Pore Formation by Equinatoxin II, a Eukaryotic Protein Toxin, Occurs by Induction of Nonlamellar Lipid Structures
2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês
10.1074/jbc.m305916200
ISSN1083-351X
AutoresGregor Anderluh, Mauro Dalla Serra, Gabriella Viero, Graziano Guella, Peter Maček, Gianfranco Menestrina,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoPore formation in the target cell membranes is a common mechanism used by many toxins in order to kill cells. Among various described mechanisms, a toroidal pore concept was described recently in the course of action of small antimicrobial peptides. Here we provide evidence that such mechanism may be used also by larger toxins. Membrane-destabilizing effects of equinatoxin II, a sea anemone cytolysin, were studied by various biophysical techniques. 31P NMR showed an occurrence of an isotropic component when toxin was added to multilamellar vesicles and heated. This component was not observed with melittin, α-staphylococcal toxin, or myoglobin. It does not originate from isolated small lipid structures, since the size of the vesicles after the experiment was similar to the control without toxin. Electron microscopy shows occurrence of a honeycomb structure, previously observed only for some particular lipid mixtures. The analysis of FTIR spectra of the equinatoxin II-lipid complex showed lipid disordering that is consistent with isotropic component observed in NMR. Finally, the cation selectivity of the toxin-induced pores increased in the presence of negatively charged phosphatidic acid, indicating the presence of lipids in the conductive channel. The results are compatible with the toroidal pore concept that might be a general mechanism of pore formation for various membrane-interacting proteins or peptides. Pore formation in the target cell membranes is a common mechanism used by many toxins in order to kill cells. Among various described mechanisms, a toroidal pore concept was described recently in the course of action of small antimicrobial peptides. Here we provide evidence that such mechanism may be used also by larger toxins. Membrane-destabilizing effects of equinatoxin II, a sea anemone cytolysin, were studied by various biophysical techniques. 31P NMR showed an occurrence of an isotropic component when toxin was added to multilamellar vesicles and heated. This component was not observed with melittin, α-staphylococcal toxin, or myoglobin. It does not originate from isolated small lipid structures, since the size of the vesicles after the experiment was similar to the control without toxin. Electron microscopy shows occurrence of a honeycomb structure, previously observed only for some particular lipid mixtures. The analysis of FTIR spectra of the equinatoxin II-lipid complex showed lipid disordering that is consistent with isotropic component observed in NMR. Finally, the cation selectivity of the toxin-induced pores increased in the presence of negatively charged phosphatidic acid, indicating the presence of lipids in the conductive channel. The results are compatible with the toroidal pore concept that might be a general mechanism of pore formation for various membrane-interacting proteins or peptides. Proteins and peptides with the capacity to increase membrane permeability have been elaborated by a large number of organisms and are used as toxins, effectors in immune response or apoptosis. One of the most commonly adopted mechanisms is the formation of pores in the targeted membrane as occurs, for example, with pore-forming toxins (PFT) 1The abbreviations used are: PFT, pore-forming toxin(s); DPhPC, 1,2-diphytanoyl-sn-glycerophosphocholine; EqtII, equinatoxin II; EM, electron microscopy; FTIR spectroscopy, Fourier-transformed infrared spectroscopy; LUV, large unilamellar vesicles; L/P, lipid/protein ratio; MLV, multilamellar vesicles; PA, phosphatidic acid; PC, phosphatidylcholine; PLM, planar lipid membranes; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; QLS, quasielastic light scattering; SM, sphingomyelin; SUV, small unilamellar vesicles. (1Menestrina G. Dalla Serra M. Lazarovici P. Pore-forming Peptides and Protein Toxins. Taylor & Francis Group, London, UK2003Crossref Google Scholar, 2van der Goot F.G. Pore-forming Toxins. Springer Verlag, Berlin2001Crossref Google Scholar). Bacterial PFT, protein molecules of M r > 30,000, usually follow two strategies; they either form a channel via insertion of a de novo generated transmembrane β barrel (examples are staphylococcal α-toxin, the cholesterol-dependent cytolysins, and the protective antigen of anthrax toxin), or they insert a bundle of preexisting α-helices through the membrane (like colicins and crystal δ-endotoxins) (3Heuck A.P. Tweten R.K. Johnson A.E. Biochemistry. 2001; 40: 9065-9073Crossref PubMed Scopus (127) Google Scholar, 4Lesieur C. Vécsey-Semjén B. Abrami L. Fivaz M. van der Goot F.G. Mol. Membrane Biol. 1997; 14: 45-64Crossref PubMed Scopus (147) Google Scholar). Smaller molecules, like antimicrobial peptides or peptide toxins with M r between 1000 and 5000, have developed a wider set of mechanisms (5Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6785) Google Scholar). In fact, besides the β-barrel (e.g. protegrin) (6Heller W.T. Waring A.J. Lehrer R.I. Huang H.W. Biochemistry. 1998; 37: 17331-17338Crossref PubMed Scopus (122) Google Scholar) and the α-helix bundle (e.g. alamethicin and other peptaibols) (7Chugh J.K. Wallace B.A. Biochem. Soc. Trans. 2001; 29: 565-570Crossref PubMed Scopus (174) Google Scholar), some alternative strategies were found, which directly modify the bilayer organization of the membrane. They range from a generic destabilization (exemplified by the carpet-like model) (8Shai Y. Oren Z. Peptides. 2001; 22: 1629-1641Crossref PubMed Scopus (323) Google Scholar) to the formation of specific mixed lipid-peptide structures, like the toroidal pore, which was observed with magainin (9Matsuzaki K. Sugishita K. Ishibe N. Ueha M. Nakata S. Miyajima K. Epand R.M. Biochemistry. 1998; 37: 11856-11863Crossref PubMed Scopus (411) Google Scholar) and melittin (10Yang L. Harroun T.A. Weiss T.M. Ding L. Huang H.W. Biophys. J. 2001; 81: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar). Actinoporins are a peculiar class of eukaryotic PFT with intermediate M r, exclusively found in sea anemones. It is a family of cysteineless proteins with M r around 18,000–20,000 and a preference for sphingomyelin (SM) (11Anderluh G. Maček P. Toxicon. 2002; 40: 111-124Crossref PubMed Scopus (346) Google Scholar). They form cation-selective pores with a diameter of ∼2 nm on cellular and model membranes (12Varanda A. Finkelstein A. J. Membr. Biol. 1980; 55: 203-211Crossref PubMed Scopus (86) Google Scholar, 13Belmonte G. Pederzolli C. Maček P. Menestrina G. J. Membr. Biol. 1993; 131: 11-22Crossref PubMed Scopus (192) Google Scholar, 14Tejuca M. Dalla Serra M. Alvarez C. Potrich C. Menestrina G. J. Membr. Biol. 2001; 183: 125-135Crossref PubMed Scopus (98) Google Scholar). The three-dimensional structure of the soluble state of one actinoporin, equinatoxin II (EqtII; from the sea anemone Actinia equina) was recently solved by x-ray (15Athanasiadis A. Anderluh G. Maček P. Turk D. Structure. 2001; 9: 341-346Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and NMR (16Hinds M.G. Zhang W. Anderluh G. Hansen P.E. Norton R.S. J. Mol. Biol. 2002; 315: 1219-1229Crossref PubMed Scopus (126) Google Scholar). The molecule is composed of a hydrophobic β-sandwich core, flanked on the opposite sides by two α-helices. The first 30 N-terminal residues, which include one helix, are the best candidates for pore formation. This part of the molecule is amphipathic in character, is well conserved in all actinoporins, is clearly similar to some membrane-interacting peptides like melittin and fusogenic viral peptides (17Belmonte G. Menestrina G. Pederzolli C. Križaj I. Gubenšek F. Turk T. Maček P. Biochim. Biophys. Acta. 1994; 1192: 197-204Crossref PubMed Scopus (105) Google Scholar), and is the only portion of the molecule that can change conformation without disrupting the general fold (15Athanasiadis A. Anderluh G. Maček P. Turk D. Structure. 2001; 9: 341-346Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Preliminary evidence of the involvement of the N-terminal part in the formation of the transmembrane channel was obtained by a low resolution cysteine-scanning mutagenesis along the molecule (18Anderluh G. Barlič A. Podlesek Z. Maček P. Pungerčar J. Gubenšek F. Zecchini M. Dalla Serra M. Menestrina G. Eur. J. Biochem. 1999; 263: 128-136Crossref PubMed Scopus (91) Google Scholar) and by N-terminal truncations (19Anderluh G. Pungerčar J. Križaj I. Štrukelj B. Gubenšek F. Maček P. Protein Eng. 1997; 10: 751-755Crossref PubMed Scopus (60) Google Scholar). More recently, a complete cysteine-scanning mutagenesis of the region encompassing residues 10–28 clearly demonstrated that this portion adopts α-helical configuration in the membrane, that the α-helix is longer than in the soluble form, that it lies on the water-lipid interface in the membrane-bound state, and that it inserts into the membrane to line the pore interior, forming an angle of around 20° with the bilayer normal (20Malovrh P. Viero G. Dalla Serra M. Podlesek Z. Lakey J.H. Maček P. Menestrina G. Anderluh G. J. Biol. Chem. 2003; 278: 22678-22685Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The slight increase in helicity that occurs upon lipid binding was already noted by FTIR spectroscopy (21Menestrina G. Cabiaux V. Tejuca M. Biochem. Biophys. Res. Commun. 1999; 254: 174-180Crossref PubMed Scopus (68) Google Scholar) and is consistent with the length of the homologous, membrane-perturbing, helical peptides. In our current model, initial binding is provided by a cluster of exposed aromatic residues (which includes Trp-112 and Trp-116) (18Anderluh G. Barlič A. Podlesek Z. Maček P. Pungerčar J. Gubenšek F. Zecchini M. Dalla Serra M. Menestrina G. Eur. J. Biochem. 1999; 263: 128-136Crossref PubMed Scopus (91) Google Scholar, 22Malovrh P. Barlič A. Podlesek Z. Menestrina G. Maček P. Anderluh G. Biochem. J. 2000; 346: 223-232Crossref PubMed Scopus (81) Google Scholar, 23Hong Q. Gutierrez-Aguirre I. Barlič A. Malovrh P. Kristan K. Podlesek Z. Maček P. Turk D. González-Mañas J.M. Lakey J.H. Anderluh G. J. Biol. Chem. 2002; 277: 41916-41924Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), followed by N-terminal helix translocation to the surface of the membrane and its insertion into the lipid bilayer (20Malovrh P. Viero G. Dalla Serra M. Podlesek Z. Lakey J.H. Maček P. Menestrina G. Anderluh G. J. Biol. Chem. 2003; 278: 22678-22685Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 23Hong Q. Gutierrez-Aguirre I. Barlič A. Malovrh P. Kristan K. Podlesek Z. Maček P. Turk D. González-Mañas J.M. Lakey J.H. Anderluh G. J. Biol. Chem. 2002; 277: 41916-41924Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The channel entity is not stable enough to be isolated and analyzed as a single unit. Therefore, the number of monomers appearing in the final pore was deduced from indirect experiments. All evidence, gathered from cross-linking (13Belmonte G. Pederzolli C. Maček P. Menestrina G. J. Membr. Biol. 1993; 131: 11-22Crossref PubMed Scopus (192) Google Scholar), steady state (12Varanda A. Finkelstein A. J. Membr. Biol. 1980; 55: 203-211Crossref PubMed Scopus (86) Google Scholar), and kinetic (24Tejuca M. Dalla Serra M. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (156) Google Scholar) partitioning experiments, consistently indicated a tetrameric structure. Notably, however, the pore diameter is too large to be formed by a simple bundle of four helices. One possibility to solve this apparent contradiction is to assume that the pore is partially lined by membrane lipids. In fact, perturbation of the lamellar lipid structure has been noted with some of the helical peptides similar to the N-terminal helix of actinoporins (e.g. mastoparan (25Hori Y. Demura M. Niidome T. Aoyagi H. Asakura T. FEBS Lett. 1999; 455: 228-232Crossref PubMed Scopus (15) Google Scholar), staphylococcal δ-lysin (26Lohner K. Staudegger E. Prenner E.J. Lewis R.N.A.H. Kriechbaum M. Degovics G. McElhaney R.N. Biochemistry. 1999; 38: 16514-16528Crossref PubMed Scopus (46) Google Scholar), the HIV-1 viral fusion peptide (27Pereira F.B. Valpuesta J.M. Basañez G. Goñi F.M. Nieva J.L. Chem. Phys. Lipids. 1999; 103: 11-20Crossref PubMed Scopus (30) Google Scholar), and some synthetic peptides with an amphipathic α-helical character (28Liu F. Lewis R.N.A.H. Hodges R.S. McElhaney R.N. Biochemistry. 2001; 40: 760-768Crossref PubMed Scopus (42) Google Scholar)). In addition, a direct effect of EqtII on the lipid phase has been recently observed and attributed to the separation of a sphingomyelin-enriched phase and to vesiculation (29Bonev B.B. Lam Y.H. Anderluh G. Watts A. Norton R.S. Separovic F. Biophys. J. 2003; 84: 2382-2392Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Given its similarity with melittin, we suspected that the N-terminal helix of actinoporins could perturb the membrane by giving rise to the formation of a toroidal protein-lipid pore. Direct functional evidence for such a structure was already provided by the toxin-strengthening effect of lipids that favor the positive curvature appearing at the center of the toroidal pore and by the fact that pore formation induced the mobilization of lipid molecules between the two leaflets of the bilayer (30Alvarez C. Dalla Serra M. Potrich C. Bernhart I. Tejuca M. Martinez D. Pazos I.F. Lanio M.E. Menestrina G. Biophys. J. 2001; 80: 2761-2774Abstract Full Text Full Text PDF PubMed Google Scholar). Here we report a new investigation of EqtII effects on membrane order, providing original evidence that it can affect the lipid phase by a peculiar mechanism, previously observed only in some specific lipid mixtures. Such a mechanism, which is consistent with the concept that its biological activity is exerted via the formation of a toroidal protein-lipid pore, represents a novel paradigm of protein-induced membrane destabilization. Toxins, Proteins, and Lipids—Native EqtII was isolated from the sea anemones as described (31Maček P. Lebez D. Toxicon. 1988; 26: 441-451Crossref PubMed Scopus (137) Google Scholar). Melittin (65–85% pure by high pressure liquid chromatography) and myoglobin (type II, minimum 90%, from whale muscle) were from Sigma; pure α-toxin from Staphylococcus aureus was a kind gift of Dr. Hungerer (Behringwerk, Marburg, Germany). Lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-diphytanoyl-sn-glycerophosphocholine (DPhPC), phosphatidic acid (PA), egg phosphatidylcholine (PC), and brain SM, were all obtained from Avanti Polar Lipids (Alabaster, AL). Lipid Vesicle Preparations—PC, POPC, and cholesterol were dissolved in chloroform, SM in chloroform/ethanol (4:1, v/v). Lipid mixtures were vacuum-dried in a film at the bottom of a round flask and further treated in a speed-vac for 1–2 h. The lipid films were resuspended at room temperature by vigorously vortexing in the appropriate buffer, followed by six cycles of freezing and thawing in liquid nitrogen. Such dispersions of multilamellar vesicles (MLV) were either immediately used as such or were extruded through polycarbonate membranes with pores of diameter 400 or 600 nm to prepare large unilamellar vesicles (LUV) of that size. Alternatively, small unilamellar vesicles (SUV) were prepared by sonicating the MLV on ice for 30 min, followed by gentle sedimentation to remove titanium particles released by the sonotrode. Lipid samples with PFT or myoglobin, were prepared similarly except that the buffer used for lipid hydration contained appropriate amounts of the proteins. For actinoporins, mixture POPC/SM or PC/SM in a molar ratio of 1:1 or 2:1 was used, since these two mixtures are the most sensitive in functional assays for binding and permeabilization (13Belmonte G. Pederzolli C. Maček P. Menestrina G. J. Membr. Biol. 1993; 131: 11-22Crossref PubMed Scopus (192) Google Scholar, 24Tejuca M. Dalla Serra M. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (156) Google Scholar). For the same reason, we used POPC/cholesterol in a molar ratio of 1:1 with α-toxin (32Forti S. Menestrina G. Eur. J. Biochem. 1989; 181: 767-773Crossref PubMed Scopus (72) Google Scholar). NMR Spectra— 31P NMR spectra of the different lipid and lipid/toxin preparations were recorded at 121.4 MHz on a Varian XL-300 spectrometer. Lipid composition was POPC/SM 2:1 (mol/mol), and lipid concentration was 30 mm in 100 mm NaCl, 30 mm Tris-HCl, 1 mm EDTA, pH 7.0 (NMR buffer). 0.4–0.7 ml of the sample were placed in 5-mm Pyrex NMR tubes. Two different protocols were used (as indicated). The phase-cycled Hahn echo pulse sequence was used on samples that were supplemented with 10% deuterated water to provide an internal lock frequency control (33Rance M. Byrd R.A. J. Magn. Reson. 1983; 52: 221-240Google Scholar). Recycle time was 2 s, 90° pulse width was 30 μs, delay between pulses was 100 μs, sweep width was 38.46 kHz, acquisition time was 0.104 s, and transient number was 2000–10,000. Alternatively, spectra were acquired by the single pulse acquisition technique (60° pulse angle and 0.4-s interpulse time) on samples without deuterated water. 2000–4000 transients were used in this case. In all measurements, 8192 complex data points were acquired and exponentially multiplied with 75 Hz prior to Fourier transformation with continuous wave 1H-decoupling during data acquisition. 31P chemical shifts were measured relative to 0.0 ppm indicated by sonicated unilamellar vesicles. Variable temperatures were obtained with a homemade temperature-control unit that was calibrated on the chemical shift difference between the residual OH and CD2H proton of 99.8% deuterated methanol (temperature accuracy ±0.2 °C). Samples were left 10 min to equilibrate after each change of temperature Measurement of Vesicle Size by Quasielastic Light Scattering (QLS)— MLV, LUV, and SUV size was determined by QLS (34Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1570) Google Scholar) at a fixed angle (90°) and room temperature, using a laser particle sizer (Malvern Z-sizer 3) equipped with a 5-milliwatt helium-neon laser. For this assay, lipid samples were diluted in NMR buffer to give similar scattering (from 25 to 250 times, depending on their size). A 64-channel correlator was used capable of particle size estimates in the range of 5–5000 nm. Data were analyzed by the cumulant method using Malvern Application Software. The first cumulant provided the apparent diffusion coefficient of the particles (from which the hydrodynamic radius can be derived by the Stokes-Einstein relation), whereas the second cumulant gave the distribution width (35Santos N.C. Castanho M.A.R.B. Biophys. J. 1996; 71: 1641-1650Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Permeabilization of Lipid Vesicles—Permeabilization was assayed by measuring the leakage of calcein (36Kayalar C. Düzgünes N. Biochim. Biophys. Acta. 1986; 860: 51-56Crossref PubMed Scopus (48) Google Scholar). MLV were prepared as above, but in the presence of 80 mm calcein (pH 7.0 with NaOH). The external calcein was removed by spinning through minicolumns (Pierce) loaded with Sephadex G50 medium preequilibrated with NMR buffer. Fluorescence was measured in a spectrofluorimeter (SPEX Fluoromax) using a 1-cm semimicroquartz cuvette with 1 ml of NMR buffer with 1 μm lipids continuously stirred. Lysins were added to vesicles at a lipid/protein (or peptide) molar ratio of 1. Excitation wavelength was set to 485 nm, and emission was set to 520 nm with both slits set to 2 nm. Release of calcein (R), a percentage, was calculated according to Equation 1, R=(Fmeas-Finit)/(Fmax-Finit)·100(Eq. 1) where F meas, F init, and F max are the measured, initial, and maximal fluorescence, respectively. F max was obtained by the addition of Triton X-100 to 1 mm final concentration. Spontaneous leakage was negligible on this time scale. The experiments were run at room temperature. Electron Microscopy (EM)—Transmission EM was performed with a Philips CM100 microscope operating at 80 kV. A drop of the sample was deposited on the copper grids coated with formvar film and negatively stained with 1% phosphotungstic acid (Sigma). Samples were dried on air before imaging. Images were taken with a BioScan Camera model 792 (Gatan) with CCD resolution of 1024 × 1024 pixels. For size estimation, samples were imaged after the NMR experiment. The dimensions were estimated by measuring the diameter of vesicles from the EM image. Averages for MLV and MLV with EqtII were obtained after fitting dimensions to Gaussian distribution. Averages for MLV with melittin were calculated from the data, since the distribution was not Gaussian in that case. Preparation of Vesicles for Infrared Spectroscopy—SUV were prepared as above with POPC/SM (1:1) (5 mg/ml) for EqtII and PC/cholesterol (1:1) (6 mg/ml) for α-toxin. EqtII was applied at a lipid/protein ratio (L/P) of 100. Unbound toxin, estimated by the residual hemolytic activity, was between 5 and 10% and was not removed. Samples were applied straight to the germanium crystal. α-Toxin was used at L/P of 200 and incubated at 37 °C for 1 h. Free toxin (∼35%, as determined by hemolytic activity) was removed by repeated ultrafiltration through polysulfone filters of 300-kDa cut-off (NMWL; Millipore Corp.) as described (37Ferreras M. Höper F. Dalla Serra M. Colin D.A. Prévost G. Menestrina G. Biochim. Biophys. Acta. 1998; 1414: 108-126Crossref PubMed Scopus (76) Google Scholar). All preparations were in 10 mm Hepes, pH 7.0. FTIR—FTIR spectra were collected in the attenuated total reflection configuration, as described (21Menestrina G. Cabiaux V. Tejuca M. Biochem. Biophys. Res. Commun. 1999; 254: 174-180Crossref PubMed Scopus (68) Google Scholar), on a Bio-Rad FTS 185 spectrometer with a deuterium triglycine sulfate detector and KBr beam splitter, at a nominal resolution of 0.5 cm–1. Three kinds of spectra were taken: toxin alone, lipid alone (SUV of either PC/SM 1:1 or PC/cholesterol 1:1), or toxin-treated SUV. The samples were spread on a 10-reflection germanium crystal (45° cut), flushed with D2O-saturated nitrogen, and housed in a vertical attenuated total reflection attachment (by Specac). Polarized spectra were collected with a rotating wire grid polarizer (fir grid; Specac), manually positioned either parallel (0°) or perpendicular (90°) to the plane of the internal reflections. The orientation of a structural element was calculated from the dichroic ratio, R = A 0°/A 90°, where A 0° and A 90° are the absorption bands of the functional group of that element in the parallel and perpendicular configuration, respectively (38Tamm L.K. Tatulian S.A. Q. Rev. Biophys. 1997; 30: 365-429Crossref PubMed Scopus (625) Google Scholar, 39Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (513) Google Scholar). The form factor, S, was derived from R using Equation 2 (40Axelsen P.H. Kaufman B.K. McElhaney R.N. Lewis R.N.A.H. Biophys. J. 1995; 69: 2770-2781Abstract Full Text PDF PubMed Scopus (85) Google Scholar), S=Ex2-REy2+Ez212(3cos2θ-1)(Ex2-REy2-2Ez2)(Eq. 2) where θ represents the angle between the long axis of the molecule under consideration and the transition moment of the investigated vibration; Ex, Ey , and Ez are the components of the electric field of the evanescent wave in the three directions (the z axis being perpendicular to the plane of the crystal), which we calculated according to Harrick expressions for thick films (40Axelsen P.H. Kaufman B.K. McElhaney R.N. Lewis R.N.A.H. Biophys. J. 1995; 69: 2770-2781Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 41Harrick N.J. Internal Reflection Spectroscopy. Harrick Scientific Corp., Ossining, NY1967Google Scholar). The following order parameters were calculated: (a) S L, for the lipid chains, using either the symmetric or asymmetric CH2 stretching (bands centered at 2850 and 2920 cm–1, respectively) and θ = 90°; (b) S amide I′, for the amide I′ band (integrated between 1600 and 1700 cm–1) with θ = 0° (42Tamm L.K. Tatulian S.A. Biochemistry. 1993; 32: 7720-7726Crossref PubMed Scopus (61) Google Scholar); and (c) S α, for the α-helix, using the Lorentzian component at 1651 ± 1 cm–1, obtained by deconvoluting and curve-fitting the amide I′ band (between 1700 and 1600 cm–1) as previously described (21Menestrina G. Cabiaux V. Tejuca M. Biochem. Biophys. Res. Commun. 1999; 254: 174-180Crossref PubMed Scopus (68) Google Scholar) and using θ = 39° (38Tamm L.K. Tatulian S.A. Q. Rev. Biophys. 1997; 30: 365-429Crossref PubMed Scopus (625) Google Scholar). To analyze the amide I′ band of the lipid-bound toxin, the spectra were previously corrected by subtracting the contribution of the lipid alone, with a weight that eliminates the stretching band of the phospholipid carboxyl groups at 1738 cm–1. From the order parameter, we calculated the average tilt angle γ of the molecular axis with respect to the z axis according to Equation 3 (40Axelsen P.H. Kaufman B.K. McElhaney R.N. Lewis R.N.A.H. Biophys. J. 1995; 69: 2770-2781Abstract Full Text PDF PubMed Scopus (85) Google Scholar), S=12(3cos2γ-1)(Eq. 3) By definition, S represents the fraction of molecules aligned with the axis direction, whereas (1 – S) is the fraction remaining disordered. The mean angle σ of the α-helix axis with respect to the direction of the lipid chains was recalculated from Equation 4 (40Axelsen P.H. Kaufman B.K. McElhaney R.N. Lewis R.N.A.H. Biophys. J. 1995; 69: 2770-2781Abstract Full Text PDF PubMed Scopus (85) Google Scholar), Sα12(3cos2γL-1)=12(3cos2σ-1)(Eq. 4) where γL is the angle formed by the lipid chains with the z axis. Finally, the lipid to bound toxin ratio (L/Pb) was calculated from A 90° according to Equation 5 (42Tamm L.K. Tatulian S.A. Biochemistry. 1993; 32: 7720-7726Crossref PubMed Scopus (61) Google Scholar), L/Pb=0.208(nres-1)(1-SamideI')(1+SL/2)·∫28002980A90∘(νL)dν∫16001690A90∘(νamideI')dν(Eq. 5) where n res represents the total number of residues in the toxin (179 for EqtII and 293 for α-toxin). Determination of Ion Selectivity of the EqtII Channel—Electrical properties of EqtII pores were studied using planar lipid membranes (PLM) exactly as described (20Malovrh P. Viero G. Dalla Serra M. Podlesek Z. Lakey J.H. Maček P. Menestrina G. Anderluh G. J. Biol. Chem. 2003; 278: 22678-22685Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). On a weight basis, PLM were made of 20% SM, (80 – x)% of DPhPC and x% PA, with x ranging from 0 to 20. EqtII was added at nanomolar concentration only to the cis side, where voltage was applied. Reversal voltages were measured in a 10-fold KCl gradient and translated into a permeability ratio P +/P – (where P + and P – refer to cation and anion permeability, respectively) by the Goldmann-Hodgkin-Katz equation. Initially, both sides (volume of 2 ml) were bathed by symmetrical solution of 10 mm Tris, 100 mm KCl, pH 8.0. Thereafter, the trans side was perfused with 10 volumes of 10 mm Tris, 1 m KCl, pH 8.0. Effects of EqtII on Lipid Isotropy: NMR—The effects of equinatoxin (EqtII) on lipid membranes were first studied by 31P NMR spectroscopy. Initially, pure lipid samples were examined in order to find the most suitable preparation. Four different kinds of vesicles were compared (SUV, LUV400, LUV600, and MLV), which share the same bilayer organization but have quite different size, as shown by QLS (Table I). Ideally, the lipid preparation should display a pure lamellar phase signal so that any toxin-induced change of lipid organization (e.g. from lamellar to hexagonal or isotropic phases) (43Seddon J.M. Biochim. Biophys. Acta. 1990; 1031: 1-69Crossref PubMed Scopus (1008) Google Scholar, 44Cullis P.R. Hope M.J. de Kruijff B. Verkleij A.J. Tilcock C.P. Kuo J.F. Phospholipids and Cellular Regulation. Vol. 1. CRC Press, Inc., Boca Raton, FL1985: 1-59Google Scholar) could be detected. Only MLV approximated this behavior, showing, even at the higher temperature, a clear predominance of the broad asymmetric 31P NMR spectra (with low field shoulder) typical of the lamellar phase over a small isomorphic signal (Fig. 1). All other preparations showed the occurrence of a peak around 0 ppm, suggesting the presence of a population of fast tumbling phospholipid molecules, which was predominant, at least at the higher temperature. Clearly, the amount of isotropic phase was inversely correlated to the size of the vesicles and directly related to the temperature. For these reasons, as in other works (25Hori Y. Demura M. Niidome T. Aoyagi H. Asakura T. FEBS Lett. 1999; 455: 228-232Crossref PubMed Scopus (15) Google Scholar, 26Lohner K. Staudegger E. Prenner E.J. Lewis R.N.A.H. Kriechbaum M. Degovics G. McElhaney R.N. Biochemistry. 1999; 38: 16514-16528Crossref PubMed Scopus (46) Google Scholar, 28Liu F. Lewis R.N.A.H. Hodges R.S. McElhaney R.N. Biochemistry. 2001; 40: 760-768Crossref PubMed Scopus (42) Google Scholar, 29Bonev B.B. Lam Y.H. Anderluh G. Watts A. Norton R.S. Separovic F. Biophys. J. 2003; 84: 2382-2392Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we used only MLV in the rest.Table IDiameter of lipid particles measured by quasielastic light scattering or electron microscopyLipid structureaLipid composition was, in all cases, POPC/SM 2:1 (mol/mol).L/PQLSEMbThe value reported is the mean ± S.D. of 69, 247, or 157 vesicles for MLV, MLV + EqtII, or MLV + melittin, respectively. In the case of MLV with melittin, the distribution was quite broad (approximately 40% of vesicles were smaller than 250 nm). Note that, in the presence of heterogeneous distributions, QLS provided slightly higher estimates than EM, probably
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