic linear antibiotic peptides such as magainin, PGLa, LL37, and melittin. Observations made with these peptides are distinct as seen from data obtained with the hydrophobic peptide alamethicin. The cationic amphipathic peptides predominantly adopt membrane alignments parallel to the bilayer surface; thus the distribution of polar and non-polar side chains of the amphipathic helices mirror the environmental changes at the membrane interface. Such a membrane partitioning of an amphipathic helix has been shown to cause considerable disruptions in the lipid packing arrangements, transient openings at low peptide concentration, and membrane disintegration at higher peptide-to-lipid ratios. The manifold supramolecular arrangements adopted by lipids and peptides are represented by the 'soft membranes adapt and respond, also transiently' (SMART) model. Whereas molecular dynamics simulations provide atomistic views on lipid membranes in the presence of antimicrobial peptides, the biophysical investigations reveal interesting details on a molecular and supramolecular level, and recent microscopic imaging experiments delineate interesting sequences of events when bacterial cells are exposed to such peptides. Finally, biophysical studies that aim to reveal the mechanisms of synergistic interactions of magainin 2 and PGLa are presented, including unpublished isothermal titration calorimetry (ITC), circular dichroism (CD) and dynamic light scattering (DLS) measurements that suggest that the peptides are involved in liposome agglutination by mediating intermembrane interactions. A number of structural events are presented in schematic models that relate to the antimicrobial and synergistic mechanism of amphipathic peptides when they are aligned parallel to the membrane surface.


Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular
11.
Sci Rep ; 6: 39450, 2016 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-28004740

RESUMO

Circular Dichroism data are often decomposed into their constituent spectra to quantify the secondary structure of peptides or proteins but the estimation of the secondary structure content fails when light scattering leads to spectral distortion. If peptide-induced liposome self-association occurs, subtracting control curves cannot correct for this. We show that if the cause of the light scattering is independent from the peptide structural changes, the CD spectra can be corrected using principal component analysis (PCA). The light scattering itself is analysed and found to be in good agreement with backscattering experiments. This method therefore allows to simultaneously follow structural changes related to peptide-liposome binding as well as peptide induced liposome self-association. We apply this method to study the structural changes and liposome binding of vectofusin-1, a transduction enhancing peptide used in lentivirus based gene therapy. Vectofusin-1 binds to POPC/POPS liposomes, causing a reversal of the negative liposome charge at high peptide concentrations. When the peptide charges exactly neutralise the lipid charges on both leaflets reversible liposome self-association occurs. These results are in good agreement with biological observations and provide further insight into the conditions required for efficent transduction enhancement.

12.
Curr Top Med Chem ; 16(1): 65-75, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26139118

RESUMO

The structural requirements for the synergistic enhancement of antimicrobial activities of the cationic linear peptides PGLa and magainin 2 were investigated. In a first step the antimicrobial activities were evaluated for a number of modifications of the sequences and equimolar mixtures thereof. In particular fluorophore labelled peptides maintain a high degree of antimicrobial activity and considerable synergism when tested conjointly. Thereafter, circular dichroism spectroscopy indicated that these extended sequences adopt helical conformations in the presence of model membranes similar to the unmodified sequences. Energy transfer between the fluorophores suggested that the peptides reside in close proximity to each other when bound to the membrane surface at high concentrations. The fluorophore interactions quickly diminish at lower peptide-to-lipid ratios indicating that the peptide-peptide interactions are weak. Furthermore, (15)N solid-state NMR measurements of the membrane topology of [(15)N-Ala14]-PGLa and of its fluorophorecarrying analogue reconstituted into supported 1, 2-didecanoyl-sn-glycero-3-phosphocholine bilayers were performed. These experiments revealed no correlation between the topological state of PGLa and the observed synergistic enhancement of antimicrobial activities due to the presence of magainins. These results suggest that lipid mediated interactions rather than the formation of tight peptide-peptide complexes in the membrane are responsible for synergistic activities of the mixtures.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Magaininas/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Sinergismo Farmacológico , Magaininas/química , Testes de Sensibilidade Microbiana , Espectrometria de Fluorescência , Relação Estrutura-Atividade
13.
Protein Expr Purif ; 109: 29-34, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25655203

RESUMO

A frequent problem of recombinant protein production is their insolubility. To address this issue, engineered Escherichiacoli strains like Arctic Express that produce an exogenous chaperone facilitating protein folding, have been designed. A drawback is the frequent contamination of the protein by chaperones. A simple method, using urea at a sub-denaturing concentration, allows unbinding of Cpn60 from expressed protein. This method was successfully used to purify 2 proteins, an enzyme and a viral protein. The enzyme was fully active. The nature of interaction forces between enzyme and Cpn60 was investigated. The method is likely applicable to purify other proteins.


Assuntos
Bioquímica/métodos , Chaperonina 60/metabolismo , Escherichia coli/metabolismo , Engenharia Genética , Proteínas Recombinantes/metabolismo , Difusão Dinâmica da Luz , Eletroforese em Gel de Poliacrilamida , Cinética
14.
Langmuir ; 30(21): 6171-9, 2014 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-24807004

RESUMO

Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos/química , Alameticina/química , Desenho de Fármacos , Glicerol/química , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Lipídeos/química , Magaininas/química , Espectroscopia de Ressonância Magnética , Conformação Proteica , Espectrometria de Fluorescência , Fatores de Tempo , Lipossomas Unilamelares/química
15.
Methods Mol Biol ; 948: 85-103, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23070765

RESUMO

Amphipathic, pH-responsive, membrane-active peptides such as LAH4 and derivatives thereof have the ability to effectively deliver genes and small interfering RNA (siRNA) into mammalian cells. Their ability to bind and protect nucleic acids and then disrupt membranes when activated at low pH enables them to harness the endocytic machinery to deliver cargo efficiently and with low associated toxicity. This chapter describes protocols for the chemical synthesis of transfection peptides of the LAH4 family, complex formation with nucleic acids, and their use for the in vitro delivery of either plasmid DNA or siRNA into mammalian cell lines.


Assuntos
DNA/metabolismo , Técnicas de Transferência de Genes , Histidina , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Plasmídeos/genética , RNA Interferente Pequeno/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/genética , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Humanos , Luz , Luciferases/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Peptídeos/síntese química , Peptídeos/toxicidade , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Espalhamento de Radiação , Transfecção
16.
Biophys J ; 98(11): 2544-53, 2010 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-20513398

RESUMO

We report on the reversible association of anionic liposomes induced by an antimicrobial peptide (LAH4). The process has been characterized for mixed membranes of POPC and POPS at molar ratios of 1:1, 3:1, and 9:1. Although the vesicles remain in suspension in the presence of excess amounts of peptide, the addition of more lipids results in surface charge neutralization, aggregation of the liposomes, and formation of micrometer-sized structures that coexist in equilibrium with vesicles in suspension. At low ratios of anionic lipids, vesicle aggregation is a reversible process, and vesicle disassembly is observed upon inversion of the surface charge by further supplementation with anionic vesicles. In contrast, a different process, membrane fusion, occurs in the presence of high phosphatidylserine concentrations. Upon binding to membranes containing low POPS concentrations, the peptide adopts an in-plane alpha-helical structure, a secondary structure that is conserved during vesicle association and dissociation. Our finding that peptides are essential for vesicle aggregation contributes to a better understanding of the activity of antimicrobial peptides, and suggests an additional layer of complexity in membrane-protein lipid interactions.


Assuntos
Lipossomos/química , Peptídeos/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Peptídeos Catiônicos Antimicrobianos , Dicroísmo Circular/métodos , Luz , Bicamadas Lipídicas/química , Estrutura Secundária de Proteína , Espalhamento de Radiação , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral/métodos
17.
FASEB J ; 24(6): 1737-46, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20103720

RESUMO

Catestatin is a natural peptide of higher organisms including humans, with a wide variety of biological functions involved in catecholamine inhibition, cardiovascular regulation, control of blood pressure, inflammation, and innate immunity. It is derived from the natural processing of chromogranin A, induced in the skin after injury, and produced by chromaffin cells and neutrophils. With neutrophils, the peptide enters the cell by crossing the plasma membrane where it interacts with internal targets to induce calcium influx. Therefore, we investigated the membrane interactions and structure of several catestatin-derived peptides. Whereas fluorescence dye release experiments are indicative of membrane permeabilization, multidimensional solution NMR and circular dichroism spectroscopies show that catestatin adopts alpha-helical conformations between Ser-6 and Tyr-12 in the presence of dodecylphosphocholine micelles. Furthermore, proton-decoupled (15)N solid-state NMR spectroscopy of sequences labeled with (15)N and reconstituted into oriented lipid bilayers indicates that this domain is aligned in a strongly tilted to inplanar alignment. Proton-decoupled (31)P NMR spectra of the same samples are indicative of conformational and/or orientational heterogeneity at the level of the lipid bilayer head groups due to the presence of catestatin. The sequence and 3-dimensional structure of catestatin exhibit homologies with penetratin, which is suggestive that they both enter the cells by related mechanisms to target internal structures.


Assuntos
Cromogranina A/química , Cromogranina A/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Cinética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
18.
J Biol Chem ; 284(1): 119-133, 2009 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-18984589

RESUMO

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.


Assuntos
Anti-Infecciosos/química , Antimaláricos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos/química , Membrana Celular/química , Histidina/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
19.
J Pept Sci ; 14(4): 488-95, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18085719

RESUMO

Members of the LAH4 family of cationic linear peptide antibiotics have been designed to form amphipathic helical structures in membrane environments and switch from alignments parallel to the bilayer surface to transmembrane orientations in a pH-dependent manner. Here the aggregation in aqueous buffer of two members of the family has been investigated by DLS. The peptides form monomers or small oligomers at pH = 5 but associate into nano-sized aggregates at physiological pH. The diameter of these latter complexes can be considerably reduced by sonication. Furthermore, the membrane interactions of the various supramolecular aggregates with POPC or mixed POPC/POPS vesicles have been investigated in calcein-release assays. In all the cases tested, the large preformed oligomeric peptide aggregates of 20-40 nm in size were more active than the structures with the smallest hydrodynamic radii in releasing the fluorescent dye from LUV. In contrast, the relative activity after sonication depends on the specific environment tested. The data suggest that these amphiphiles form micellar structures and support the notion that they can act in a manner comparable to detergents.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Histidina/química , Proteínas de Membrana/química , Peptídeos/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Permeabilidade da Membrana Celular , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Luz , Bicamadas Lipídicas/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peptídeos/síntese química , Fosfolipídeos/química , Estrutura Secundária de Proteína , Espalhamento de Radiação
20.
Biochemistry ; 46(51): 15175-87, 2007 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-18052076

RESUMO

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


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Histidina/metabolismo , Dourada , Sequência de Aminoácidos , Animais , Linhagem Celular , Dicroísmo Circular , Cor , Fibroblastos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Triptofano/química
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