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• W2074366880 abstract "The simian immunodeficiency virus fusion peptide constitutes a 12-residue N-terminal segment of the gp32 protein that is involved in the fusion between the viral and cellular membranes, facilitating the penetration of the virus in the host cell. Simian immunodeficiency virus fusion peptide is a hydrophobic peptide that in Me2SO forms aggregates that contain β-sheet pleated structures. When added to aqueous media the peptide forms large colloidal aggregates. In the presence of lipidic membranes, however, the peptide interacts with the membranes and causes small changes of the membrane electrostatic potential as shown by fluorescein phosphatidylethanolamine fluorescence. Thioflavin T fluorescence and Fourier transformed infrared spectroscopy measurements reveal that the interaction of the peptide with the membrane bilayer results in complete disassembly of the aggregates originating from an Me2SO stock solution. Above a lipid/peptide ratio of about 5, the membrane disaggregation and water precipitation processes become dependent on the absolute peptide concentration rather than on the lipid/peptide ratio. A schematic mechanism is proposed, which sheds light on how peptide-peptide interactions can be favored with respect to peptide-lipid interactions at various lipid/peptide ratios. These studies are augmented by the use of the fluorescent dye 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine that shows the interaction of the peptide with the membranes has a clear effect on the magnitude of the so-called dipole potential that arises from dipolar groups located on the lipid molecules and oriented water molecules at the membrane-water interface. It is shown that the variation of the membrane dipole potential affects the extent of the membrane fusion caused by the peptide and implicates the dipolar properties of membranes in their fusion. The simian immunodeficiency virus fusion peptide constitutes a 12-residue N-terminal segment of the gp32 protein that is involved in the fusion between the viral and cellular membranes, facilitating the penetration of the virus in the host cell. Simian immunodeficiency virus fusion peptide is a hydrophobic peptide that in Me2SO forms aggregates that contain β-sheet pleated structures. When added to aqueous media the peptide forms large colloidal aggregates. In the presence of lipidic membranes, however, the peptide interacts with the membranes and causes small changes of the membrane electrostatic potential as shown by fluorescein phosphatidylethanolamine fluorescence. Thioflavin T fluorescence and Fourier transformed infrared spectroscopy measurements reveal that the interaction of the peptide with the membrane bilayer results in complete disassembly of the aggregates originating from an Me2SO stock solution. Above a lipid/peptide ratio of about 5, the membrane disaggregation and water precipitation processes become dependent on the absolute peptide concentration rather than on the lipid/peptide ratio. A schematic mechanism is proposed, which sheds light on how peptide-peptide interactions can be favored with respect to peptide-lipid interactions at various lipid/peptide ratios. These studies are augmented by the use of the fluorescent dye 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine that shows the interaction of the peptide with the membranes has a clear effect on the magnitude of the so-called dipole potential that arises from dipolar groups located on the lipid molecules and oriented water molecules at the membrane-water interface. It is shown that the variation of the membrane dipole potential affects the extent of the membrane fusion caused by the peptide and implicates the dipolar properties of membranes in their fusion. Simian immunodeficiency virus (SIV) 1The abbreviations used are: SIVsimian immunodeficiency virusSIVwtSIV fusion peptideHIVhuman immunodeficiency virusFPEfluorescein phosphatidylethanolaminedi-8-ANEPPS1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaineThTthioflavin TgpglycoproteinPEphosphatidylethanolaminePCphosphatidylcholineLUVlarge unilamellar vesiclesATRattenuated total reflectanceFTIRFourier transform infrared spectrometryFRETfluorescence resonance energy transferKC6-keto-cholestanolNBD12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diaz-4-ol-yl)). entry in target cells is mediated by the viral envelope glycoproteins, designated gp120 and gp32, which are derived by proteolytic cleavage of the gp160 precursor. SIV gp120 and gp32 play an equivalent role to that of gp120 and gp41 in the human immunodeficiency virus, type 1 (HIV-1), which has a structure and biological properties very similar to SIV (1Bosch M.L. Earl P.L. Fargnoli K. Picciafuoco S. Giombini F. Wong-Staal F. Franchini G. Science. 1989; 244: 694-697Crossref PubMed Scopus (194) Google Scholar, 2Kowalski M. Potz J. Basiripour L. Dotfman T. Chun Goh W. Terwilliger E. Dayton A. Rosen C. Haseltine W. Sodroski J. Science. 1987; 237: 1351-1355Crossref PubMed Scopus (613) Google Scholar, 3Lasky L.A. Nakamura G. Smith D. Fennie C. Shimlasaki C. Patzer E. Berman T. Gregory T. Capon D. Cell. 1987; 50: 975-985Abstract Full Text PDF PubMed Scopus (606) Google Scholar). gp32 and gp41 appear to possess one transmembrane domain and are thought to exhibit a multi-role function that involves anchoring the envelope glycoprotein complex to the viral membrane, oligomerization of the envelope glycoprotein, and for the putative membrane fusion between viral and cell membranes. simian immunodeficiency virus SIV fusion peptide human immunodeficiency virus fluorescein phosphatidylethanolamine 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine thioflavin T glycoprotein phosphatidylethanolamine phosphatidylcholine large unilamellar vesicles attenuated total reflectance Fourier transform infrared spectrometry fluorescence resonance energy transfer 6-keto-cholestanol 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diaz-4-ol-yl)). During the entry of the virus into the target cell, gp120 is known to bind to CD4 that serves as a primary receptor for the virus on a target membrane surface (4Dalgleish A.G. Beverley P.C. Clapham P.R. Crawford D.M. Graeves M.F. Weiss R.A. Nature. 1984; 312: 763-767Crossref PubMed Scopus (2585) Google Scholar, 5Maddon P.J. Dalgleish A.G. McDougal J.S. Clapham P.R. Weiss R.A. Axel R. Cell. 1986; 47: 333-348Abstract Full Text PDF PubMed Scopus (1511) Google Scholar). Members of the chemokine receptor family are also known to be necessary to facilitate the entry of the virus (6Alkhatib G. Combadiere C. Brader C.C. Feng Y. Murphy P.M. Berger E. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2452) Google Scholar, 7Dragic T. Litwin V. Allaway G.P. Martin S. Huang Y. Nagashima K.A. Cayanan C. Maddon P.J. Koup R.A. Moore J.P. Paxton W.A. Nature. 1996; 381: 667-673Crossref PubMed Scopus (2821) Google Scholar). Thus consistent with this model, direct interactions have been demonstrated between gp120-CD4 complexes and specific chemokine receptors (8Trkola A. Paxton W.A. Monard S.P. Hoxie J.A. Siami M.A. Thompson D.A. Wu L.J. Mackay C.R. Horuk R. Moore J.P. J. Virol. 1998; 72: 396-404Crossref PubMed Google Scholar). The binding of gp120 to CD4 appears to induce major conformational changes of the gp120 complex that leads to the exposure of the gp32 N-terminal fusogenic domain in the case of SIV or gp41 in the case of HIV (2Kowalski M. Potz J. Basiripour L. Dotfman T. Chun Goh W. Terwilliger E. Dayton A. Rosen C. Haseltine W. Sodroski J. Science. 1987; 237: 1351-1355Crossref PubMed Scopus (613) Google Scholar, 9Gallaher W.R. Cell. 1987; 50: 327-328Abstract Full Text PDF PubMed Scopus (348) Google Scholar). Exposure of this structure therefore, is thought to facilitate the fusion of the juxtaposed viral and plasma membranes and leads to intracellular infection. Membrane fusion, therefore, is one of the key events during viral infection and is thought to facilitate the incorporation of the viral capsid into the cytoplasm of host cell. Many studies using model membranes, such as liposomes and synthetic peptides corresponding to the fusion regions of enveloped virus proteins, have revealed the essential role of the secondary structure and the orientation of the peptides when inserted in a lipid bilayer (for a review see Ref. 10Durell S.R. Martin I. Ruysschaert J.M. Shai Y. Blumenthal R. Mol. Membr. Biol. 1997; 14: 97-112Crossref PubMed Scopus (191) Google Scholar). The data presently available suggest that the HIV/SIV fusion peptide assumes extended and disordered forms in the non-fusogenic state and transforms into an α-helix as it penetrates into the target cell membrane as a prelude to or actually during the fusion process. There are strong indications that unique oblique orientations of the viral fusion peptides modify the average orientation of phospholipid acyl chains, giving rise either to inverted lipid phases or to intermediates in membrane destabilization and lipid mixing (11Martin I. Schaal H. Scheid A. Ruysschaert J.-M. J. Virol. 1996; 70: 298-304Crossref PubMed Google Scholar, 12Colotto A. Martin I. Ruysschaert J.M. Sen A. Hui W. Epand R.M. Biochemistry. 1996; 35: 980-990Crossref PubMed Scopus (84) Google Scholar, 13Colotto A. Epand R.M. Biochemistry. 1997; 36: 7644-7651Crossref PubMed Scopus (80) Google Scholar, 14Siegel D. Epand R.M. Biophys. J. 1997; 73: 3089-3111Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Although a lot of information on the correlation of the viral synthetic fusion peptides lytic and fusogenic activity with the peptide secondary structure is available (see e.g. Ref. 10Durell S.R. Martin I. Ruysschaert J.M. Shai Y. Blumenthal R. Mol. Membr. Biol. 1997; 14: 97-112Crossref PubMed Scopus (191) Google Scholar), detailed studies on the nature of the sequence of specific interactions of fusion peptides with simple phospholipid membranes are still lacking. Thus information relating to the initial interactions of fusion peptides with membranes may shed more light on the fusion process itself. Many membrane binding assays of viral peptides involve radio derivatives or their conjugation to a chromophoric indicator (15Lüneberg J. Martin I. Nußler F. Ruysschaert J.-M. Herrmann A. J. Biol. Chem. 1995; 270: 27606-27614Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 16Rapaport D. Shai Y. J. Biol. Chem. 1994; 269: 15124-15131Abstract Full Text PDF PubMed Google Scholar, 17Shai Y. Trends Biochem. Sci. 1995; 20: 460-464Abstract Full Text PDF PubMed Scopus (284) Google Scholar). In the former case no kinetic data are accessible, and inevitably, elements of doubt exist in the latter case that such chemical modification may interfere with the interaction of the peptide with the membrane surface. It has been established, however, that localization of fluorescent probes such as fluorescein phosphatidylethanolamine (FPE) at the membrane surface offers the possibility of measuring, in real time, the interactions of peptides or proteins with membranes in a virtually non-invasive manner (18Wall J. Golding C. van Venn M. O'Shea P. Mol. Membr. Biol. 1995; 12: 183-192Crossref PubMed Scopus (91) Google Scholar, 19Golding C. Senior S. Wilson M.T. O'Shea P. Biochemistry. 1996; 35: 10931-10937Crossref PubMed Scopus (38) Google Scholar). Thus, we report studies of fusion peptide-membrane interactions approached by a utilization of this novel fluorescence technique. The technique involves labeling membranes with very small amounts (<1 mol%) of FPE which is sensitive to the membrane surface electrostatic potential (ψs). Changes in ψs caused by the net addition or removal of charged species induce a corresponding increase or decrease in the fluorescence intensity of the probe. This simple but highly sensitive technique allows determination of the time evolution of the binding of such peptides to membranes, but additional interactions such as conformational changes of the peptides may also be identified (18Wall J. Golding C. van Venn M. O'Shea P. Mol. Membr. Biol. 1995; 12: 183-192Crossref PubMed Scopus (91) Google Scholar, 19Golding C. Senior S. Wilson M.T. O'Shea P. Biochemistry. 1996; 35: 10931-10937Crossref PubMed Scopus (38) Google Scholar). Whereas the present study is directed toward simple membrane systems, the additional virtue of the FPE-based technique is that it facilitates almost identical experiments with lymphocytes (20Wall J. Ayoub F. O'Shea P. J. Cell Sci. 1995; 108: 2673-2682Crossref PubMed Google Scholar). On the other hand, we have shown recently (21Cladera J. O'Shea P. Biophys. J. 1998; 74: 2434-2442Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) that peptide-membrane interactions can be affected by variations of the membrane dipole potential. This is a relatively recently understood membrane property that is generated by the presence of electrical dipoles on the phospholipid molecules and the presence of orientated water molecules at the membrane-water interface (22Brockman H. Chem. Phys. Lipids. 1994; 73: 57-59Crossref PubMed Scopus (344) Google Scholar). Following a dual-wavelength fluorescence method, it has been shown that the potential sensitive dye di-8-ANEPPS can be used to measure changes in the dipole potential produced by dipolar compounds, such as phloretin or ketocholestanol, interacting with the membrane (23Gross E. Bedlack R.S. Loew L.M. Biophys. J. 1994; 67: 208-216Abstract Full Text PDF PubMed Scopus (230) Google Scholar, 24Clarke R.J. Kane D.J. Biochim. Biophys. Acta. 1997; 1323: 223-239Crossref PubMed Scopus (122) Google Scholar, 25Clarke R.J. Biochim. Biophys. Acta. 1997; 1327: 269-278Crossref PubMed Scopus (112) Google Scholar) and to monitor the peptide membrane interaction and its effect and dependence on the magnitude of the dipole potential (21Cladera J. O'Shea P. Biophys. J. 1998; 74: 2434-2442Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). A number of complications are evident with studies of the free fusion peptides as opposed to whole viral particles. It is known that the SIV fusion peptides, which sequence is highly hydrophobic, are very insoluble in water and not even totally soluble in Me2SO, according the spectroscopic data reported by Martin et al. (26Martin I. Dubois M.C. Defrise-Quertain F. Saermark T. Burny A. Brasseur R. Ruysschaert J.-M. J. Virol. 1994; 68: 1139-1148Crossref PubMed Google Scholar). This is a situation very different from that of the fusion peptides on the virus, where for example in the case of HIV the fusion peptide constitutes just the N terminus of the gp41 protein, which is attached to the rest of the virus and seems to form trimeric complexes (27Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1848) Google Scholar). Thus, it is important to try to assess the influence of the aggregation state of the fusion peptide in the Me2SO suspension on the interaction of the peptide with the lipidic membranes and take into account the existence of peptide-peptide interactions in addition to the lipid-peptide interactions. In recent years, the use of the dye thioflavin T (ThT) for the study of the fibrillogenesis process triggered by the β-amyloid peptide of Alzheimer's disease (28Castaño E.M. Prelli F. Wisniewski T. Golabeck A. Kumar R.A. Soto C. Frangione B. Biochem. J. 1995; 306: 599-604Crossref PubMed Scopus (214) Google Scholar) has opened the possibility of using such dyes to study other aggregation-disaggregation processes. In the present study, membrane systems with well defined lipid compositions were used in an attempt to characterize the sequence of interactions of the membrane binding and insertion of the simian viral fusion peptide. The synthetic peptide corresponding to the 12-residue N-terminal region of SIV- gp32 (NH2-Gly-Val-Phe-Val-Leu-Gly-Phe-Leu-Gly-Phe-Leu-Ala) was added to the lipid membrane of specified phospholipid compositions (50 mol % PC and 50 mol % PE), and their interactions were followed using the FPE-, ThT-, and di-8-ANEPPS-based techniques (18Wall J. Golding C. van Venn M. O'Shea P. Mol. Membr. Biol. 1995; 12: 183-192Crossref PubMed Scopus (91) Google Scholar). A number of factors such as peptide concentration and lipid composition, variation of the dipole potential, and peptide aggregation were investigated. The implications of these studies for the biological activity of the immunodeficiency virus are discussed. Egg phosphatidylethanolamine (PE) and thioflavin T (ThT) were purchased from Sigma. FPE was synthesized as described previously according to Wall et al. (18Wall J. Golding C. van Venn M. O'Shea P. Mol. Membr. Biol. 1995; 12: 183-192Crossref PubMed Scopus (91) Google Scholar). 1-(3-Sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine (di-8-ANEPPS) was purchased from Molecular Probes Inc. High pressure liquid chromatography-purified synthetic peptides prepared with the C terminus in amide form were purchased from Quality Controlled Biochemical, Inc. (Hopkinton, MA). Stock solutions of these peptides were made up in Me2SO, typically at a concentration of 4 mg ml−1. Phospholipids dissolved in chloroform, di-8-ANEPPS (when required), and the appropriate additive (6-ketocholestanol or phloretin in methanol) were mixed in a round bottom flask, and the solution was dried under a stream of nitrogen to deposit a thin lipid film on the inside of a glass tube. Large unilamellar vesicles (LUV) were prepared by hydrating the dried lipid film with the sucrose buffer (280 mm sucrose, 10 mm Tris, pH 7.5), then repeatedly freezing and thawing the suspension 5 times, and finally extruding it 10 times through two polycarbonate filters of pore size 0.1 μm (Nucleopore Corp., Pleasanton, CA) using an extruder (Lipex Biomembranes Inc., Vancouver, Canada) according to the extrusion procedure of Mayer et al. (29Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1576) Google Scholar). LUVs were labeled exclusively in the outer bilayer leaflet with FPE as described by Wallet al. (18Wall J. Golding C. van Venn M. O'Shea P. Mol. Membr. Biol. 1995; 12: 183-192Crossref PubMed Scopus (91) Google Scholar). Briefly, LUVs were incubated with FPE dissolved in ethanol (never more than 0.1% of the total aqueous volume) at 37 °C for 1 h in the dark. Any remaining unincorporated FPE was removed by gel filtration on a PD10 Sephadex column equilibrated with the appropriate buffer. Such a procedure leads to the incorporation of 30–50% of the externally added FPE to the preformed LUV. Furthermore, there was no observed transmembrane “flipping” of the FPE, at least over time scales of 1 week, FPE-liposomes were stored at 4 °C until use. Fluorescence time courses were obtained by adding the desired amount of peptide to 2-ml lipid suspensions (200 μm lipid) on an SLM-Aminco model spectrofluorimeter. For FPE experiments excitation and emission wavelengths were set at 490 and 518 nm, respectively. Dual wavelength recordings with the dye di-8-ANEPPS were obtained by exciting the samples at two different wavelength (460 and 520 nm) and measuring their emission intensity ratio, R(460/520), at 580 nm (21Cladera J. O'Shea P. Biophys. J. 1998; 74: 2434-2442Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 23Gross E. Bedlack R.S. Loew L.M. Biophys. J. 1994; 67: 208-216Abstract Full Text PDF PubMed Scopus (230) Google Scholar). Assessment of peptide aggregation was determined using thioflavin T at a dye concentration of 35 μm in the fluorescence cuvette containing buffer or a membrane suspension (200 μmlipid). Typically, spectroscopic data were downloaded in ASCII file format and analyzed with the aid of commercial data analysis packages such as EasyplotTM by Stuart Karon copyright by Spiral Software & MIT (for Windows NT 32-bit (version 4) published by Cherwell Scientific), e.g. both the FPE and ThT fluorescence time courses were found to be best described by double exponential processes according to Equation 1.observed signal=A1e−k1t+A2e−k2t+offsetEq. 1 where A 1 and A 2are the amplitudes, and k 1 andk 2 are the rate constants of the biexponential process. The contribution of light scattering to the fluorescence signals was corrected by recordings made with vesicles without the respective fluorescence dyes at the same vesicle concentration and subtracting from the traces obtained with the dye present. Lipid mixing was determined by measuring the fluorescence intensity change resulting from the fluorescence resonance energy transfer (FRET) between two probes, NBD-PE and rhodamine-PE, inserted into the lipid bilayer as described by Struck et al. (30Struck D.K. Hoekstra D. Pagano R. Biochemistry. 1981; 20: 4093-4099Crossref PubMed Scopus (1142) Google Scholar). Fluorescence was monitored by using an SLM 8000 spectrofluorimeter with excitation and emission slits at 4 nm. Probes were added to the lipid film, and membrane vesicles were prepared as described above. Liposomes containing both probes at 0.6% (molar ratio) each were mixed with probe-free liposomes at 1/9 molar ratio at a final lipid concentration of 300 μm. The initial fluorescence at the 1/9 (labeled/unlabeled) suspension was taken as 0% fluorescence, and the 100% fluorescence was determined by using an equivalent concentration of vesicles prepared with 0.06% fluorescent phospholipid each. The suspensions were excited at 470 nm, and any NBD fluorescence resulting from FRET was recorded at 530 nm. Phospholipid vesicles (with 15 mol % phloretin or 6-ketocholestanol when required) were prepared as described previously (21Cladera J. O'Shea P. Biophys. J. 1998; 74: 2434-2442Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), using D2O-based media containing 280 mm sucrose, 10 mm Tris, pD 7.5. 300 μl of liquid suspension containing SIVwt 100 μm (9 μl SIVwt 3.3 mm in Me2SO added to 291 μl of PC/PE vesicles 2 mm) were placed in a SeZn plate (SpectraTech contact sampler, HATR) for attenuated total reflectance (ATR) data acquisition. ATR infrared spectra were acquired on a Nicolet 410 or 710 spectrometer equipped with an MTC detector, working at an instrumental resolution of 2 cm−1. Typically, a total of 1000 scans were averaged at room temperature, apodized with a triangle function, and Fourier-transformed. To obtain the pure spectra of the protein, spectra of the solvent were collected under identical conditions, and subtractions were done with the computer. Residual water vapor bands were also subtracted using a water vapor spectrum. Following the addition of SIVwt to FPE-labeled PC/PE vesicles, a significant increase of the fluorescence occurred, indicating the interaction of the fusion peptide with the membrane surface, as shown in Fig.1. SIVwt was manufactured in its amide form and is composed of uncharged amino acids; the only charge existing at pH 7.5 on the peptide is that arising from the positive N terminus. An increase of the fluorescence of the FPE-labeled vesicles is consistent with the increased electropositive surface potential caused by the binding of the positively charged peptide to the membrane surface. The incremental phase of the trace shown in Fig. 1 was found to fit a double exponential process (Equation 1), and the calculated rate constants are reported in TableI.Table ISummary of the rates of membrane interaction of the SIV peptideRate constants k 1 andk 2 (s−1)FPE measurementsThT measurementsdi-8ANEPPS measurements 0.4/0.04 0.4/0.040.34/0.02Rate constants are derived from fitting of data in Figs. 1 (FPE measurements), 3 (ThT measurements), and 5A (di-8-ANEPPS measurements) to Equation 1 (see “Experimental Procedures” section). Open table in a new tab Rate constants are derived from fitting of data in Figs. 1 (FPE measurements), 3 (ThT measurements), and 5A (di-8-ANEPPS measurements) to Equation 1 (see “Experimental Procedures” section). In contrast with several other peptides we have studied (e.g. Refs. 19Golding C. Senior S. Wilson M.T. O'Shea P. Biochemistry. 1996; 35: 10931-10937Crossref PubMed Scopus (38) Google Scholar, 21Cladera J. O'Shea P. Biophys. J. 1998; 74: 2434-2442Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 31Wolfe C. Cladera J. O'Shea P. Mol. Membr. Biol. 1998; 15: 221-227Crossref PubMed Scopus (17) Google Scholar), no slow fluorescence decrease (following the initial increase) indicating insertion of the positive charge into the membrane was observed for SIVwt. The positively charged N terminus must remain at the membrane surface but does not preclude the possibility of a more remote segment (i.e. with no net charge) of the peptide penetrating the lipidic bilayer as reported for other such amphipathic peptides. The inset in Fig. 1 shows the titration of PC/PE vesicles with the SIVwt peptide. An interesting observation from attempts to determine the dose-response characteristics was the fact that above 30 μm for PC/PE membranes, it was not possible to acquire more experimental points as above such concentrations significant peptide aggregation takes place. The results shown in Fig. 1 clearly indicate that SIVwt interacts with the membrane surface of PC/PE vesicles. The aggregation observed above the critical concentration described directed our attention to the aggregation state of the peptide. SIVwt is highly hydrophobic, relatively insoluble in water, and even forms soluble aggregates in Me2SO (the infrared spectrum indicates β-sheet structure typical of protein aggregates), according to the structural data reported in Martin et al. (26Martin I. Dubois M.C. Defrise-Quertain F. Saermark T. Burny A. Brasseur R. Ruysschaert J.-M. J. Virol. 1994; 68: 1139-1148Crossref PubMed Google Scholar). When added to the vesicle suspensions, these peptide aggregates present in the Me2SO solution have to partition between the aqueous medium and the membranes. To try to obtain some information about the evolution of the peptide aggregates when they interact with the membranes we used the fluorescent dye thioflavin T (ThT) as an indicator of soluble and colloidal peptide aggregates. It is worth emphasizing that the addition of Me2SO had virtually no effect on the fluorescence originating from FPE. This indicates that given the nature of Me2SO, there appears to be no significant interaction between the solvent and the various membrane moieties. Thioflavin T has been shown to indicate the presence of β-amyloid peptide aggregates (i.e. so-called fibril formation) (28Castaño E.M. Prelli F. Wisniewski T. Golabeck A. Kumar R.A. Soto C. Frangione B. Biochem. J. 1995; 306: 599-604Crossref PubMed Scopus (214) Google Scholar). The dye appears to intercalate within the β-sheet type secondary structure of the aggregates, and this interaction causes an increase of its fluorescence. Fig. 2 shows how the fluorescence of ThT dissolved in aqueous buffer changed when different amounts of SIVwt were added into the buffer from a Me2SO solution. The addition of the peptide was always followed by a very fast fluorescence increase, the magnitude of which increased with the peptide concentration. This fluorescence increase results from the very fast interaction of ThT with the peptide aggregates. Up to SIVwt 5 μm after the initial increase the fluorescence remains stable. At peptide concentration of 10 μm or higher, however, a fluorescence decrease follows the initial increase. The higher the peptide concentration, the faster the decrease and the noisier became the signal. The noisiest part of the traces corresponds to the formation of huge aggregates which, as was the case for Fig. 1(inset), are clearly visible by the naked eye and are buoyant. In Fig. 3, the ThT fluorescence time evolution when 10 μm SIV was added to a PC/PE vesicle suspension (200 μm lipid) is shown. The most striking feature is that in the presence of vesicles, after the initial fluorescence increase, the fluorescence decays in an exponential fashion, reaches a stable final level without the formation of large aggregates. Some care must be employed, however, for as shown in Fig. 4if the concentration of peptide is increased beyond 30 μm(at a constant lipid concentration) then aggregation dominates the behavior of the peptide.Figure 4A, ATR-FTIR spectrum of SIVwt peptide (100 μm) mixed 50 mol % PC, 50 mol % PE membranes (2 mm lipid). B, ThT fluorescence variation after adding 100 μm SIV wt to a 2 mm lipid, PC/PE vesicle suspension (trace 1), and 10 μm SIVwt to a 200 μm lipid, PC/PE suspension (trace 2). ThT was 35 μm. Temperature was 25 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ThT fluorescence decay in the presence of membranes was found to fit a double exponential rate equation (Equation 1), as reported in Table I. The two rate constants are found to be very similar to those calculated for the binding of the peptide to the membrane surface (Fig.1 and Table I). It is interesting to note the fact that after the initial fast fluorescence increase the fluorescence decreases to the same level of fluorescence prior to the peptide addition (Fig. 3). Since the ThT fluorescence reflects the degree of aggregation of the peptide, it can be deduced from Fig. 3 that in the presence of PC/PE membranes the peptide dis-aggregates completely. Previous structural studies with SIVwt or similar peptides (HIV fusion peptides) have shown, however, that a significant amount of aggregated β-structure is detected when the peptides are mixed with vesicles (11Martin I. Schaal H. Scheid A. Ruysschaert J.-M. J. Virol. 1996; 70: 298-" @default.
• W2074366880 created "2016-06-24" @default.
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• W2074366880 date "1999-10-01" @default.
• W2074366880 modified "2023-09-30" @default.
• W2074366880 title "Characterization of the Sequence of Interactions of the Fusion Domain of the Simian Immunodeficiency Virus with Membranes" @default.
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