ABSTRACT
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.
Subject(s)
Lipid Bilayers , Melitten/chemistry , Phosphatidylcholines/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Drug Stability , Kinetics , Light , Magnetic Resonance Spectroscopy , Molecular Conformation , Phosphorus , Scattering, Radiation , Structure-Activity Relationship , ThermodynamicsABSTRACT
In order to gain some insight into the mechanism of insertion into membranes of the pore-forming domain of colicin A and the structure of its membrane-bound form, circular dichroism (in the near and far ultraviolet), fluorescence and ultraviolet spectroscopy experiments were carried out. Because the structure of the water-soluble form of this fragment has been determined by X-ray crystallography, these spectroscopic methods provided valuable information on the secondary structure and the environment of aromatic residues within the two forms of the peptide. These results strongly suggest that the pore-forming domain of colicin A does not undergo drastic unfolding upon insertion into membrane. The conformational change associated with this process is triggered by the negatively charged lipids and probably consists of a reorientation of helix pairs with respect to each other. Exposure of the aromatic residues to the aqueous phase decreases on binding to lipids whilst the exposure of the tryptophans to the membrane phase increases. This cannot occur without a reorientation of helices 3-10. All data from this study support the model presented previously in which the known crystal structure opens like an 'umbrella' inserting the hydrophobic hairpin (helix 8-9) perpendicular to the membrane plane and the helical pair 1-2 and the domain containing the three tryptophans (helices 3-7) lying more or less parallel to the membrane plane. Lipids are bound more tightly to the protein at acidic pH than at neutral pH although a similar lipid protein complex is formed with 1,2-dimyristoyl-sn-glycero(3)-phospho(1)- -sn-glycerol at both pH values.
Subject(s)
Colicins/chemistry , Membrane Proteins/chemistry , Circular Dichroism , Fluorescence Polarization , Hydrogen-Ion Concentration , Models, Molecular , Protein Conformation , Spectrum Analysis , Tryptophan , TyrosineABSTRACT
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.
Subject(s)
Bee Venoms/pharmacology , Fluorescence Polarization , Melitten/pharmacology , Phospholipids , Spectrum Analysis, Raman , 1,2-Dipalmitoylphosphatidylcholine , Diphenylhexatriene , Phosphatidylcholines , Phosphatidylglycerols , Temperature , ThermodynamicsABSTRACT
Melittin is known to self-associate as tetramers in solutions of high ionic strength. Here, an N-bromosuccinimide oxidized-Trp19 melittin is prepared. This derivative can act as an acceptor of the fluorescence of native melittin and is used in order to observe a possible self-association of melittin in phospholipid bilayers. Resonance energy transfer was shown to occur in solutions of high ionic strength, showing that oxidized melittin can associate with native melittin. In phospholipid bilayers, no association is detected in the absence of NaCl. In its presence, an equilibrium between monomeric melittin and oligomeric species is observed. These species are not dimers, but any other degree of association may account for our experimental results. Significant differences in characteristic transfer efficiency reveal differences in the structure of these oligomers according to the length or state of phospholipids (fluid or at the transition temperature). These bound complexes are also different from the soluble hetero-oligomer. Some models of bound complexes are proposed which may explain the leakage and the further disruption of vesicles or cells induced by melittin.
Subject(s)
Bee Venoms , Lipid Bilayers , Melitten , Phosphatidylcholines , Phosphatidylglycerols , 1,2-Dipalmitoylphosphatidylcholine , Bromosuccinimide , Energy Transfer , Macromolecular Substances , Models, Molecular , Molecular Conformation , Protein Conformation , Spectrometry, Fluorescence , SpectrophotometryABSTRACT
Frequency-domain fluorometry was used to investigate the intensity and anisotropy decays of diphenylhexatriene (DPH) in melittin-lipid complexes. Simulated and experimental data indicate that correlation times ranging from 0.3 to 500 ns can be determined using data from 1 to 200 MHz. For the melittin-lipid complexes the hindered rotator model was not adequate to account for the anisotropy decays, especially at temperatures above the transition temperatures. At high protein-to-lipid ratios the data revealed the formation of small particles (100 A) of melittin and dipalmitoylphosphatidylcholine and the disruption of membrane order in bilayers of dipalmitoylphosphatidic acid.
Subject(s)
Bee Venoms/metabolism , Diphenylhexatriene/metabolism , Melitten/metabolism , Phospholipids/metabolism , Polyenes/metabolism , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Fluorescence , Fluorometry , Lipid Bilayers/metabolismABSTRACT
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.
Subject(s)
Bee Venoms , Melitten , Phosphatidylcholines , Biophysical Phenomena , Biophysics , Chromatography, Gel , Freeze Fracturing , Light , Lipid Bilayers , Membrane Fusion , Membrane Lipids , Membrane Proteins , Microscopy, Electron , Models, Biological , Scattering, RadiationABSTRACT
Factors II, X and IX are blood-clotting proteins which bind to phospholipid interfaces in the presence of Ca2+ to activate coagulation. The topology of their binding site on the membrane was investigated in two ways. First, the transition temperature changes of equimolar mixtures of dipalmitoylglycerophosphocholine/phosphatidylserine and dimyristoylglycerophosphocholine/dipalmitoylglycerophosphoserin e were examined by the fluorescence polarization of 1,6-diphenylhexatriene. Results show that Ca2+ triggers a shift of about 3-4 degrees C and that blood-clotting factors further increase this shift by about 1.5 degree C. This suggests that in the gel phase, Ca2+ induces some aggregation of the phosphatidylserine molecules which is reinforced by blood proteins. Second, isothermal energy transfer experiments were performed with natural lipids in their fluid phase. The tryptophan residues of the factors were the energy donors, and pyrene covalently bound to a fatty acid chain of either phosphatidylcholine or phosphatidic acid was the energy acceptor. These pyrene-phospholipids probe either the neutral or the acidic component of phospholipid mixtures. It is concluded that the binding sites of the factors are constituted by both types of lipids and that their composition depends on the membrane. Factor II exhibits some specificity for acidic phospholipids and seems to be surrounded by non-interacting zwitterionic lipids. Factor IX appears to be surrounded by statistically the same amount of charged and zwitterionic lipids. We also demonstrate that binding can also occur without Ca2+. This Ca2+-independent binding probably involves electrostatic and hydrophobic forces but its physiological significance remains to be elucidated.
Subject(s)
Blood Coagulation Factors/metabolism , Membrane Lipids/blood , Phospholipids/blood , Binding Sites , Calcium/physiology , Diphenylhexatriene , Energy Transfer , Factor IX/metabolism , Factor X/metabolism , Fluorescence Polarization , Humans , Membrane Fluidity , Prothrombin/metabolism , Spectrometry, Fluorescence , TemperatureABSTRACT
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.
Subject(s)
Bee Venoms , Dimyristoylphosphatidylcholine , Melitten , Chemical Phenomena , Chemistry, Physical , Fluorescence Polarization , Hot Temperature , Macromolecular Substances , Phospholipases A/metabolism , Protein Conformation , Spectrometry, FluorescenceABSTRACT
The effects of a Naja mossambica mossambica cardiotoxin on the thermotropic properties of charged phospholipids have been studied by fluorescence polarization, differential scanning calorimetry, and Raman spectroscopy. The binding of the toxin is only governed by the net charge at the interface and is not affected by the polar head group structure of the phospholipids or by the acyl chains physical state, degree of insaturation, or length. The effect of the toxin on the phospholipid structure is drastic. In all cases, the gel to liquid-crystalline phase transition monitored by fluorescence and Raman spectroscopies is progressively abolished without notable shift in temperature as the proportion of toxin is increased. The endothermic peaks detected by differential scanning calorimetry decrease in intensity as the toxin content is increased but always remain sharp. All the techniques used give complementary results, and none of them reveals the presence of secondary transitions at higher or lower temperatures. We thus believe that the lipid molecules that are perturbed by the toxin, approximately 10 +/- 2 molecules, do not undergo a phase transition. Raman results demonstrate that these "boundary" lipids display a population of gauche rotamers that is as high as the one found in the liquid-crystalline phase of the pure phospholipid and this even well below the phase transition temperature. On the other hand, fluorescence results are interpreted as due to a partial immobilization of the lipids in contact with the toxin above the transition temperature. Thus, even though the interaction is governed by electrostatic forces, the toxin penetrates at least partially into the bilayers, inducing a disorganization of the aliphatic chains and changes in their mobility; this could explain their lytic activity.
Subject(s)
Cobra Cardiotoxin Proteins , Elapid Venoms , Liposomes , Phospholipids , Animals , Calorimetry, Differential Scanning , Kinetics , Molecular Conformation , Spectrometry, Fluorescence , Spectrum Analysis, Raman , Structure-Activity Relationship , TemperatureABSTRACT
Cardiotoxins are small basic proteins (7 000 daltons) that are found in the venoms of Elapidae snakes. Although they are structurally close to alpha-neurotoxins present in the same secretions, their activity is related to their ability to interact with every cell membrane inducing, near micromolar concentration, the modification of its biological properties and/or physical structure. The mode of action of cardiotoxins, on a molecular level, is still under investigation. However, lipid-protein interactions are more and more involved in their binding to membrane and in their activities. Using new experimental data a better definition of phospholipid-cardiotoxin interaction is arrived at and a tentative molecular explanation of the pharmacological activities of these proteins is presented and discussed.
Subject(s)
Cobra Cardiotoxin Proteins/toxicity , Elapid Venoms/toxicity , Phospholipids/metabolism , Amino Acid Sequence , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cobra Cardiotoxin Proteins/isolation & purification , Elapid Venoms/metabolism , Iodine , Peptide HydrolasesABSTRACT
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.
Subject(s)
Bee Venoms , Melitten , Phospholipids , Kinetics , Molecular Conformation , Osmolar Concentration , Protein Binding , Protein Conformation , Spectrometry, Fluorescence , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Bee Venoms , Melitten , Membrane Fluidity/drug effects , Phospholipids , Anions , Bee Venoms/pharmacology , Calorimetry , Fluorescence Polarization , Lipid Bilayers , Melitten/pharmacology , Phosphatidic Acids , Phosphatidylglycerols , Phosphatidylserines , Structure-Activity RelationshipABSTRACT
Four cardiotoxins (CTX I-IV) from Naja mossambica mossambica were compared for their ability to interact with phospholipid vesicles and their capacity to bind erythrocytes. It is concluded that the affinity of the toxins always increases in the order: I approximately equal to II less than III less than IV. The binding is specific for charged lipids even in lipid mixtures. Proteolytic attack of the free and lipid-bound cardiotoxin indicates that at least the first loop Leu1-Thr13 is at the lipid contact. Tryptic and synthetic peptides constitutive of this loop are shown to interact with lipids. Arg5 residue increases the affinity toward the bilayer. The Raman spectra of lipid-bound cardiotoxin indicate a secondary and tertiary structure mainly similar to that of the free toxin. On charged lipids cardiotoxins induce a decrease of the enthalpy and an increase of disorder without change in the transition temperature; at saturating amounts of toxin the transition is abolished. In binary mixtures of phosphatidylcholine and charged lipids the observed effects can be accounted by a phase separation induced by the toxin.
Subject(s)
Cobra Cardiotoxin Proteins , Elapid Venoms , Phospholipids , Amino Acid Sequence , Erythrocyte Membrane/metabolism , Kinetics , Liposomes , Neurotoxins , Spectrometry, FluorescenceABSTRACT
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.