A temperature-dependent inhibitory activity of serum on the capacity of Saccharomyces cerevisiae-derived hepatitis B surface antigen to bind to monocytes.

Hepatitis B surface antigen, when produced in yeast (rHBsAg), is capable of binding to cells that express the lipopolysaccharide coreceptor CD14. This interaction is enhanced by a serum protein, the lipopolysaccharide binding protein (LBP). Here we report that most of the rHBsAg particles that attached to monocytes at 0 degrees C, were not endocytosed but were released back into the serum-containing binding buffer at 37 degrees C. Additionally, serum-dependent binding at 37 degrees C was weak when compared to the serum-dependent attachment at 0 degrees C. Pre-incubation at 37 degrees C of cells together with serum did not abolish binding of freshly added rHBsAg at 0 degrees C. However, pre-incubation of rHBsAg with serum at 37 degrees C reduced attachment to cells following incubation at 0 degrees C. Soluble CD14 and LBP, two serum proteins which can act as phospholipid transfer molecules, were shown not to be responsible for the inhibitory effect. Pre-incubation at 37 degrees C of rHBsAg in serum-free hepatoma cell line-conditioned media resulted in a pronounced reduction in subsequent binding to cells at 0 degrees C. These observations suggest that the temperature-dependent inhibitory effect is caused by serum factors that are probably secreted by hepatocytes.

Probing the channel-bound shaker B inactivating peptide by stereoisomeric substitution at a strategic tyrosine residue.

A synthetic peptide patterned after the sequence of the inactivating ball domain of the Shaker B K(+) channel, the ShB peptide, fully restores fast inactivation in the deletion Shaker BDelta6-46 K(+) channel, which lacks the constitutive ball domains. On the contrary, a similar peptide in which tyrosine 8 is substituted by the secondary structure-disrupting d-tyrosine stereoisomer does not. This suggests that the stereoisomeric substitution prevents the peptide from adopting a structured conformation when bound to the channel during inactivation. Moreover, characteristic in vitro features of the wild-type ShB peptide such as the marked propensity to adopt an intramolecular beta-hairpin structure when challenged by anionic phospholipid vesicles, a model target mimicking features of the inactivation site in the channel protein, or to insert into their hydrophobic bilayers, are lost in the d-tyrosine-containing peptide, whose behavior is practically identical to that of noninactivating peptide mutants. In the absence of high resolution crystallographic data on the inactivated channel/peptide complex, these latter findings suggest that the structured conformation required for the peptide to promote channel inactivation, as referred to above, is likely to be beta-hairpin.

Tyrosine phosphorylation of the inactivating peptide of the shaker B potassium channel: a structural-functional correlate.

A synthetic peptide patterned after the sequence of the inactivating "ball" domain of the Shaker B K(+) channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K(+) channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation-dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.

A protein G fragment from the salmonid viral hemorrhagic septicemia rhabdovirus induces cell-to-cell fusion and membrane phosphatidylserine translocation at low pH.

The fusion-related properties of segments p9, p3, p4, and p9 + p2 surrounding the p2 phospholipid-binding domain of the protein G (pG) of the salmonid rhabdovirus of viral hemorrhagic septicemia (VHS) (Nuñez, E., Fernandez, A. M., Estepa, A., Gonzalez-Ros, J. M., Gavilanes, F., and Coll, J. M. (1998) Virology 243, 322-330; Estepa, A., and Coll, J. M. (1996) Virology 216, 60-70), have been studied at neutral and fusion (low) pH values by using its derived peptides. Cell-to-cell fusion, translocation of phosphatidylserine, and inhibition of fusion of pG-transfected cells defined the p9 + p2 (fragment 11, sequence 56-110) as a fragment with higher specific activity for anionic phospholipid aggregation than the previously reported p2. While fragment 11, p2, and p3 showed interactions with anionic phospholipids, p9 and p4 showed no interactions with any phospholipids. When added to a cell monolayer model at low pH, fragment 11 induced pH-dependent cell-to-cell fusion and translocated phosphatidylserine from the inner to the outer leaflet of the membrane. At low pH and in the presence of anionic phospholipids, fragment 11 showed more than 80% beta-sheet conformation (IR and CD spectroscopies). Finally, anti-fragment 11 antibodies inhibited low pH-dependent pG-transfected cell-to-cell fusion. All of the data support the conclusion that fragment 11 is a primary determinant of some of the viral cell fusion events in VHSV.

Identification of novel cellular proteins that bind to the LC8 dynein light chain using a pepscan technique.

Dynein is a minus end-directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid-1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection.

Structure and interaction with membrane model systems of a peptide derived from the major epitope region of HIV protein gp41: implications on viral fusion mechanism.

The HIV-1 gp41 envelope protein mediates entry of the virus into the target cell by promoting membrane fusion. With a view toward possible new insights into viral fusion mechanisms, we have investigated by infrared, fluorescence, and nuclear magnetic resonance spectroscopies and calorimetry a fragment of 19 amino acids corresponding to the immunodominant region of the gp41 ectodomain, a highly conserved sequence and major epitope. Information on the structure of the peptide both in solution and in the presence of model membranes, its incorporation and location in the phospholipid bilayer, and the modulation of the phase behavior of the membrane has been gathered. Here we demonstrate that the peptide binds and interacts with negatively charged phospholipids, changes its conformation in the presence of a membraneous medium, and induces leakage of vesicle contents as well as a new phospholipid phase. These characteristics might be important for the formation of the fusion-active gp41 core structure, promoting the close apposition of the two viral and target-cell membranes and therefore provoking fusion.

Urea equilibrium unfolding of the major core protein of the retrovirus feline immunodeficiency virus and its tryptophan mutants.

Circular dichroism and fluorescence spectroscopy have been employed to study the urea unfolding mechanism of a recombinant form of the major core protein of feline immunodeficiency virus (FIV-rp24) and its native tryptophan mutants. The equilibrium denaturation curves indicate the existence of two transitions. The first unfolding transition most likely reflects the denaturation of the carboxy-terminal region of FIV-rp24. Consequently, the second transition, where the changes in fluorescence are produced, should reflect the denaturation of the amino-terminal region. If the intermediate observed upon urea denaturation is an on-pathway species, the data described herein can reflect the sequential and independent loss of structure of the two domains that this type of proteins possesses.

Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus.

The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.

Thermally induced conformational changes in horseradish peroxidase.

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme N -->k D where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Two-dimensional crystallization on lipid monolayers and three-dimensional structure of sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus.

Sticholysin II (Stn II), a potent cytolytic protein isolated from the sea anemone Stichodactyla helianthus, has been crystallized on lipid monolayers. With Fourier-based methods, a three-dimensional (3D) model of Stn II, up to a resolution of 15 A, has been determined. The two-sided plane group is p22(1)2, with dimensions a = 98 A, b = 196 A. The 3D model of Stn II displays a Y-shaped structure, slightly flattened, with a small curvature along its longest dimension (51 A). This protein, with a molecular mass of 19. 2 kDa, is one of the smallest structures reconstructed with this methodology. Two-dimensional (2D) crystals of Stn II on phosphatidylcholine monolayers present a unit cell with two tetrameric motifs, with the monomers in two different orientations: one with its longest dimension lying on the crystal plane and the other with this same axis leaning at an angle of approximately 60 degrees with the crystal plane.

Compact residual structure in lentil lectin at pH 2.

Lentil lectin obtained from Lens culinaris collected in the La Armuña area (Salamanca, Spain) was examined by high-sensitivity differential scanning calorimetry, fluorimetry and measurements of circular dichroism at pH 2.0 and 7.4. At pH 2.0 the lentil lectin is not in the native state; however, at this pH it does show signs of a residual structure that breaks down upon heating. The lentil lectin at pH 2 shares some similarities with what has become known as the molten globule state. The thermal denaturation of intact (pH 7.4) and partially unfolded (pH 2.0) lentil lectin was irreversible and strongly dependent upon the scan rate, suggesting that its denaturation is under kinetic control. The process of lentil lectin denaturation is interpreted in terms of the simple kinetic model, Nk --> D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state.

Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide.

A peptide corresponding to the N-terminal region of the S protein of hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously demonstrated to perform aggregation and destabilization of acidic liposome bilayers and to adopt a highly stable beta-sheet conformation in the presence of phospholipids. The changes in the lipid moiety produced by this peptide have been followed by fluorescence depolarization and electron microscopy. The later was employed to determine the size and shape of the peptide-vesicle complexes, showing the presence of highly aggregated and fused structures only when negatively charged liposomes were employed. 1,6-Diphenyl-1,3,5-hexatriene depolarization measurements showed that the interaction of the peptide with both negatively charged and zwitterionic liposomes was accompanied by a substantial reduction of the transition amplitude without affecting the temperature of the gel-to-liquid crystalline phase transition. These data are indicative of the peptide insertion inside the bilayer of both types of liposomes affecting the acyl chain order, though only the interaction with acidic phospholipids leads to aggregation and fusion. This preferential destabilization of the peptide towards negatively charged phospholipids can be ascribed to the electrostatic interactions between the peptide and the polar head groups, as monitored by 1-(4-(trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene fluorescence depolarization analysis.

Circular dichroism and fluorescence spectroscopic properties of the major core protein of feline immunodeficiency virus and its tryptophan mutants. Assignment of the individual contribution of the aromatic sidechains.

The gene coding for the major capsid protein of feline immunodeficiency virus (FIV) has been cloned into the expression vector pQE60, which allows protein purification by affinity chromatography on a nitrilotriacetic acid/Ni/agarose column. The gene was expressed in Escherichia coli and the resultant soluble protein (FIV-rp24) purified to electrophoretic homogeneity. The amino-acid composition of the recombinant protein is almost identical to that predicted from the DNA sequence. This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site-directed mutagenesis to obtain two single mutants and a double mutant. Circular dichroism and fluorescence spectroscopy were employed to study the structural features of FIV-rp24 protein and its tryptophan mutants. The analysis of the CD spectra indicated that alpha-helix is the major secondary structural element (48-52%) and that the overall three-dimensional structure is not modified by the mutations. The fluorescence emission spectra showed that both tryptophan residues occupy a highly hydrophobic environment. Moreover, the different tyrosine fluorescence intensities of wild-type and mutant proteins are indicative of the existence of resonance energy transfer processes to nearby tryptophan. The individual contributions of each tryptophan residue to the spectroscopic properties of the wild-type protein were obtained from the spectra of all these proteins. Thermal denaturation studies indicate that the two tryptophan residues do not contribute equally to the stabilization of the three-dimensional structure.

Fusogenic activity of hepadnavirus peptides corresponding to sequences downstream of the putative cleavage site.

Sequence homology between the amino-terminal region of the S protein of hepatitis B Virus (HBV) and known fusion peptides from retroviruses and paramyxoviruses led us to propose that this region might be equally involved in the initial infective steps of hepadnaviruses. In fact, we showed that a synthetic peptide corresponding to the N-terminus region of the S protein of HBV had membrane-interacting properties and was able to induce liposome fusion adopting an extended (beta-sheet) conformation (Rodríguez-Crespo et al., 1996, 1995). We describe herein studies on the interaction of peptides derived from the N-terminal region of the S protein of duck (DHBV: Met-Ser-Gly-Thr-Phe-Gly-Gly-Ile-Leu-Ala-Gly-Leu-Ile-Gly-Leu-Leu) and woodchuck hepatitis B viruses (WHV: Met-Ser-Pro-Ser-Ser-Leu-Leu-Gly-Leu-Leu-Ala-Gly-Leu-Gln-Val-Val) with liposomes. These peptides were able to induce to a different extent aggregation, lipid mixing, and leakage of internal aqueous contents from both neutral and negatively charged phospholipid vesicles in a concentration-dependent and pH-independent manner. Fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene-labeled vesicles indicated that both peptides become inserted into the hydrophobic core of the lipid bilayer. Circular dichroism studies indicated that the DHBV peptide adopts an extended conformation in the presence of lipids, whereas the WHV peptide displays a high content of alpha-helical conformation. Therefore, these results extend our previous findings obtained for human hepatitis B virus to other members of the hepadnavirus family and suggest that this region of the S protein is important in the initial steps of the infective cycle.

Binding of dynein light chain (PIN) to neuronal nitric oxide synthase in the absence of inhibition.

PIN, an 89-amino-acid polypeptide found in a rat hippocampal cDNA library using the yeast two-hybrid system and various neuronal nitric oxide synthase (nNOS) fragments as bait, was reported to be an inhibitor of nNOS (Science 274, 774-778, 1996). PIN reportedly inhibited nNOS selectively and did not interact with either the endothelial or inducible nitric oxide synthase isoforms. Inhibition was attributed to the ability of PIN to dissociate the catalytically active nNOS homodimer. PIN is a dynein light chain (J. Biol. Chem. 271, 19358-19366, 1996), which suggested that PIN may serve as an axonal transport protein for nNOS. We have synthesized a rat PIN cDNA by recursive polymerase chain reaction and have expressed the protein in Escherichia coli. Recombinant PIN is a folded dimeric, mostly alpha-helical protein with a single deeply buried tryptophan residue. We have also expressed and purified the nNOS fragment to which PIN reportedly binds (residues 163-245). This recombinant peptide has a disordered secondary structure. Gel-filtration experiments show that PIN binds to both the full-length nNOS and nNOS fragment. However, PIN neither inhibits nNOS activity nor dissociates the nNOS dimer into monomeric species. PIN thus possibly functions as a dynein light chain involved in nNOS axonal transport but is not an inhibitor of the enzyme. Our results agree with the proposal (Cell 82, 743-752, 1995) that the PIN recognition sequence in nNOS both lies outside the catalytic core and is not part of the monomer-monomer contact region.

Inactivating peptide of the Shaker B potassium channel: conformational preferences inferred from studies on simple model systems.

Previous studies on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide, H2N-MAAVAGLYGLGEDRQHRKKQ) and anionic phospholipid vesicles, used as model targets, have shown that the ShB peptide: (i) binds to the vesicle surface with high affinity; (ii) readily adopts a strongly hydrogen-bonded beta-structure; and (iii) becomes inserted into the hydrophobic bilayer. We now report fluorescence studies showing that the vesicle-inserted ShB peptide is in a monomeric form and, therefore, the observed beta-structure must be intramolecularly hydrogen-bonded to produce a beta-hairpin conformation. Also, additional freeze-fracture and accessibility-to-trypsin studies, which aimed to estimate how deeply and in which orientation the folded monomeric peptide inserts into the model target, have allowed us to build structural models for the target-inserted peptide. In such models, the peptide has been folded near G6 to configure a long beta-hairpin modelled to produce an internal cancellation of net charges in the stretch comprising amino acids 1-16. As to the positively charged C-terminal portion of the ShB peptide (RKKQ), this has been modelled to be in parallel with the anionic membrane surface to facilitate electrostatic interactions. Since the negatively charged surface and the hydrophobic domains in the model vesicle target may partly imitate those present at the inactivation 'entrance' in the channel protein [Kukuljan, M., Labarca, P. and Latorre, R. (1995) Am. J. Physiol. Cell Physiol. 268, C535-C556], we believe that the structural models postulated here for the vesicle-inserted peptide could help to understand how the ShB peptide associates with the channel during inactivation and why mutations at specific sites in the ShB peptide sequence, such as that in the ShB-L7E peptide, result in non-inactivating peptide variants.

Phospholipid interactions of a peptide from the fusion-related domain of the glycoprotein of VHSV, a fish rhabdovirus.

Previous studies mapped a p2 domain (aa 82-109) which binds phosphatidylserine (PS) (Estepa and Coll, 1996a) and contains three contiguous hydrophobic amino acid heptad repeats followed by a positively charged stretch (Coll, 1995b) in the glycoprotein G of the viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. Anti-p2 antibodies inhibited low-pH VHSV-induced fusion (Estepa and Coll, 1997) and low-pH PS binding to VHSV (Estepa and Coll, 1996a). We report here further studies on the interaction of the synthetic peptide p2 with phospholipid vesicles. The synthetic p2 peptide was able to mediate aggregation, lipid mixing, and leakage of contents only with negatively charged phospholipid vesicles and in a concentration-dependent manner. As shown by its effect on lipid phase transitions deduced from data with fluorescence polarization and differential scanning calorimetry, the p2 peptide becomes inserted into the hydrophobic negatively charged phospholipid vesicle bilayers. In addition, data based on circular dichroism showed that the p2 peptide folds as a structure with a high content of beta-sheets stabilized by interaction with anionic phospholipids. These studies are potentially relevant to viral fusion in VHSV.

Incubation of superoxide dismutase with malondialdehyde and 4-hydroxy-2-nonenal forms new active isoforms and adducts. An evaluation of xenobiotics in fish.

The effects in fish (Sparus aurata) of dieldrin, previously reported to be an inducer of peroxisomal enzymes (Pedrajas et al., Comp. Biochem. Physiol. 115C (1996) 125-131), were compared with those of clofibrate. Although dieldrin provoked the more severe peroxisomal changes, both compounds induced oxidative stress as detected by the increased levels of microsomal thiobarbituric acid reactive substances; however the malondialdehyde (MDA) content, determined after HPLC separation of the MDA-TBA complex, was not significantly altered. These results suggest that, besides MDA, other aldehydes were formed in xenobiotic-injected fish, leading us to assess the oxidative effects of such xenobiotics by following changes in superoxide dismutase (SOD) pattern. New active SOD isoforms were detected by isoelectrofocusing in the light mitochondrial (LMF) and cytosolic (CF) fractions. Most of the new SOD bands could be reproduced in vitro by incubation of fish liver cell-free extracts with MDA. To clarify the effects of aldehydes, Cu,Zn- and Mn-SOD isoforms were purified and amino acid analysis was carried out. The new bands found in LMF and CF fractions were reproduced in vitro after incubation of pure SODs with MDA and 4-hydroxy-2-nonenal (HNE), the new SOD bands formed being coincident with the loss of Lys or His residues. Lysine residues were preferentially derivatized after treatment of Cu,Zn-SOD with MDA, but in Mn-SOD the lysine residues were modified only after treatment with MDA, while the histidine residues were modified only by HNE. No change of SOD activity was detected after MDA or HNE exposure, although at the higher aldehyde concentrations used protein aggregates were formed. Therefore, the appearance of new active SOD bands, after isoelectrofocusing separation, can be proposed as a biomarker of oxidative stress.

Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus.

The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that the protein occurs as a monomer in solution. Labeling of free sulphydryl groups with [1-14C]iodoacetamide suggests that none of the three cysteine residues of rp26 is involved in intramolecular disulfide bonds. The circular dichroism spectrum of rp26 was consistent with the following assignment of secondary structure elements: 51% a-helix, 15% beta-turn, and 34% aperiodic. Fluorescencespectroscopy revealed that the three tryptophan residues in rp26 occupy two different environments. These data support the conclusion that the recombinant protein is folded into an ordered and probably native conformation. Immunoblotting and enzyme immunoassay with EIAV infected sera demonstrated that recombinant p26 protein may be useful for diagnostic purposes.

Structural properties of the putative fusion peptide of hepatitis B virus upon interaction with phospholipids. Circular dichroism and Fourier-transform infrared spectroscopy studies.

A peptide corresponding to the N-terminal sequence of the S protein from hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously shown to interact with phospholipids and promote vesicle aggregation, phospholipid mixing, and liposome leakage, as well as erythrocyte lysis [Rodríguez-Crespo, I., Núñez, E., Gómez-Gutiérrez, J., Yélamos, B., Albar, J. P., Peterson, D. L. & Gavilanes, F. (1995) J. Gen. Virol. 76, 301-308]. The conformation of this putative fusion peptide has been studied, both at low and high peptide concentrations, by means of circular dichroism and Fourier-transform infrared spectroscopy, respectively. When the peptide is dissolved in trifluoroethanol, a significant population of alpha-helical structure is found in spite of the proline residue at position 11. In contrast, this hydrophobic oligopeptide has a high tendency to form large beta-sheet aggregates in aqueous buffers. Most of these aggregates can be eliminated by centrifugation. The peptide remaining in the supernatant adopts a non-ordered conformation. The aggregates can be dissociated by the anionic detergent sodium cholate, but the peptide still maintains an extended conformation. In the presence of acidic phospholipid vesicles, the putative fusion peptide adopts a highly stable beta-sheet conformation. Thus, unlike the fusion peptides of other viruses, an extended conformation seems to be the preferred structure when interacting with phospholipids. Such a conformation should be responsible for its membrane destabilization properties.