RESUMEN
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.
Asunto(s)
Luz , Proteínas Fluorescentes Verdes/químicaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Proteína Huntingtina/metabolismo , Péptidos/metabolismo , Benzotiazoles/química , Dicroismo Circular , Dispersión Dinámica de Luz , Exones , Fluorescencia , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Membrana Dobles de Lípidos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Péptidos/químicaRESUMEN
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Membrana Celular/química , Biofisica , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos , Magaininas/química , Espectroscopía de Resonancia MagnéticaRESUMEN
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.
Asunto(s)
Bioquímica/métodos , Chaperonina 60/metabolismo , Escherichia coli/metabolismo , Ingeniería Genética , Proteínas Recombinantes/metabolismo , Dispersión Dinámica de Luz , Electroforesis en Gel de Poliacrilamida , CinéticaRESUMEN
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos/química , Alameticina/química , Diseño de Fármacos , Glicerol/química , Humanos , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Lípidos/química , Magaininas/química , Espectroscopía de Resonancia Magnética , Conformación Proteica , Espectrometría de Fluorescencia , Factores de Tiempo , Liposomas Unilamelares/químicaRESUMEN
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.
Asunto(s)
Cromogranina A/química , Cromogranina A/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Humanos , Cinética , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Bacterias Gramnegativas , Membrana Dobles de Lípidos , Magaininas/química , Rastreo Diferencial de Calorimetría , Membrana Celular , Simulación de Dinámica Molecular , TermodinámicaRESUMEN
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.
Asunto(s)
Liposomas/química , Péptidos/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Péptidos Catiónicos Antimicrobianos , Dicroismo Circular/métodos , Luz , Membrana Dobles de Lípidos/química , Estructura Secundaria de Proteína , Dispersión de Radiación , Espectroscopía Infrarroja por Transformada de Fourier , Análisis Espectral/métodosRESUMEN
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.
Asunto(s)
Péptidos de Penetración Celular/química , Técnicas de Transferencia de Gen , Membrana Dobles de Lípidos/química , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/química , Sustitución de Aminoácidos , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/metabolismo , Células HEK293 , VIH-1/química , Humanos , Isoleucina/química , Isoleucina/genética , Prolina/química , Prolina/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
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°.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , ADN/genética , Histidina/genética , Membranas/efectos de los fármacos , Alanina/genética , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/farmacología , ADN/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Lentivirus/genética , Leucina/genética , Membranas/metabolismo , Porosidad , TransfecciónRESUMEN
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.
Asunto(s)
Péptidos de Penetración Celular/química , Transfección/métodos , Cationes/química , Línea Celular , Membrana Celular/metabolismo , ADN/administración & dosificación , ADN/genética , Histidina/química , Humanos , Concentración de Iones de Hidrógeno , Plásmidos/genética , ARN Interferente Pequeño/genéticaRESUMEN
[This corrects the article DOI: 10.3389/fncel.2018.00464.].
RESUMEN
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.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Animales , Membrana Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica MolecularRESUMEN
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.
RESUMEN
Cationic amphipathic alpha-helical peptides preferentially disrupt anionic lipids in mixed model membranes, potentially causing a catastrophic release of the cell contents or attenuation of the membrane potential. The effective role of such peptides requires considerable discrimination between target and host cells, which is likely to occur at the level of the cell membrane. Here, we explore the roles of a variety of common membrane constituents in mediating the interaction between the antimicrobial peptide pleurocidin and model membranes. We employ intrinsic tryptophan fluorescence and circular dichroism to observe the effect of increasing concentrations of sterol in the membrane on peptide binding, using (2)H solid-state NMR of chain deuterated lipids simultaneously to probe the effective chain disruption of the anionic phospholipid component of the membrane. We show that the degree of ordering of the lipid acyl chains in the membrane is dependent on the nature of the zwitterionic phospholipid headgroup in mixed anionic membranes. Furthermore, the presence of cholesterol and ergosterol increases acyl chain order in the liquid crystalline model membranes, but to differing degrees. Our results show how sterols can protect even negatively charged membranes from the disruptive effects of antimicrobial peptides, thereby providing a molecular view of the differences in sensitivity of various target membranes to linear cationic antibiotic peptides where bacteria (no sterols) are most susceptible, lower eukaryotes including fungi (containing ergosterol) exhibit an intermediate degree of sensitivity, and higher organisms (containing cholesterol) are largely resistant to antimicrobial peptides.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Modelos Químicos , Modelos Moleculares , Fosfolípidos/química , Esteroles/química , Simulación por Computador , Iones , Conformación MolecularRESUMEN
Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x(L) is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-x(L) associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-x(L) its membrane interactions are weak. In the presence of membranes Bcl-x(L) can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-x(L) hydrophobic binding pocket becomes accessible for protein-protein interactions whilst the structure of the N-terminal region remains intact.
Asunto(s)
Membrana Dobles de Lípidos/química , Espectroscopía de Resonancia Magnética/métodos , Fluidez de la Membrana , Modelos Químicos , Modelos Moleculares , Proteína bcl-X/química , Proteína bcl-X/ultraestructura , Simulación por Computador , Isótopos de Nitrógeno/análisis , Isótopos de Nitrógeno/química , Conformación ProteicaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli/efectos de los fármacos , Magaininas/farmacología , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Sinergismo Farmacológico , Magaininas/química , Pruebas de Sensibilidad Microbiana , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
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.
RESUMEN
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.
Asunto(s)
ADN/metabolismo , Técnicas de Transferencia de Gen , Histidina , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Plásmidos/genética , ARN Interferente Pequeño/metabolismo , Secuencia de Aminoácidos , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN/química , ADN/genética , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Portadores de Fármacos/toxicidad , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Humanos , Luz , Luciferasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Péptidos/síntesis química , Péptidos/toxicidad , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Dispersión de Radiación , TransfecciónRESUMEN
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.