RESUMO
Designed to model ideally amphipathic beta-sheets, the minimalist linear (KL)(m)K peptides (m=4-7) were synthesized and proved to form stable films at the air/water interface, they insert into compressed dimyristoylphosphatidylcholine monolayers and interact with egg phosphatidylcholine vesicles. Whatever the interface or the lateral pressure applied to the films, FT-IR and polarization-modulated IRRAS spectroscopy developed in situ on the films indicated that all the peptides totally fold into intermolecular antiparallel beta-sheets. Calculated spectra of the amide region allowed us to define the orientation of the beta-strands compared to the interface. It is concluded that such beta-sheets remain flat-oriented without deep perturbation of zwitterionic phospholipids. Dansyl labelling at the N-terminus indicates that all the peptides are monomeric at a low concentration in aqueous buffer and bind to lipids with similar Dns burying. The affinities for zwitterionic lecithin mono- and bilayers, quantitatively estimated from buffer to lipid partition constants, monotonically increased with peptide length, indicating that hydrophobicity is a limiting parameter for lipid and membrane affinities. Peptides induced permeability increases on zwitterionic liposomes, they are strongly hemolytic towards human erythrocytes and their activity increases concurrently with length. Taking into account the lipid affinity, a hemolytic efficiency can be defined: at the same amount of peptide bound, this efficiency strongly increases with the peptide length. It is proposed that the first determinant step of membrane disturbance is the invasion of the outer membrane leaflet by these ideally amphipathic beta-sheeted structures lying flat at the interface, like large rafts depending on the number of beta-strands.
Assuntos
Hemólise/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Dimiristoilfosfatidilcolina , Desenho de Fármacos , Humanos , Técnicas In Vitro , Metabolismo dos Lipídeos , Lipossomos , Dados de Sequência Molecular , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade , ÁguaRESUMO
Proton NMR of melittin differs according to the association state of the peptide in the monomer or tetramer. Melittin interacts with lysophosphatidyl-choline micelles, whatever the association state of melittin; well resolved superimposed spectra from both components for all the lipid to peptide molar ratios are observed. Within the complexes, local mobility and fast exchange occurs. On binding concomitant shifts on Trp19 indole lines and on the aliphatic CH2 protons of the lipids are detected. The lipid perturbation is maximum for methylene groups in a alpha and beta of the ester bond, this could allow positionning of Trp19 in the hydrophobic core of the lipids.
Assuntos
Venenos de Abelha , Lisofosfatidilcolinas , Meliteno , Cinética , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Conformação Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical rate of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C14 lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10-20 degrees C. This second group of lipids is constituted by monoacid C16 lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C16 aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C14 chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.
Assuntos
Venenos de Abelha , Meliteno , Fluidez de Membrana/efeitos dos fármacos , Fosfolipídeos , Ânions , Venenos de Abelha/farmacologia , Calorimetria , Polarização de Fluorescência , Bicamadas Lipídicas , Meliteno/farmacologia , Ácidos Fosfatídicos , Fosfatidilgliceróis , Fosfatidilserinas , Relação Estrutura-AtividadeRESUMO
The behavior of lytic ideally amphipathic peptides of generic composition LiKj(i=2j) and named LKn, n=i+j, is investigated in situ by the monolayer technique combined with the recently developed polarization modulation IR spectroscopy (PMIRRAS). A change in the secondary structure occurs versus peptide length. Peptides longer than 12 residues fold into alpha-helices at interfaces as expected from their design, while enough shorter peptides, from 9 down to 5 residues, form intermolecular antiparallel beta-sheets. Analysis of experimental and calculated PMIRRAS spectra in the amide I and II regions show that peptides are flat oriented at the interfaces. Structures and orientation are preserved whatever the nature of the interface, air/water or DMPC monolayer, and the lateral pressure. Peptide partition constants, KaffPi, are estimated from isobar surface increases of DMPC monolayers. They strongly increase when Pi decreases from 30 mN/m to 8 mN/m and they vary with peptide length with an optimum for 12 residues. This non-monotonous dependence fits with data obtained in bilayers and follows the hemolytic activity of the peptides. Lipid perturbations due to peptide insertion essentially detected on the PO4- and CO bands indicate disorder of the lipid head groups. Lysis induced on membranes by such peptides is proposed to first result from their flat asymmetric insertion.
Assuntos
Proteínas Hemolisinas/química , Lipídeos/química , Peptídeos/química , Ar , Sequência de Aminoácidos , Compostos de Dansil/química , Dados de Sequência Molecular , Pressão , Conformação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Água/químicaRESUMO
The effect of melittin on different binary mixtures of phospholipids has been studied by polarization of DPH fluorescence in order to determine if melittin can induce phase separation. Since the interaction between lipids and melittin is sensitive to both electrostatic and hydrophobic forces, we have studied the effect of the acyl chain length and of the polar head group of the lipids. In spite of the difference of the chain length between dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), no phase separation occurs in an equimolar mixture of these lipids in presence of melittin. However, when the charged lipid dipalmitoylphosphatidylglycerol (DPPG) is mixed with either DPPC or DSPC, the addition of melittin leads to phase separation. The DSPC/DPPG/melittin system, which shows a very complex thermotropism, has also been studied by Raman spectroscopy using DPPG with deuteriated chains in order to monitor each lipid independently. The results suggest that the higher affinity of melittin for DPPG leads to a partial phase separation. We propose the formation of DPPG-rich domains perturbed by melittin and peptide-free regions enriched in DSPC triggered by the head group charge and chain-length differences.
Assuntos
Venenos de Abelha/farmacologia , Polarização de Fluorescência , Meliteno/farmacologia , Fosfolipídeos , Análise Espectral Raman , 1,2-Dipalmitoilfosfatidilcolina , Difenilexatrieno , Fosfatidilcolinas , Fosfatidilgliceróis , Temperatura , TermodinâmicaRESUMO
The binding of a dansylated analogue of melittin (DNC-melittin) to natural membranes is described. The cytolytic peptide from honey bee venom melittin was enzymatically labelled in its glutamine-25 with the fluorescent probe monodansylcadaverine using guinea pig liver transglutaminase. The labelled peptide was characterised functionally in cytolytic assays, and spectroscopically by circular dichroism and fluorescence. The behaviour of DNC-melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. We used resonance energy transfer to measure the state of aggregation of melittin on the membrane plane in synthetic and natural lipid bilayers. When bound to erythrocyte ghost membranes, the extent of energy transfer was found to be equivalent to when bound to small unilamellar vesicles of phosphatidylcholine. Our results correlate best with a proposed model in which the initial interaction between melittin and the red blood cells could be merely electrostatic and the peptide remains in a low alpha-helical conformation. The next step would be a peptide stabilisation in the membrane in a monomeric alpha-helical conformation that would imply the collapse of the membrane structure and liberation of the cell contents.
Assuntos
Bicamadas Lipídicas , Meliteno/análogos & derivados , Animais , Sítios de Ligação , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Dicroísmo Circular , Transferência de Energia , Corantes Fluorescentes , Glutamina , Cobaias , Hemólise/efeitos dos fármacos , Humanos , Fígado/enzimologia , Meliteno/química , Meliteno/metabolismo , Meliteno/farmacologia , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , Transglutaminases/metabolismoRESUMO
Light scattering and 31P-NMR have been used to monitor the effect of the bee-toxin, melittin, on phosphatidylcholine (PC) bilayers of variable acyl chain length (from C16:0 to C20:0). Melittin interacts with all lipids provided the interaction is initiated in the lipid fluid phase. For low-to-moderate amounts of toxin (lipid-peptide molar ratios, Ri > or = 15), the system takes the form of large spheroidal vesicles, in the fluid phase, whose radius increases from 750 A with dipalmitoyl-PC (DPPC) to 1500 A with diarachinoyl-PC (DAPC). These vesicles fragment into small discoids of 100-150 A radius when the system is cooled down below Tc (the gel-to-fluid phase transition temperature). Little chain length dependence is observed for the small objects. Small structures are also detected independently of the physical state of lipids (gel or fluid) when Ri < or = 5 and provided the interaction has been made above Tc. Small discs clearly characterized for DPPC and distearoyl-PC (DSPC) lipids are much less stable with DAPC. However in the long term, all these small structures fuse into large lipid lamellae. Discs are thermodynamically unstable and kinetics of disappearance of the small lipid-toxin complexes increases as the chain length increases in the sense: DAPC >> DSPC > DPPC. Kinetics of fusion of the small discs into extended bilayers is described by a pseudo-first-order law involving a lag time after which fusion starts. Increasing the chain length decreases the lag time and increases the rate of fusion. Formation of both the large vesicles in the fluid phase and the small discs in the gel phase as well as their stability is discussed in terms of relative shapes and dynamics of both lipids and toxin.
Assuntos
Bicamadas Lipídicas , Meliteno/química , Fosfatidilcolinas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Estabilidade de Medicamentos , Cinética , Luz , Espectroscopia de Ressonância Magnética , Conformação Molecular , Fósforo , Espalhamento de Radiação , Relação Estrutura-Atividade , TermodinâmicaRESUMO
According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5. Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very sensitive to the binding of proteins, and so is a useful method to study lipid-protein interactions. The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of bilayer, only due to the interaction of the hydrophobic peptide tail. Interaction of dipalmitoly phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 degrees C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected. Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.
Assuntos
Citocromos , Membranas Artificiais , Fosfatidilcolinas , Fosfatidiletanolaminas , Animais , Sítios de Ligação , Bovinos , Concentração de Íons de Hidrogênio , Cinética , Modelos Biológicos , Conformação Molecular , Ligação Proteica , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
In solutions of increasing ionic strength, the molecular weight of melittin varies from 2840 (monomeric melittin) to 11200. This polymerization, concomitant with an important change in conformation (Talbot, J.C., Dufourcq, J., De Bony, J., Faucon, J.F. and Lussan, C. (1979) FEBS Lett. 102, 191-193), is accompanied by a significant alteration in the partial specific volume of the molecule. The binding of melittin to phospholipids (phosphatidylserine, lysolecithin, dihexanoyl-, dioctanoyl- and lysolauroylphosphatidylcholine) depends on the state of association of the toxin and on the critical micelle concentration of lipids. No interaction is observed between monomeric melittin and free lipids, whereas tetrameric melittin can bind free lipids to form mixed micelles. At phospholipid concentrations above the critical micelle concentration, melittin in any state of self-association can bind lipids. The mixed micelles formed at saturation appear to be independent of the initial state of association of melittin.
Assuntos
Venenos de Abelha , Meliteno , Fosfolipídeos , Cinética , Conformação Molecular , Concentração Osmolar , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and/or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures T greater than or equal to Tm efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and/or non-hydrolysable lipids. Melittin monomer does not change Tm. When melittin tetramer is stabilized, it decreases Tm by 10-15 deg. C. The transition broadens, and is finally abolished for Ri less than or equal to 2. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For T less than or equal to Tm, fluorescence and Raman show that melittin decreases the amount of CH2 groups in 'trans' conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at T greater than or equal to Tm, while from Raman the positional intermolecular order decreases without significant change in the CH2 groups 'trans'/'gauche' ratio.
Assuntos
Venenos de Abelha , Dimiristoilfosfatidilcolina , Meliteno , Fenômenos Químicos , Físico-Química , Polarização de Fluorescência , Temperatura Alta , Substâncias Macromoleculares , Fosfolipases A/metabolismo , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
In a minimalist approach to modeling lytic toxins, amphipathic peptides of LiKj with i=2j composition and whose length varies from 5 to 22 residues were studied for their ability to induce hemolysis and lipid vesicle leakage. Their sequences were designed to generate ideally amphipathic alpha helices with a single K residue per putative turn. All the peptides were lytic, their activities varying by more than a factor of 103 from the shortest 5-residue-long peptide (5-mer) to the longest 22-mer. However, there was no monotonous increase versus length. The 15-mer was as active as the 22-mer and even more than melittin which is used as standard. Partition coefficients from the buffer to the membrane increased in relation to length up to 12 residues, then weakly decreased to reach a plateau, while they were expected to increase monotonously with peptide length and hydrophobicity as revealed from HPLC retention times. Fluorescence labeling by a dansyl group at the N-terminus, or by a W near the CO-terminus, show that up to 12 residues, the peptides were essentially monomeric while longer peptides strongly aggregated in the solution. Lipid affinity was then controlled by peptide length and was found to be limited by folding and self-association in buffer. The lytic activity resulted both from lipid affinity, which varied by a factor of 20-fold, and from efficiency in disturbing the membrane when bound, the latter steeply and monotonously increasing with length. The 15-residue-long peptide, KLLKLLLKLLLKLLK, had the optimal size for highest lytic activity. The shallow location of the fluorescent labels in the lipids is further evidence for a model of peptides remaining flat at the interface.
Assuntos
Proteínas Hemolisinas/química , Hemólise , Sequência de Aminoácidos , Compostos de Dansil/química , Eritrócitos/efeitos dos fármacos , Humanos , Lipossomos/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Espectrometria de FluorescênciaRESUMO
Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.
Assuntos
Venenos de Abelha , Meliteno , Fosfatidilcolinas , Fenômenos Biofísicos , Biofísica , Cromatografia em Gel , Técnica de Fratura por Congelamento , Luz , Bicamadas Lipídicas , Fusão de Membrana , Lipídeos de Membrana , Proteínas de Membrana , Microscopia Eletrônica , Modelos Biológicos , Espalhamento de RadiaçãoRESUMO
Ion channel formation by three analogues of staphylococcal delta-toxin, an amphipathic and alpha-helical channel-forming peptide, has been evaluated by measurement of ionic currents across planar lipid bilayers. Replacement of beta-branched, hydrophobic residues by leucine and movement of a tryptophan residue from the hydrophilic to the hydrophobic face of the helix does not significantly alter ion channel activity. Removal of the N-terminal blocking group combined with the substitution of glycine-10 by leucine changes the single channel properties of delta-toxin, without altering macroscopic conductance/voltage behaviour. Truncation of the N-terminus by three residues results in complete loss of channel-forming activity. These changes in channel-forming properties upon altering the peptide sequence do not mirror changes in haemolytic activity. The results lend support to the proposal that channel formation and haemolysis are distinct events. Channel properties are discussed in the context of a model in which the pore is formed by a bundle of approximately parallel transbilayer helices.
Assuntos
Proteínas de Bactérias/farmacologia , Proteínas Hemolisinas/farmacologia , Canais Iônicos/síntese química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Bicamadas Lipídicas/química , Dados de Sequência MolecularRESUMO
Possible interactions between calpain II and phospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylcholine were studied using fluorescence and gel filtration techniques. Changes in fluorescence intensity of purified calpain II show that the enzyme strongly interacts with phosphatidylinositol and phosphatidylserine and to a lesser extent with phosphatidylcholine. These results are corroborated by the gel filtration technique which permits the isolation of the enzyme phospholipid complex. Association between calpain II and various phospholipid vesicles can occur in the absence of calcium. Such binding occurs without any observable change of the molecular mass of the two subunits on SDS-polyacrylamide gel electrophoresis.
Assuntos
Calpaína/metabolismo , Lipossomos , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Animais , Cálcio/farmacologia , Calpaína/isolamento & purificação , Cinética , Peso Molecular , Músculos/enzimologia , Ligação Proteica , CoelhosRESUMO
The self-assembling of double-stranded DNA with short synthetic peptides has been analysed using the fluorescent properties of the intercalating dye, ethidium bromide. Two membrane-active peptides with appropriate sequences of lysine and leucine amino acids and a short polylysine have been probed. The results revealed that the secondary structure of the peptide decisively aimed the peptide-DNA complex formation: only the longest peptide, which is the only one to exhibit an alpha-helical structure in solution, could achieve DNA compacting before charge neutralisation. The obtained complex retained a significant membrane activity as demonstrated by calcein leakage experiments. This shows that short synthetic peptides of elementary sequence can combine both membrane activity and DNA-condensing properties. The potential of these constructs as DNA carriers will be discussed.
Assuntos
DNA/química , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Etídio , Corantes Fluorescentes , Luz , Lipossomos , Conformação Proteica , Espalhamento de Radiação , Espectrometria de FluorescênciaRESUMO
Melittin, a C-terminal glutamine peptide, incorporated the fluorescent probe monodansylcadaverine (DNC) when catalysed by guinea-pig liver transglutaminase and Ca2+, as determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A 1:1 adduct DNC-melittin was identified in which a single glutamine residue out of two, i.e. Gln25, acts as acyl donor. Incubation of melittin with transglutaminase in the absence of DNC originated high molecular mass complexes indicative that the peptide lysine residue can act as an acyl acceptor. The DNC-melittin was about 3 times more active in the lysis of red cell membranes than native melittin. Fluorescence study of the labelled melittin in the submicromolar range where it is active on cells showed that while totally exposed to solvent in methanol solution, both Trp and dansyl groups are buried in buffer solution. This strongly suggests that DNC-melittin is self-associated and indeed more active than the native melittin in the same conditions.
Assuntos
Cadaverina/análogos & derivados , Meliteno/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Catálise , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Corantes Fluorescentes , Cobaias , Hemólise , Humanos , Fígado/enzimologia , Meliteno/farmacologia , Dados de Sequência MolecularRESUMO
An original series of 12- to 22-residue-long peptides was developed, they are only constituted by apolar Leu and charged Lys residues periodically located in the sequence in order to general ideal highly amphipathic alpha-helices. By circular dichroism, the peptides are proven to be mainly alpha-helical in organic and aqueous solvents and in the presence of lipids. The peptides are highly hemolytic, their activity varies according to the peptide length. The 15-, 20-, and 22-residue-long-peptides have LD50 approximately 5 x 10(-8) M for 10(7) erythrocytes, i.e. they are 5-10 times more active than melittin, and are indeed several orders of magnitude more active than magainin or mastoparan.
Assuntos
Proteínas Hemolisinas/farmacologia , Meliteno/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Dicroísmo Circular , Proteínas Hemolisinas/química , Humanos , Leucina/química , Lisina/química , Meliteno/química , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The actions of bee venom melittin and delta-lysin from Staphylococcus aureus on membranes have been monitored by solid-state deuterium and phosphorus NMR and shown to differ depending on temperature and on the lipid-to-peptide molar ratio Ri. In the gel phase of phosphatidylcholine model membranes, for lipid-to-peptide ratios Ri greater than 15, melittin induces isotropic lines interpreted as reflecting the presence of small discoidal structures, whereas delta-lysin does not. These small objects are metastable, that is, within a time-scale of hours they return to large lipid bilayers. The kinetics of this process depend on the lecithin chain length. In the fluid phases, at temperatures greater than that of the gel-to-fluid transition Tc, analysis of the quadruplar splittings in terms of chain ordering indicates that both melittin and delta-lysin similarly disorder the membrane. At temperatures above but close to Tc, melittin preferentially orders the center of the bilayer, while delta-lysin promotes ordering throughout the entire bilayer thickness. These effects are interpreted as reflecting different locations of the peptides with respect to the membrane surface. The addition of greater amounts of toxins, Ri = 4, on phosphatidylcholine model membranes induces very small structures irrespective of the temperature in the case of melittin, but only above Tc for delta-lysin. NMR spectral features similar to those characterizing the small fast-tumbling objects with phosphatidylcholine are also observed with egg phosphatidylethanolamine and erythrocyte membranes. The formation of small structures is thus inferred as a general process which reflects membrane supramolecular reorganization.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Bactérias/farmacologia , Venenos de Abelha/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Meliteno/farmacologia , 1,2-Dipalmitoilfosfatidilcolina , Deutério , Proteínas Hemolisinas , Espectroscopia de Ressonância Magnética , Membranas Artificiais , Estrutura Molecular , Radioisótopos de FósforoRESUMO
Quenching of fluorescence was used to monitor adsorption of thrombin (T), antithrombin (AT) and their inactive complex (T-AT) onto three anticoagulant biomaterials made of polystyrene beads bearing the functional groups of heparin. An adsorption capacity of 0.12 mumol of T per mg of polymer allowed the formation of a monolayer of protein at the polymer surface. An affinity constant of 3 x 10(7) l.mol-1 between thrombin and polymer was estimated, whatever the polymer used. The affinity of T-AT was similar although weaker. Desorption of proteins from the polymeric interface by means of polycations (polybrene and polylysine) showed that the inactive complex T-AT is more quantitatively and easily released than thrombin.
Assuntos
Anticoagulantes , Antitrombinas/metabolismo , Materiais Biocompatíveis , Poliestirenos , Trombina/metabolismo , Adsorção , Venenos Elapídicos , Humanos , Cinética , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
The binding of melittin to phospholipid bilayers and micelles depends on its quaternary structure and on the state of association of lipids. Monomeric melittin only binds to lipids above their cmc, whereas tetrameric melittin exhibits a biphasic binding; the interaction with monomeric lipids being possible without dissociation of the tetramer. In lipid excess, the bound state observed by fluorescence, polarization and ORD are always very similar. We propose the following model: the presence of a lipidic interface is necessary for the binding of monomeric melittin, while the tetramer may interact with lipid monomers without any dissociation: it might increase in size by addition of lipid molecules to form a micelle-like particle. The perturbations induced by melittin on the thermotropic behaviour of charged phospholipids are detected by calorimetry (DSC) and fluorescence polarization of DPH. For the first group of lipids, constituted of mono or divalent C14 and of divalent C16 lipids, the transitions are progressively abolished in the presence of melittin, without any shift of the temperature. For a second group of lipids, essentially constituted of monovalent C16 lipids, a cooperative transition is always observed. Moreover, at lipid to protein molar ratios higher than 8, there are two distinct well-defined transitions, at the same temperature as for pure lipid and 10 degrees C to 15 degrees C lower. All these results are interpreted by a phase separation occurring between quasi-pure lipid regions and the lipid-melittin complex. These last ones either could, or not, give rise to a phase transition, according to the cohesion of the initial bilayer. In the case of binary mixtures, there would be a phase separation between enriched phosphatidylcholine regions and negative lipid-melittin complexes.