Biomedical subjects

F M Goñi

Publications and source records attributed to F M Goñi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Ceramides in phospholipid membranes: effects on bilayer stability and transition to nonlamellar phases.

The effects of ceramides of natural origin on the gel-fluid and lamellar-inverted hexagonal phase transitions of phospholipids (mainly dielaidoylphosphatidylethanolamine) have been studied by differential scanning calorimetry, with additional support from infrared and 31P nuclear magnetic resonance (NMR) spectroscopy. In the lamellar phase, ceramides do not mix ideally with phospholipids, giving rise to the coexistence of domains that undergo the gel-fluid transition at different temperatures. The combination of differential scanning calorimetry and infrared spectroscopy, together with the use of deuterated lipids, allows the demonstration of independent melting temperatures for phospholipid and ceramide in the mixtures. In the lamellar-hexagonal phase transitions, ceramides (up to 15 mol %) decrease the transition temperature, without significantly modifying the transition enthalpy, thus facilitating the inverted hexagonal phase formation. 31P-NMR indicates the coexistence, within a certain range of temperatures, of lamellar and hexagonal phases, or hexagonal phase precursors. Ceramides from egg or from bovine brain are very similar in their effects on the lamellar-hexagonal transition. They are also comparable to diacylglycerides in this respect, although ceramides are less potent. These results are relevant in the interpretation of certain forms of interfacial enzyme activation and in the regulation and dynamics of the bilayer structure of cell membranes.

Animals↗

PubMed1999

Interbilayer lipid mixing induced by the human immunodeficiency virus type-1 fusion peptide on large unilamellar vesicles: the nature of the nonlamellar intermediates.

A peptide corresponding to the 23 N-terminal amino acid residues of the human immunodeficiency virus type-1 (HIV-1) gp41 has the capacity to induce intervesicular lipid mixing in large unilamellar liposomes composed of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol (CHOL) (molar ratio, 1:1:1). Cryo-transmission electron microscopy (cryo-TEM) of diluted vesicles to which peptides has been externally added reveals a morphology that is compatible with the formation of nonlamellar lipidic aggregates during the time-course of lipid mixing. 31P-nuclear magnetic resonance and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMADPH) steady-state anisotropy data at equilibrium indicate that the peptide is able to modulate the lipid polymorphism in pelletted membranes by: (i) promoting the thermotropic formation of inverted phases; and (ii) driving the lamellar-to-nonlamellar transition towards the formation of isotropic phases. Therefore, our combined morphological and spectroscopic data reveal the existence of a direct correlation between the ability of the externally added peptide to induce lipid-mixing in dilute liposome samples and its capacity to modulate lipid polymorphism in stacked bilayers.

Amino Acid Sequence↗

PubMed1999

Structure and dynamics of membrane proteins as studied by infrared spectroscopy.

Infrared (IR) spectroscopy is a useful technique in the study of protein conformation and dynamics. The possibilities of the technique become apparent specially when applied to large proteins in turbid suspensions, as is often the case with membrane proteins. The present review describes the applications of IR spectroscopy to the study of membrane proteins, with an emphasis on recent work and on spectra recorded in the transmission mode, rather than using reflectance techniques. Data treatment procedures are discussed, including band analysis and difference spectroscopy methods. A technique for the analysis of protein secondary and tertiary structures that combines band analysis by curve-fitting of original spectra with protein thermal denaturation is described in detail. The assignment of IR protein bands in H2O and in D2O, one of the more difficult points in protein IR spectroscopy, is also reviewed, including some cases of unclear assignments such as loops, beta-hairpins, or 3(10)-helices. The review includes monographic studies of some membrane proteins whose structure and function have been analysed in detail by IR spectroscopy. Special emphasis has been made on the role of subunit III in cytochrome c oxidase structure, and the proton pathways across this molecule, on the topology and functional cycle of sarcoplasmic reticulum Ca(2+)-ATPase, and on the role of lipids in determining the structure of the nicotinic acetylcholine receptor. In addition, shorter descriptions of retinal proteins and references to other membrane proteins that have been studied less extensively are also included.

Bacteriorhodopsins↗

PubMed1999

Structure and functional properties of diacylglycerols in membranes.

1. 1,2-Diacyl-sn-glycerols (DAG) are minor components of cell membranes (about 1 mole% of the lipids) and yet they are potent regulators of both the physical properties of the lipid bilayer and the catalytic behaviour of several membrane-related enzymes. 2. In the pure state DAG's present a considerable polymorphism, with several crystalline phases in addition to the neat fluid phase. The most stable crystalline phase is the so-called beta' phase, a monoclinic crystalline form with orthorhombic perpendicular subcell chain packing, in which both acyl chains lie parallel to each other in a hairpinlike configuration about the sn-1 and sn-2 glycerol carbon atoms. The molecules are organized in a bilayer, with the glycerol backbone roughly parallel to the plane of the bilayer, and the acyl chains tilted at approximately 60 degrees with respect to that plane. Acyl chain unsaturation, and particularly a single cis unsaturation, impairs chain packing in mixed-chain DAG's, and this results in an increased number of metastable crystalline phases. 3. DAG's mix with phospholipids in fluid bilayers when their melting temperature is below or close enough to the melting temperature of the bilayer system. When incorporated in phospholipid bilayers, the conformation of DAG is such that the glycerol backbone is nearly perpendicular to the bilayer, with the sn-1 chain extending from the glycerol Cl carbon into the hydrophobic matrix of the bilayer and the sn-2 chain first extending parallel to the bilayer surface, then making a 90 degrees bend at the position of the sn-1 carbonyl to become parallel to the sn-1 chain. DAG's are located in phospholipid bilayers about two CH2 units deeper than the adjacent phospholipids. DAG's mix nonideally with phospholipids, giving rise to in-plane separations of DAG-rich and -poor domains, even in the fluid state. DAG molecules also increase the separation between phospholipid headgroups, and decrease the hydration of the bilayer surface. Also, because the transversal section of the DAG headgroup is small when compared to that of the acyl chains, DAG favours the (negative) curvature of the lipid monolayers, and DAG-phospholipid mixtures tend to convert into inverted nonlamellar hexagonal or cubic phases. 4. A number of membrane enzyme activities are modulated (activated) by DAG, most notably protein kinase C, phospholipases and other enzymes of lipid metabolism. Protein kinase C activation (and perhaps that of other enzymes as well) occurs as the combined result of a number of DAG-induced modifications of lipid bilayers that include: changes in lipid headgroup conformation, interspacing and hydration, changes in the bilayer propensity to form inverted nonlamellar phases, and lateral phase separations of DAG-rich and -poor domains. Among the DAG-activated enzymes, phospholipases C show the peculiarity of yielding the activator DAG as their reaction product, and this allows the self-induced transition from a low- to a high-activity status. 5. DAG's induce or enhance membrane fusion in a number of ways, mainly through partial dehydration of the bilayer surface, increase in lipid monolayer curvature and perhaps lateral phase separation. DAG-increased fusion rates have been demonstrated in several instances of cation-induced fusion of model membranes, as well as in Ca(2+)-induced fusion of chromaffin granules with plasma membrane vesicles. Also phospholipase C has been shown to induce vesicle aggregation and fusion through the catalytic generation of DAG in the bilayers. A rather general property of DAG is that it promotes vesicular or interparticle aggregation. 6. In the living cell, DAG is often generated through phospholipid degradation in response to an extracellular agonist binding a specific receptor in the cell surface. DAG is said to act as an intracellular second messenger. (ABSTRACT TRUNCATED)

Cell Membrane↗

PubMed1999

Insertion of Escherichia coli alpha-haemolysin in lipid bilayers as a non-transmembrane integral protein: prediction and experiment.

alpha-Haemolysin is an extracellular protein toxin (approximately 107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential. In this work, we have studied the perturbation produced by purified alpha-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy. All the data concur in indicating that alpha-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted alpha-haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic alpha-helices, appropriate for protein-lipid interaction, but no hydrophobic transmembrane helices are predicted in alpha-haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by alpha-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of alpha-haemolysin as a pore-forming toxin.

Amino Acid Sequence↗

PubMed1998

Vesicle membrane fusion induced by the concerted activities of sphingomyelinase and phospholipase C.

When vesicles composed of an equimolar mixture of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with phospholipase C, phospholipid hydrolysis occurs without major changes in vesicle architecture. In the same way, addition of sphingomyelinase leads only to sphingomyelin cleavage. However, when both enzymes are added together, their joint hydrolytic activities give rise to leakage-free vesicle aggregation, lipid mixing, and aqueous contents mixing, i.e. vesicle fusion. The contribution of both enzymes is unequal, the main role of sphingomyelinase being the production of relatively large amounts of ceramide that will facilitate the lamellar-to-nonlamellar transition in the formation of the fusion pore, whereas phospholipase C provides mainly a localized, asymmetric, high concentration of diacylglycerol that constitutes the trigger for the fusion process. The lipidic end-products of both enzymes cooperate in destabilizing and fusing the membranes in a way that is never achieved through the action of any of the enzymes individually, nor by the products themselves when premixed with the other lipids during liposome preparation. Thus the enzymes appear to be coupled through their reaction products. This is the first observation of membrane fusion induced by the concerted activities of two enzymes. Besides, considering that both diacylglycerol and ceramide are important metabolites involved in cell signaling, it may also provide new ideas in the exploration of "cross-talk" phenomena between different signal transduction pathways.

Ceramides↗

PubMed1998

Phospholipase C hydrolysis of phospholipids in bilayers of mixed lipid compositions.

Phosphatidylcholine phospholipase C (EC 3.1.4.3) from Bacillus cereus has been assayed with substrates in the form of large unilamellar vesicles. Phosphatidylcholine, phosphatidylethanolamine (also a substrate for the enzyme), sphingomyelin, and cholesterol have been mixed in various proportions, in binary, ternary, and quaternary mixtures. A lag period, followed by a burst of enzyme activity, has been found in all cases. The activity burst was always accompanied by an increase in turbidity of the vesicle suspension. Varying lipid compositions while keeping constant all the other parameters leads to a range of lag times extending over 2 orders of magnitude (from 0.13 to 38.0 min), and a similar variability is found in maximal enzyme rates (from 0.40 to 55.9 min-1). Meanwhile, the proportion of substrate that is hydrolyzed during the lag period remains relatively constant at 0.10% moles of total lipid, in agreement with the idea that enzyme activation is linked to vesicle aggregation through diacylglycerol-rich patches. Phosphatidylethanolamine and cholesterol enhance the enzyme activity in a dose-dependent way: they reduce the lag times and increase the maximal rates. The opposite is true of sphingomyelin. These lipids exert each its own peculiar effect, positive or negative, either alone or in combination, so that the susceptibility of a given mixture to the enzyme activity can be to some extent predicted from its composition. Phospholipase C activity is not directly influenced by the formation of nonlamellar structures. However, the presence of lipids with a tendency to form nonlamellar phases, such as phosphatidylethanolamine or cholesterol, stimulates the enzyme even under conditions at which purely lamellar phases exist. Conversely sphingomyelin, a well-known stabilizer of the lamellar phase, inhibits the enzyme. Thus phospholipase C appears to be regulated by the overall geometry and composition of the bilayer.

Bacillus cereus↗

PubMed1998

Detergent solubilisation of phospholipid bilayers in the gel state: the role of polar and hydrophobic forces.

Testing the solubilisation of phosphatidylcholine (PC) bilayers by Triton X-100 reveals that in the gel state, but not in the fluid state, the amount of detergent required to solubilise the phospholipid is highly dependent on the chain length. Saturated C16 and C18 PC are virtually insoluble at 4 degreesC. However, addition of water-soluble reagents that perturb hydrogen bonding, e.g. urea, or of small proportions of non-bilayer lipids, make the bilayers amenable to detergent solubilisation, even at low temperatures. These results are relevant in the explanation of the origin of detergent-resistant membrane fragments as found, e.g. in caveolae or 'rafts'.

Detergents↗

PubMed1998

Phospholipase cleavage of glycosylphosphatidylinositol reconstituted in liposomal membranes.

Glycosylphosphatidylinositol (GPI) purified from rat liver lipids was incorporated into lipid bilayers of defined compositions, in the form of large unilamellar vesicles. The GPI concentration in the bilayers was kept constant at 25 mole%, whereas the remaining lipids being phosphatidylcholine, phosphastidylethanolamine, sphingomyelin and/or cholesterol were varied. The resulting liposomes consisted of spherical vesicles, approximately 100 nm in diameter, that could keep their aqueous contents separated from the extravesicular medium. When these liposomes were treated with either Bacillus cereus phosphatidylinositol-phospholipase C, Trypanosoma brucei GPI-phospholipase C, or bovine serum GPI-phospholipase D, GPI was hydrolyzed at different rates, depending on the enzyme and the bilayer lipid composition. These observations open the way to biophysical and biochemical studies of enzymic GPI cleavage under defined conditions. Extensive GPI hydrolysis was observed in certain cases that could allow the use of these systems for the preparation of inositol phosphoglycans, proposed second messengers of a wide variety of hormones, cytokines and growth factors.

Animals↗

PubMed1998

Reversible denaturation, self-aggregation, and membrane activity of Escherichia coli alpha-hemolysin, a protein stable in 6 M urea.

Escherichia coli alpha-hemolysin (HlyA) is an extracellular protein toxin (107 kDa) whose cell lytic activity may be preserved for months at -20 degreesC in the presence of 6 M urea, although it decays rapidly in urea-free buffers. This paper describes experiments addressed to unravel the role of urea in HlyA stabilization. Urea up to 8 M inhibits the Ca2+-binding and hemolytic activities of the protein, alters its secondary and tertiary structures, and reduces its tendency to self-aggregation. All these changes are largely reversed upon urea removal by dilution or dialysis, suggesting that they are interrelated. Furthermore, the extent of recovery of the native activities and structural features of alpha-hemolysin that follows urea removal increases with the concentration of urea during the previous phase. Thus, it seems that urea elicits the reversible transition of HlyA to a less active but more stable state whose structure differs significantly from that of the native protein. Moreover dialysis equilibration of the protein with buffers containing 3 M urea induces the formation of a molecular form of HlyA 5-10 times more active than the native protein in the absence of urea. This hyperactive intermediate appears to keep the native secondary structure of HlyA, but with a less compact tertiary structure, that increases the number of exchangeable Ca2+ ions under these conditions. Changes in the intrinsic fluorescence of HlyA also support the notion of a conformational change in the high-activity intermediate. The intermediate is only detected when assayed in the presence of Ca2+ and 3 M urea and can bind a large number of calcium ions (approximately 12 vs approximately 3 for the native protein); it shows a large tendency to self-aggregation and presumably, in the presence of membranes, a similar tendency to irreversible insertion, which may be the reason for its high lytic activity.

Amino Acid Sequence↗

PubMed1998

Effect of single chain lipids on phospholipase C-promoted vesicle fusion. A test for the stalk hypothesis of membrane fusion.

The effect of low proportions (up to 5 mol %) of single-chain lipids on phospholipase C-promoted fusion of large unilamellar vesicles has been investigated with the aim of testing the so-called stalk model of membrane fusion. This model is known in two main versions, the one originally published by Kozlov and Markin [Kozlov, M. M. and Markin, V. S. (1983) Biofizika 28, 255-261] and what is known as the "modified stalk model" [Siegel, D. P. (1993) Biophys. J. 65, 2124-2140], that differ in a number of predictions. In the view of the latter author, hydrocarbons or other nonpolar lipids should help fusion by decreasing the interstitial energy of the stalk connecting the two apposed bilayers. We show that small amounts of hexadecane or squalene increase significantly the fusion rates in our system. Changes in monolayer curvature are the object of different predictions by the original and modified stalk theories. According to the original form, fusion would be promoted by lipids inducing a negative curvature in the closest (cis) monolayers of the fusing membranes and inhibited by the same lipids in the trans monolayers; the opposite would happen with lipids inducing a positive curvature. The modified stalk model predicts that fusion is helped by increasing the negative curvature of both monolayers. In our system, symmetrically distributed arachidonic acid, which increases the negative curvature, enhances lipid and content mixing, and the opposite is found with symmetrically distributed lysophosphatidylcholine or palmitoylcarnitine, which facilitate a positive monolayer curvature. In addition, fluorescence polarization and 31P NMR studies of the lamellar-to-isotropic (Q224 cubic) thermotropic transition of a lipid mixture corresponding to our liposomal composition reveal that all lipids that facilitate fusion decrease the transition temperature, while fusion inhibitors increase the transition temperature. Moreover, fusion (content mixing) rates show a maximum at the lamellar-to-isotropic transition temperature. These observations support the involvement of inverted lipid structures, as occurring in the inverted cubic phases, in membrane fusion. All these data are in full agreement with the stalk model of membrane fusion, particularly in its modified version.

Alkanes↗

PubMed1998

Calcium-dependent conformation of E. coli alpha-haemolysin. Implications for the mechanism of membrane insertion and lysis.

Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.

Bacterial Proteins↗

PubMed1998

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.

Animals↗

PubMed1998

Interaction of wheat alpha-thionin with large unilamellar vesicles.

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.

Antimicrobial Cationic Peptides↗

PubMed1998

Infrared studies of protein-induced perturbation of lipids in lipoproteins and membranes.

The paper reviews the main recent publications concerning infrared (IR) spectroscopy as applied to the study of lipid-protein interactions in model and cell membranes, lipoproteins, and related systems (e.g. lung surfactant). The review focuses mainly on transmission IR. Based on the available data, a number of general conclusions are presented on the perturbations caused by proteins on either the hydrocarbon chains, the polar headgroups or the interface region. Lipid-protein interactions in native cell membranes do not reveal significant differences from what is observed in semisynthetic model systems.

Animals↗

PubMed1998

Interfacial enzyme activation, non-lamellar phase formation and membrane fusion. Is there a conducting thread?

Previous studies from this laboratory have shown that the enzymic generation of diacylglycerol in bilayers by phospholipase C may lead to membrane fusion through the formation of transient non-lamellar lipidic intermediates. The present paper intends to explore the correlations existing among the three main processes involved, namely (a) the induction (or inhibition) of lamellar-to-non-lamellar phase transitions in lipid mixtures through the addition of small (< 5 mol%) proportions of other lipids, (b) the promotion, by the latter lipids, of fusion in otherwise stable phospholipid vesicles (large unilamellar liposomes) under conditions leading to inverted hexagonal/inverted cubic phase formation in bulk lipid systems, and (c) the modulation, by the same small proportions of lipids, of phospholipase C hydrolysis of phosphatidylcholine in liposome bilayers. It is concluded that phospholipase C may give rise to non-lamellar lipidic structures that in turn permit liposomal fusion to occur, but neither enzyme activity is directly modulated by non-lamellar phase formation, nor will whatever kind of enzyme-induced non-lamellar structure give rise to fusion. Moreover, only under certain kinetic conditions will the enzyme give rise to the organization of non-lamellar structures that are conducive to the fusion event.

Animals↗

PubMed1998

Phosphatidylinositol-dependent membrane fusion induced by a putative fusogenic sequence of Ebola virus.

The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBO(GE) (GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBO(GE) was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBO(EA) (EGAAIGLAWIPYFGPAA) and EBO(EE) (EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBO(GE) except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBO(GE) sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBO(GE) peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.

Amino Acid Sequence↗

PubMed1998

E. coli alpha-hemolysin: a membrane-active protein toxin.

alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca(2+)-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can alpha-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for alpha-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Bacterial Proteins↗