FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2081846733> ?p ?o ?g. }

• W2081846733 endingPage "22685" @default.
• W2081846733 startingPage "22678" @default.
• W2081846733 abstract "Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10–28 are organized as an α-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation. Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10–28 are organized as an α-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation. Pore-forming toxins (PFT) 1The abbreviations used are: PFT, pore-forming toxins; DPhPC, 1,2 diphytanoyl-sn-3-glycerophosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DTT, dithiothreitol; EqtII, equinatoxin II; IANBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; L/T, lipid/toxin; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl) methanethiosulfonate hydrobromide; MTSES, sodium (2-sulfonatoethyl) methanethiosulfonate; 7-NO-PC, 1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-phosphocholine; PLM, planar lipid membrane; SM, sphingomyelin; SUV, small unilamellar vesicles; WT, wild-type. are considered as a link between soluble and membrane proteins (1Gouaux E. Curr. Opin. Struct. Biol. 1997; 7: 566-573Crossref PubMed Scopus (151) Google Scholar). These proteins, produced in a soluble form, act upon target cell membranes by forming transmembrane channels. During this toxic action they adapt their structure to expose the hydrophobic parts needed for membrane association and pore formation. PFT can be divided according to the transmembrane structural elements of the final inserted state. Some bacterial PFT form stable oligomeric β-barrels (2Heuck A.P. Tweten R.K. Johnson A.E. Biochemistry. 2001; 40: 9065-9073Crossref PubMed Scopus (128) Google Scholar). Examples include α-toxin from Staphylococcus aureus (3Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1962) Google Scholar), a family of cholesterol-dependent cytolysins from Gram-positive bacteria (4Rossjohn J. Feil S.C. McKinstry W.J. Tweten R.K. Parker M.W. Cell. 1997; 89: 685-692Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), anthrax protective antigen (5Petosa C. Collier R.J. Klimpel K.R. Leppla S.H. Liddington R.C. Nature. 1997; 385: 833-838Crossref PubMed Scopus (683) Google Scholar), and others. PFT pores lined by α-helices (similar to colicins) (6Lakey J.H. Slatin S.L. Curr. Top. Microbiol. Immunol. 2001; 257: 131-161PubMed Google Scholar) are fewer and less well understood, largely because of the inherent instability of the resulting pores. One interesting family of PFT is that of the actinoporins of sea anemones (7Anderluh G. Maček P. Toxicon. 2002; 40: 111-124Crossref PubMed Scopus (348) Google Scholar). They are closely related cysteine-less proteins that form pores in natural and artificial lipid membranes. Their peculiar three-dimensional structure and biochemical properties reveal them to form an entirely novel class of PFT (8Athanasiadis A. Anderluh G. Maček P. Turk D. Structure (Camb.). 2001; 9: 341-346Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 9Hinds M.G. Zhang W. Anderluh G. Hansen P.E. Norton R.S. J. Mol. Biol. 2002; 315: 1219-1229Crossref PubMed Scopus (126) Google Scholar). They are one-domain proteins; smaller than bacterial PFT, they lyse erythrocytes at picomolar concentrations and exhibit sphingomyelin dependence. The most studied representative is equinatoxin II (EqtII), an actinoporin from Actinia equina. EqtII is composed of a tightly folded β-sandwich flanked on two sides by α-helices (Fig. 1A) (8Athanasiadis A. Anderluh G. Maček P. Turk D. Structure (Camb.). 2001; 9: 341-346Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 9Hinds 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 first 30 N-terminal residues, encompassing the N-terminal α-helix (residues 16–26 from the crystal and NMR structures), is the only part that can be dislocated from the body of the molecule without disrupting the β-sandwich (Fig. 1B). Pore formation by EqtII is a multistep process (Fig. 1C). It was shown recently that the toxin binds to the lipid bilayer with the aromatic amino acid cluster located on a broad loop at the bottom of the molecule and on the C-terminal α-helix (10Malovrh P. Barlič A. Podlesek Z. Maček P. Menestrina G. Anderluh G. Biochem. J. 2000; 346: 223-232Crossref PubMed Scopus (81) Google Scholar, 11Hong 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). In the next step an N-terminal segment translocates to the lipid-water interface (10Malovrh P. Barlič A. Podlesek Z. Maček P. Menestrina G. Anderluh G. Biochem. J. 2000; 346: 223-232Crossref PubMed Scopus (81) Google Scholar, 12Anderluh G. Barlič A. Podlesek Z. Maček P. Pungerčar J. Gubenšek F. Zecchini M.L. Dalla Serra M. Menestrina G. Eur. J. Biochem. 1999; 263: 128-136Crossref PubMed Scopus (91) Google Scholar), and, finally, a transmembrane pore is formed by helices from three or four monomers as proposed by Belmonte et al. (13Belmonte G. Pederzolli C. Maček P. Menestrina G. J. Membr. Biol. 1993; 131: 11-22Crossref PubMed Scopus (192) Google Scholar). The final pore is not a rigid structure, like the stable β-barrels of bacterial PFT that can be studied by x-ray crystallography or electron microscopy (3Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1962) Google Scholar, 5Petosa C. Collier R.J. Klimpel K.R. Leppla S.H. Liddington R.C. Nature. 1997; 385: 833-838Crossref PubMed Scopus (683) Google Scholar). Actinoporin pores are not resistant to SDS and have not yet been directly visualized. The number of monomers in the pore was deduced by cross-linking and kinetic experiments (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. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (157) Google Scholar). According to helical wheel analysis, the whole region of residues 10–28, including the N-terminal α-helix, is amphipathic and could be involved in forming the walls of the functional pore (Fig. 1B) (15Belmonte 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). This region is highly conserved in all known actinoporins. In particular, the hydrophobic face is almost completely conserved, while on the polar face the most conserved are negatively charged residues (i.e. Asp-10, Asp-17, and Asp-24), which could account for the cation selectivity of actinoporins. We decided to study the topology of this important region in its membrane environment by employing cysteine-scanning mutagenesis. This approach proved to be useful in obtaining detailed structural information of membrane regions of some PFT (16Valeva A. Walev I. Pinkernell M. Walker B. Bayley H. Palmer M. Bhakdi S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11607-11611Crossref PubMed Scopus (98) Google Scholar, 17Shepard L.A. Heuck A.P. Hamman B.D. Rossjohn J. Parker M.W. Ryan K.R. Johnson A.E. Tweten R.K. Biochemistry. 1998; 37: 14563-14574Crossref PubMed Scopus (271) Google Scholar, 18Shatursky O. Heuck A.P. Shepard L.A. Rossjohn J. Parker M.W. Johnson A.E. Tweten R.K. Cell. 1999; 99: 293-299Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). A desired amino acid is changed to a cysteine by site-directed mutagenesis in a protein that normally does not possess any surface-exposed, and therefore reactive, cysteines. Extrinsic labels can then be covalently attached to this unique thiol group; thus giving the site-specific information about their immediate environment (19Heuck A.P. Johnson A.E. Cell Biochem. Biophys. 2002; 36: 89-101Crossref PubMed Scopus (35) Google Scholar). We have already performed low resolution cysteine-scanning mutagenesis, where regions of EqtII that interact with the lipid membranes were determined (12Anderluh G. Barlič A. Podlesek Z. Maček P. Pungerčar J. Gubenšek F. Zecchini M.L. Dalla Serra M. Menestrina G. Eur. J. Biochem. 1999; 263: 128-136Crossref PubMed Scopus (91) Google Scholar). In the current work we performed a detailed scanning of the region from Asp-10 to Asn-28. Mutants were modified with thiol reactive reagents and assayed by fluorescence spectroscopy and electrophysiological methods to determine the structural arrangement of this N-terminal region at each stage of pore formation. Results indicate that the whole region is transferred to the lipid-water interface during the initial binding and that the same region forms the walls of the final pore in an α-helical arrangement. Cloning, Expression, and Isolation of Cysteine Mutants—Single cysteine mutants were produced by substituting the corresponding wild-type (WT) amino acid residue with cysteine as described (12Anderluh G. Barlič A. Podlesek Z. Maček P. Pungerčar J. Gubenšek F. Zecchini M.L. Dalla Serra M. Menestrina G. Eur. J. Biochem. 1999; 263: 128-136Crossref PubMed Scopus (91) Google Scholar). Mutants were expressed from a T7-based expression vector in Escherichia coli BLR(DE3) strain and recombinant proteins purified from bacterial supernatants as described (20Anderluh G. Pungerčar J. Štrukelj B. Maček P. Gubenšek F. Biochem. Biophys. Res. Comm. 1996; 220: 437-442Crossref PubMed Scopus (109) Google Scholar). S13C, D17C, and E24C were prepared as fusion proteins using His-tagged third domain of TolA (TolAIII, a bacterial periplasmic protein) as a fusion partner. They were expressed in E. coli using the pTolT plasmid and purified from bacterial supernatants by Ni-chelate chromatography (21Anderluh G. Gokce I. Lakey J.H. Prot. Exp. Purif. 2002; 28: 173-181Crossref Scopus (28) Google Scholar). All mutants were purified to homogeneity as observed on SDS-PAGE gels. Characterization of Mutants—Mutants were tested for hemolytic activity using a microplate reader (MRX; Dynex Technologies, Dekendorf, Germany). The percentage of hemolysis was determined as described (10Malovrh P. Barlič A. Podlesek Z. Maček P. Menestrina G. Anderluh G. Biochem. J. 2000; 346: 223-232Crossref PubMed Scopus (81) Google Scholar). In short, bovine red blood cells (100 μl, OD630 = 0.5) were added to 2-fold serially diluted mutants in 100 μl of 0.13 m NaCl, 20 mm Tris-HCl, pH 7.4, and hemolysis was monitored by measuring absorbance at 630 nm for 20 min at room temperature. The ability of mutants to bind to bovine red blood cells and small unilamellar vesicles (SUV), with or without 1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-phosphocholine (7-NO-PC), at a lipid/toxin (L/T) ratio of 1:1000 was determined from the residual hemolytic activity as described (10Malovrh P. Barlič A. Podlesek Z. Maček P. Menestrina G. Anderluh G. Biochem. J. 2000; 346: 223-232Crossref PubMed Scopus (81) Google Scholar). In this test, unbound protein is detected by hemolysis assay by adding fresh erythrocytes at the end of the incubation. Hemolytic activity is observed only, when unbound protein is present in the sample (10Malovrh P. Barlič A. Podlesek Z. Maček P. Menestrina G. Anderluh G. Biochem. J. 2000; 346: 223-232Crossref PubMed Scopus (81) Google Scholar). The amount of unbound toxin was determined from the calibration curves obtained using purified mutants. For the bovine erythrocytes assay toxins, at a final 76 nm concentration, were preincubated with erythrocytes for 10 min. Thereafter, a fresh suspension of erythrocytes was added, and hemolysis measured as described above. To assay binding to SUV, 100 μl of sample after fluorescence assays (see below) was mixed with 100 μl of erythrocytes, and activity was measured as described above. IANBD Labeling—Typically, 0.5–1 mg of protein was incubated in 50 mm Tris-HCl, pH 7.1, with dithiothreitol (DTT) (toxin/DTT molar ratio 1:1) for 30 min at room temperature. Thereafter, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD) (Molecular Probes, Eugene, OR), in 5× molar excess over DTT and toxin, was added. The mixture was incubated on a magnetic stirrer at 4 °C overnight. Labeled proteins were separated from unlabeled by using a hydrophobic interaction chromatography (HIC) column (Brownlee, Aquapore) as described. The extent of protein modification was calculated from the relative areas of the two peaks that appeared at 280 nm. The first one corresponded to the unmodified and the second one to the labeled mutant. S13C, D17C, and E24C were “on-column” labeled as TolAIII fusion proteins. 0.5 ml of Ni-NTA gel slurry (Qiagen, Crawley) was placed in the column and washed with 5 ml of 20 mm β-mercaptoethanol, 20 mm NaH2PO4, 300 mm NaCl, pH 7.4. For each mutant a few milligrams of protein were incubated in the above buffer for 1 h at 4 °C. Afterwards, they were applied to the column and washed with 10 ml of the same buffer without β-mercaptoethanol. Bound mutant protein was washed with 4 ml of 5 mm IANBD and incubated in the dark at room temperature for 4 h. After labeling, the column was washed with 15 ml of enterokinase buffer (20 mm Tris-HCl, 50 mm NaCl, 2 mm CaCl2, pH 7.4). Enterokinase (Novagen) was then added (30 units for 1 mg of protein) and incubated at 30 °C for 24 h. Cleaved labeled mutants were washed from the column by 20 mm NaH2PO4, 200 mm NaCl, pH 7.4, and purified using FPLC and a MonoS ion-exchange chromatography column (Amersham Biosciences). Chemical Modification Using Methanethiosulfonate Derivatives (MTS)—Mutants were chemically modified with MTS reagents to introduce, at the thiol group, either a positive or a negative charge with (2-aminoethyl) MTS hydrobromide (MTSEA) and sodium (2-sulfonatoethyl) MTS (MTSES), respectively (both from Biotium, Inc. Fremont, CA). Mutants were preincubated overnight in a 20 molar excess of DTT. MTS reagents, freshly dissolved in H2O, were then added at 1000 molar excess to 10–50 μm mutants. After a 1-h incubation at room temperature, after which all the excess reagent had hydrolyzed, the modified samples were used for PLM experiments. Preparation of Lipid Vesicles—All lipids were from Avanti Polar Lipids (Alabaster, AL). Small unilamellar vesicles (SUV) were prepared from brain sphingomyelin (SM) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a 1:1 molar ratio. Liposomes used in lipophilic quenching experiments were prepared by using SM and DPPC, except that 20 mol% of DPPC were replaced by 7-NO-PC. Chloroform was removed by a rotary evaporator. Vesicle buffer (140 mm NaCl, 20 mm Tris-HCl, 1 mm EDTA, pH 8.5) was added to the lipid film, and the suspension was vigorously vortex-mixed in the presence of glass beads. The resulting multilamellar vesicles were converted to SUV by sonication (MSE 150W ultrasonic disintegrator) of suspension at room temperature. The SUV suspension was centrifuged at 12,000 × g for 15 min to remove titanium particles released from the probe. Vesicles were stored at 4 °C immediately after the preparation and used the next day. Fluorescence Spectroscopy—All fluorescence measurements were performed on a Jasco FP-750 spectrofluorimeter (Jasco Corporation, Japan). The sample compartment was equipped with a Peltier thermostatted single-cell holder. All experiments were done at 25 °C at a protein concentration of 250 nm. The excitation and emission slits were set to 5 nm. Tryptophan emission spectra were measured in 50 mm Tris-HCl, pH 8.0. The excitation wavelength was 295 nm, and spectra were recorded from 310–400 nm. NBD-labeled mutants in 140 mm NaCl, 20 mm Tris-HCl, 1 mm EDTA, pH 8.5 were excited at 470 nm, and the emission was scanned from 500–600 nm. Subsequently, the appropriate amount of SUV, with or without 7-NO-PC, at the final L/T ratio of 1000 was added, and the NBD emission was scanned again. The spectra were corrected for the dilution factor, and the background was subtracted using the appropriate blank with SUV. No further correction for wavelength dependence of the photomultiplier tube was done. The λmax was determined from the corrected spectra by Spectra Analysis software (part of Spectra Manager 1.53.00; Jasco Corporation). Quenching with 7-NO-PC and Iodide—The quenching efficiency, E, for the quenching of NBD emission by 7-NO-PC was calculated as follows in Equation 1, E=1−(Fquench/Fsuv)(Eq. 1) where Fquench and Fsuv represent the normalized fluorescence of IANBD at 530 nm in the presence of SUV with or without 7-NO-PC, respectively. For quenching with iodide ions, a 2.5 m stock solution of KI in 1 mm Na2S2O3 was freshly prepared prior to each experiment. The aliquots from the stock solution were added to 250 nm final NBD-labeled protein with or without the presence of SUV (L/T ratio 1000). NBD emission spectra were measured as described above. They were corrected for the emission of buffer or buffer with vesicles and for the dilution factor. The data were analyzed according to the Stern-Volmer equation shown in Equation 2, F0/F=1+KSV[Q](Eq. 2) where F0 is the fluorescence of the probe in the absence of the quencher, F is the observed fluorescence, [Q] is the concentration of iodide ions, and KSV is the collisional quenching constant. Electrical Recordings of Ion Channel Activity—Electrical properties of unmodified and MTS-modified mutants were studied on planar lipid membrane (PLM) made of 1,2 diphytanoyl-sn-glycerophosphocholine (DPhPC) and 20% (w/w) of SM, both from Avanti Polar Lipids. Mutants were added on one side (cis) to stable preformed bilayers. All experiments were started in symmetrical solutions (10 mm Tris-HCl, 100 mm KCl, pH 8.0). For the selectivity determination a 10-fold KCl gradient was formed, whereby the higher concentration was on the trans side, which was held at virtual ground. Macroscopic currents were recorded by a patch clamp amplifier (Axopatch 200, Axon Instruments). A PC equipped with a DigiData 1200 A/D converter (Axon Instruments) was used for data acquisitions. The current traces were filtered at 0.1 kHz and acquired by the computer using Axoscope 8 software (Axon Instruments). Measurements were performed at room temperature. Derivation of Helix Orientation Inside the Pore—The selectivity of the pore in MTS modification experiments was analyzed in terms of a model that relates it to the potential generated by the introduced charges. In fact these charges generate a local field at the entrance of the pore that attracts and concentrates counter ions, and therefore the partial conductance Gi of the ion species i through the pore, can be written as in Equation 3 (22Schultz S.G. Basic Principles of Membrane Transport. Cambridge University Press, New York1980Google Scholar), Gi=Kui|zi|eci(Eq. 3) where ui, zi, and ci are the mobility, valence, and local concentration of the ion i, e is the elementary charge, and K a geometrical constant (in the simplest form K = πr2/l where r and l are radius and length of the pore). The local concentration ci derives from the bulk concentration cio through Equation 4, ci=cioexp(−ziψ'pore)(Eq. 4) where Ψ′pore is the reduced potential at the center of the pore entrance (i.e. Ψ′pore/(kT/e), with k, Boltzmann constant, T, absolute temperature). From this, in the case of 1:1 salt like KCl we used, one can derive the selectivity index in Equation 5, P+/P−=G+/G−=(u+/u−)exp(−2ψ'pore)(Eq. 5) where P+, P–, G+, G–, u+, and u– are permeability, partial conductance, and mobility of cation and anion, respectively. Each mutant will have a certain Ψ′pore, that will be different depending on the residue that was substituted by the cysteine. Upon modification with MTSEA, however, there will be an additional potential generated by the new positive charge introduced at the cysteine, so that we can write Equation 6. (P+/P−)EA=(u+/u−)exp(−2(ψ'pore+ψ'EA))(Eq. 6) When the same mutant is modified with MTSES, instead, the additional potential will be generated by a negative charge as in Equation 7. (P+/P−)ES=(u+/u−)exp(−2(ψ'pore+ψ'ES))(Eq. 7) By combining Equations 6 and 7 we obtain Equation 8, (P+/P−)ES/(P+/P−)EA=exp(2(ψ'EA−ψ'ES))(Eq. 8) which is a quantity independent from the variable entrance potential of the unmodified mutant Ψ′pore. For the potential generated by a fixed charge in solution, at a distance R, we can take the Debye-Hückel expression (23Bockris J.O.M. Reddy A.K.N. Modern electrochemistry. Plenum Press, New York1970Google Scholar) in Equation 9, ψ=(Q/4πεε0)exp(−κR)/R(Eq. 9) where κ is the Debye-Hückel coefficient, Q is the fixed charge in elementary units, ϵ and ϵo the dielectric constants of water (relative) and vacuum (absolute). Because the two modifications carry one, opposite, elementary charge we can write Equation 10. (ψEA−ψES)=(e/2πεε0)exp(−κR)/R(Eq. 10) Finally, if we combine equations 8 and 10, and we take into account that the pore is formed by a tetrameric aggregate of α-helices, which we assume to be symmetrically distributed in their contribution to the field, we can write Equation 11. (P+/P−)ES/(P+/P−)EA=exp((4e2/kTπεε0)exp(−κR)/R)(Eq. 11) To calculate the distance R of the introduced charge from z, the central axis of the pore, we have to take into account its location around the α-helix and its position along the α-helix. In fact, if the helix forms an angle θ with the normal to the membrane (assumed to be parallel to the pore axis), the pore would have a funnel shape, as shown in Fig. 2. If Asp-10 is the first residue of the α-helix, located at the narrowest point of the pore, then R is given by Equation 12, R=r+d sinθ+a(1−cosα)cosθ(Eq. 12) where d = nδ (n is the position of the successive residues along the α-helix with respect to Asp-10 and δ is the pitch of the α-helix, i.e. 0.15 nm per residue), a is the radius of the α-helix (taken as 0.5 nm), α is the angular position of the n residue in the plane perpendicular to the helix axis (α = 2πn/3.6), and r is the radius of the pore at the narrowest position, where Asp-10 is located (n = 0). If we substitute Equation 12 into Equation 11, and we give to the constants their corresponding values, we obtain an expression for (P+/P–)ES/(P+/P–)EA versus n with only two free parameters, r and θ as shown in Equations 13 and 14. (P+/P−)ES/(P+/P−)EA=exp(12exp(−1.1R)/R)(Eq. 13) R=r+0.15nsinθ+0.5(1−cos1.7n)cosθ(Eq. 14) This expression was used to fit the experimental results in Fig. 8. Experimental Design—Pore formation by actinoporins is a multistep process that leads from the monomeric state in solution to a membrane-inserted aggregate composed of three or four monomers (Fig. 1C). This process includes at least three conformationally different membrane-bound states: an initial complex stabilized by the aromatic cluster (M1-state), a monomer-membrane complex with the N terminus inserted into the lipid-water interface (M2-state) (11Hong 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), and a final functional oligomeric complex (P-state). Calcein release experiments have indicated that the number of pores formed in a single vesicle depends strongly on the L/T lipid to toxin ratio (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. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (157) Google Scholar). However, even at very high toxin densities (low L/T) the proportion of pores formed was still very low in comparison to the amount of bound monomeric toxin (14Tejuca M. Dalla Serra M. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (157) Google Scholar). It is, therefore, not reasonable to study the topology of the N-terminal helix in the oligomeric pore by fluorescence techniques, as there will be always a contribution from the monomeric forms also present. For this reason, we decided to study by fluorescence only the topology of the N-terminal region 10–28 of the monomeric membrane-bound toxin. We used high L/T ratios, in order to obtain homogeneous population of the M2-state. At the conditions chosen for all subsequent experiments (SUV DPPC/SM 1:1; 250 nm EqtII; L/T molar ratio 1000) the release of calcein was only 0.4% ± 0.2 (n = 2 ± S.D.), indicating that the amount of functional pores (P-state) was negligible. There was no residual hemolytic activity in the sample after the calcein release experiment; therefore, EqtII was fully bound to the membranes (10Malovrh P. Barlič A. Podlesek Z. Maček P. Menestrina G. Anderluh G. Biochem. J. 2000; 346: 223-232Crossref PubMed Scopus (81) Google Scholar). According to the reported equilibrium constants for lipid-bound monomeric states (11Hong 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) and oligomeric P-state (14Tejuca M. Dalla Serra M. Ferreras M. Lanio M.E. Menestrina G. Biochemistry. 1996; 35: 14947-14957Crossref PubMed Scopus (157) Google Scholar) we can reasonably assume prevalence of the M2-state (about 92% of the total EqtII) over all other toxin forms. PLM experiments were employed to study the topology of the N-terminal region in the final oligomeric pore conformation, since this technique is only sensitive to ion flow through open pores. Any change in ion selectivity observed through the channels should therefore be the result of the side chain alterations introduced by cysteine mutagenesis and by subsequent chemical modifications of the thiol groups. Production and IANBD Labeling of Cysteine Mutants—Cysteine mutants were prepared by using an E. coli expression system. Intrinsic tryptophan fluorescence was measured in order to check for any conformational changes induced by the mutation. The value of the tryptophan emission maxima (λmax) for all mutants was 339 ± 1 nm, similar to that of the WT; hence we assumed that the conformation of mutants remained unaltered. Mutants were further assayed for hemolytic activity and binding to bovine red blood cells. The majority of the mutants retained >50% of the WT activity. A12C, L26C, G27C, and N28C were exceptions having only 16, 15, 17, and 0.7% of the WT activity, respectively. The reason for the decreased activity of N28C may be a lower conformational stability. As this mutant could be produced only in extremely small amounts and also precipitated over time it was not used in fluorescence studies. The lower activity of A12C and G27C was not due to low binding to the bovine erythrocytes membranes, as they bound fully, but obviously later steps in pore formation were inhibited. Of all mutants only L26C exhibited reduced binding, which resulted in reduced hemolytic activity. However, this mutant bound fully to SUV under the conditions employed for fluorescence studies. Mutants were labeled with a thiol-specific probe IANBD. G11C, S15C, and F16C were not used in fluorescence experiments due to insufficient expression levels in bacteria. No reaction between IANBD and WT was detected. The extent of labeling was mutant-dependent and ranged from 10% for L26C to 100% for I18C. L19C could not be labeled. The side chain of Leu-19 is oriented from the hydrophobic face of the helix toward the β-sandwich ensuring that it is completely buried and thus inaccessible to IANBD. Most of the labeled mutants were slightly less hemolytically active; however, the majority retained more than 50% of the hemolytic activity" @default.
• W2081846733 created "2016-06-24" @default.
• W2081846733 creator A5002934973 @default.
• W2081846733 creator A5010054488 @default.
• W2081846733 creator A5015912542 @default.
• W2081846733 creator A5016501395 @default.
• W2081846733 creator A5023705459 @default.
• W2081846733 creator A5045080266 @default.
• W2081846733 creator A5065320734 @default.
• W2081846733 creator A5085784790 @default.
• W2081846733 date "2003-06-01" @default.
• W2081846733 modified "2023-10-14" @default.
• W2081846733 title "A Novel Mechanism of Pore Formation" @default.
• W2081846733 cites W1503300581 @default.
• W2081846733 cites W1673735507 @default.
• W2081846733 cites W1965217295 @default.
• W2081846733 cites W1965642539 @default.
• W2081846733 cites W1970026235 @default.
• W2081846733 cites W1991146845 @default.
• W2081846733 cites W1995562471 @default.
• W2081846733 cites W1997256189 @default.
• W2081846733 cites W2001276190 @default.
• W2081846733 cites W2002066211 @default.
• W2081846733 cites W2005040236 @default.
• W2081846733 cites W2008045895 @default.
• W2081846733 cites W2015148410 @default.
• W2081846733 cites W2018359124 @default.
• W2081846733 cites W2018532807 @default.
• W2081846733 cites W2019333610 @default.
• W2081846733 cites W2020031720 @default.
• W2081846733 cites W2022777594 @default.
• W2081846733 cites W2023054692 @default.
• W2081846733 cites W2030683115 @default.
• W2081846733 cites W2032877221 @default.
• W2081846733 cites W2038497192 @default.
• W2081846733 cites W2039617999 @default.
• W2081846733 cites W2052589221 @default.
• W2081846733 cites W2058269689 @default.
• W2081846733 cites W2060250212 @default.
• W2081846733 cites W2070839892 @default.
• W2081846733 cites W2072220895 @default.
• W2081846733 cites W2077156283 @default.
• W2081846733 cites W2090415455 @default.
• W2081846733 cites W2090821297 @default.
• W2081846733 cites W2094095233 @default.
• W2081846733 cites W2102902733 @default.
• W2081846733 cites W2131909172 @default.
• W2081846733 cites W2146244930 @default.
• W2081846733 cites W2148131811 @default.
• W2081846733 cites W2159152993 @default.
• W2081846733 cites W2164186503 @default.
• W2081846733 cites W2169373287 @default.
• W2081846733 cites W2228156232 @default.
• W2081846733 doi "https://doi.org/10.1074/jbc.m300622200" @default.
• W2081846733 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12676945" @default.
• W2081846733 hasPublicationYear "2003" @default.
• W2081846733 type Work @default.
• W2081846733 sameAs 2081846733 @default.
• W2081846733 citedByCount "128" @default.
• W2081846733 countsByYear W20818467332012 @default.
• W2081846733 countsByYear W20818467332013 @default.
• W2081846733 countsByYear W20818467332014 @default.
• W2081846733 countsByYear W20818467332015 @default.
• W2081846733 countsByYear W20818467332016 @default.
• W2081846733 countsByYear W20818467332017 @default.
• W2081846733 countsByYear W20818467332018 @default.
• W2081846733 countsByYear W20818467332019 @default.
• W2081846733 countsByYear W20818467332020 @default.
• W2081846733 countsByYear W20818467332021 @default.
• W2081846733 countsByYear W20818467332022 @default.
• W2081846733 crossrefType "journal-article" @default.
• W2081846733 hasAuthorship W2081846733A5002934973 @default.
• W2081846733 hasAuthorship W2081846733A5010054488 @default.
• W2081846733 hasAuthorship W2081846733A5015912542 @default.
• W2081846733 hasAuthorship W2081846733A5016501395 @default.
• W2081846733 hasAuthorship W2081846733A5023705459 @default.
• W2081846733 hasAuthorship W2081846733A5045080266 @default.
• W2081846733 hasAuthorship W2081846733A5065320734 @default.
• W2081846733 hasAuthorship W2081846733A5085784790 @default.
• W2081846733 hasBestOaLocation W20818467331 @default.
• W2081846733 hasConcept C111472728 @default.
• W2081846733 hasConcept C12554922 @default.
• W2081846733 hasConcept C138885662 @default.
• W2081846733 hasConcept C185592680 @default.
• W2081846733 hasConcept C86803240 @default.
• W2081846733 hasConcept C89611455 @default.
• W2081846733 hasConceptScore W2081846733C111472728 @default.
• W2081846733 hasConceptScore W2081846733C12554922 @default.
• W2081846733 hasConceptScore W2081846733C138885662 @default.
• W2081846733 hasConceptScore W2081846733C185592680 @default.
• W2081846733 hasConceptScore W2081846733C86803240 @default.
• W2081846733 hasConceptScore W2081846733C89611455 @default.
• W2081846733 hasIssue "25" @default.
• W2081846733 hasLocation W20818467331 @default.
• W2081846733 hasOpenAccess W2081846733 @default.
• W2081846733 hasPrimaryLocation W20818467331 @default.
• W2081846733 hasRelatedWork W1531601525 @default.
• W2081846733 hasRelatedWork W1964722019 @default.

• next