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• W2023756559 abstract "Hemolysin E (HlyE) is a 34 kDa protein toxin, recently isolated from a pathogenic strain of Escherichia coli, which is believed to exert its toxic activity via formation of pores in the target cell membrane. With the goal of understanding the involvement of different segments of hemolysin E in the membrane interaction and assembly of the toxin, a conserved, amphipathic leucine zipper-like motif has been identified. In order to evaluate the possible structural and functional roles of this segment in HlyE, a 30-residue peptide (H-205) corresponding to the leucine zipper motif (amino acid 205-234) and two mutant peptides of the same size were synthesized and labeled by fluorescent probes at their N termini. The results show that the wild-type H-205 binds to both zwitterionic (PC/Chol) and negatively charged (PC/PG/Chol) phospholipid vesicles and also self-assemble therein. Detailed membrane-binding experiments revealed that this synthetic motif (H-205) formed large aggregates and inserted into the bilayer of only negatively charged lipid vesicles but not of zwitterionic membrane. Although both the mutants bound to zwitterionic and negatively charged lipid vesicles, neither of them inserted into the lipid bilayers nor assembled in any of these lipid vesicles. Furthermore, H-205 adopted a significant helical structure in membrane mimetic environments and induced the permeation of monovalent ions and release of entrapped calcein across the phospholipid vesicles more efficiently than the mutant peptides. The results presented here indicate that this H-205 (amino acid 205-234) segment may be an important structural element in hemolysin E, which could play a significant role in the binding and assembly of the toxin in the target cell membrane and its destabilization. Hemolysin E (HlyE) is a 34 kDa protein toxin, recently isolated from a pathogenic strain of Escherichia coli, which is believed to exert its toxic activity via formation of pores in the target cell membrane. With the goal of understanding the involvement of different segments of hemolysin E in the membrane interaction and assembly of the toxin, a conserved, amphipathic leucine zipper-like motif has been identified. In order to evaluate the possible structural and functional roles of this segment in HlyE, a 30-residue peptide (H-205) corresponding to the leucine zipper motif (amino acid 205-234) and two mutant peptides of the same size were synthesized and labeled by fluorescent probes at their N termini. The results show that the wild-type H-205 binds to both zwitterionic (PC/Chol) and negatively charged (PC/PG/Chol) phospholipid vesicles and also self-assemble therein. Detailed membrane-binding experiments revealed that this synthetic motif (H-205) formed large aggregates and inserted into the bilayer of only negatively charged lipid vesicles but not of zwitterionic membrane. Although both the mutants bound to zwitterionic and negatively charged lipid vesicles, neither of them inserted into the lipid bilayers nor assembled in any of these lipid vesicles. Furthermore, H-205 adopted a significant helical structure in membrane mimetic environments and induced the permeation of monovalent ions and release of entrapped calcein across the phospholipid vesicles more efficiently than the mutant peptides. The results presented here indicate that this H-205 (amino acid 205-234) segment may be an important structural element in hemolysin E, which could play a significant role in the binding and assembly of the toxin in the target cell membrane and its destabilization. Proteinaceous toxins are often major weapons of pathogenic organisms to invade their target cells and overcome the host defense system. Hemolysin E, also known as cytolysin A (ClyA) and silent hemolysin A (SheA), is a recently identified cytotoxic agent from a pathogenic strain of Escherichia coli (1Oscarsson J. Mizunoe Y. Uhlin B.E. Haydon D.J. Mol. Microbiol. 1996; 20: 191-199Crossref PubMed Scopus (118) Google Scholar, 2Del Castillo F.J. Leal S.C. Moreno F. del Castillo I. Mol. Microbiol. 1997; 25: 107-115Crossref PubMed Scopus (90) Google Scholar, 3Green J. Baldwin M.L. Microbiology. 1997; 143: 3785-3793Crossref PubMed Scopus (56) Google Scholar). This 34 kDa protein toxin is believed to be involved in the intra- and extra-intestinal infection caused by the pathogenic strain of E. coli (3Green J. Baldwin M.L. Microbiology. 1997; 143: 3785-3793Crossref PubMed Scopus (56) Google Scholar). Hemolysin E is expressed during anaerobic growth of E. coli in mammalian intestine (3Green J. Baldwin M.L. Microbiology. 1997; 143: 3785-3793Crossref PubMed Scopus (56) Google Scholar, 4Ludwig A. Bauer S. Benz R. Bergmann B. Goebel W. Mol. Microbiol. 1999; 31: 557-567Crossref PubMed Scopus (96) Google Scholar). It is interesting to mention that hemolysin E gene although found in non-pathogenic strains of E. coli, it remains silent under laboratory conditions. However, it is positively regulated by several regulatory proteins, which include SlyA and MprA (1Oscarsson J. Mizunoe Y. Uhlin B.E. Haydon D.J. Mol. Microbiol. 1996; 20: 191-199Crossref PubMed Scopus (118) Google Scholar, 3Green J. Baldwin M.L. Microbiology. 1997; 143: 3785-3793Crossref PubMed Scopus (56) Google Scholar, 5Libby S.J. Goebel W. Ludwig A. Buchmeier N. Bowe F. Fang F.C. Guiney D.G. Songer J.G. Heffron F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 489-493Crossref PubMed Scopus (165) Google Scholar, 6Uhlin B.E. Mizunoe Y. J. Cell. Biochem. 1994; 18A: 71Google Scholar, 7Ludwig A. Tengel C. Bauer S. Bubert A. Benz R. Mollenkopf H.J. Goebel W. Mol. Gen. Genet. 1995; 249: 474-486Crossref PubMed Scopus (99) Google Scholar, 8Gomez-Gomez J.M. Biazquez J. Baquero F. Martinez J.L. Mol. Microbiol. 1996; 19: 909-910Crossref PubMed Scopus (16) Google Scholar)). This probably suggests that hemolysin E is expressed in response to appropriate environmental signals. Bacteria expressing hemolysin E are able to lyse erythrocytes from several mammalian species including human in both solid and liquid media (1Oscarsson J. Mizunoe Y. Uhlin B.E. Haydon D.J. Mol. Microbiol. 1996; 20: 191-199Crossref PubMed Scopus (118) Google Scholar, 2Del Castillo F.J. Leal S.C. Moreno F. del Castillo I. Mol. Microbiol. 1997; 25: 107-115Crossref PubMed Scopus (90) Google Scholar). Macrophages grown in tissue culture media are also lysed by an E. coli strain expressing HlyE (9Oscarsson J Mizunoe Y. Li L. Lai L.H. Wieslander A. Uhlin B.E. Mol. Microbiol. 1999; 32: 1226-1238Crossref PubMed Scopus (103) Google Scholar). Sequence comparisons show that typhoid-causing bacterium Salmonella typhi and dysentery-causing organism Shigella flexneri express highly homologous proteins to HlyE of E. coli (10Wallace A.J Stillman T.J. Atkins A. Jamieson S.J. Bullough P.A. Green J. Artymiuk P.J. Cell. 2000; 100: 265-276Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). It has been shown that the expression of HlyE in the absence of the RTX-family toxins is sufficient to give a hemolytic phenotype of E. coli (11Ralph E.T. Guest J.R. Green J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10449-10452Crossref PubMed Scopus (43) Google Scholar). Recently hlye homologous genes have been cloned from human-specific Salmonella enterica serovars Typhi and Paratyphi A and the hemolytic activity of the expressed proteins have been characterized in detail (12Oscarsson J. Westermark M. Löfdahl S. Olsen B. Palmgren H. Mizunoe Y. Wai S.N. Uhlin B.E. Infect. Immun. 2002; 70: 5759-5769Crossref PubMed Scopus (87) Google Scholar). All these data indicate that HlyE similar to other hemolysins (13Welch R.A. Dellinger E.P. Minshew B. Falkow S. Nature. 1981; 294: 665-667Crossref PubMed Scopus (199) Google Scholar, 14Welch R.A. Falkow S. Infect. Immun. 1984; 43: 156-160Crossref PubMed Google Scholar) is likely to be a significant virulence factor responsible for pathogenic infection in human. Experiments in lipid bilayer indicated that hemolysin E forms pores in membrane (4Ludwig A. Bauer S. Benz R. Bergmann B. Goebel W. Mol. Microbiol. 1999; 31: 557-567Crossref PubMed Scopus (96) Google Scholar, 7Ludwig A. Tengel C. Bauer S. Bubert A. Benz R. Mollenkopf H.J. Goebel W. Mol. Gen. Genet. 1995; 249: 474-486Crossref PubMed Scopus (99) Google Scholar). This pore-forming activity of the toxin is considered to be associated with the infection of the pathogenic organism in the target cell. Crystal structure of the water-soluble form of the toxin has been solved at high resolution, which showed that the toxin mainly consists of long helical structures (10Wallace A.J Stillman T.J. Atkins A. Jamieson S.J. Bullough P.A. Green J. Artymiuk P.J. Cell. 2000; 100: 265-276Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). In a recent structure-function study, it has been reported that the deletion of 37 amino acids, by site-directed mutagenesis, from the C-terminal rendered the toxin non-hemolytic (15Atkins A. Wyborn N.R. Wallace A.J. Stillman T.J. Black L.K. Fielding A.B. Hisakado M Artymiuk P.J. Green J. J. Biol. Chem. 2000; 275: 41150-41155Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, this loss of hemolytic activity of hemolysin E may be due to misfolding of the protein, resulted from the deletion of amino acids (15Atkins A. Wyborn N.R. Wallace A.J. Stillman T.J. Black L.K. Fielding A.B. Hisakado M Artymiuk P.J. Green J. J. Biol. Chem. 2000; 275: 41150-41155Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Although hemolysin E has been characterized biochemically and biophysically by several groups, a number of questions remain to be answered. For example, it is not known how this toxin assembles in membrane and which part of the toxin is involved in membrane interaction and pore formation. With the primary objective to find out possible membrane-interacting domains in hemolysin E, a conserved, amphipathic leucine zipper-like motif has been identified. This leucine zipper-like motif (amino acid 205-234) contains three heptads with two heptadic positions occupied by leucine and isoleucine and other two positions occupied by valine and phenylalanine. Three peptides including the wild type (H-205) and two mutants have been designed and synthesized from this hemolysin E segment. One of the mutants (Mu1-H-205) is a scrambled peptide in which the positions of two heptadic amino acids, leucine and isoleucine, were interchanged with the positions of two hydrophilic amino acids lysine and serine keeping the amino acid composition of the peptide the same as the wild type. In the other mutant (Mu2-H-205), a heptadic leucine was replaced by another hydrophobic and helix-forming amino acid alanine. In order to detect its membrane interaction and assembly in aqueous and in membrane environment, peptides were labeled by fluorescent probes NBD 1The abbreviations used are: NBD7-nitrobenz-2-oxa-1,3-diazoleFmocN-(9-fluorenyl)methoxycarbonylPBSphosphate-buffered salineTFEtrifluoroethanolSUVsmall unilamellar vesiclesPCphosphatidylcholinePGphosphatidylglycerolCholcholesterol. (7-nitrobenz-2-oxa-1,3-diazole) and rhodamine (tetramethylrhodamine, Rho). Secondary structures, membrane interaction, assembly and membrane bilayer destabilization ability of H-205 and its analogues were studied. The results obtained from these studies have been discussed in terms of the role of this conserved segment in hemolysin E-mediated binding and destabilization of the target cell membrane. 7-nitrobenz-2-oxa-1,3-diazole N-(9-fluorenyl)methoxycarbonyl phosphate-buffered saline trifluoroethanol small unilamellar vesicles phosphatidylcholine phosphatidylglycerol cholesterol. Materials—Rink amide MBHA resin (loading capacity: 0.4-0.8 mmol/g) and all the N-α-Fmoc and necessary side-chain protected amino acids were purchased from Novabiochem, Switzerland. Coupling reagents for peptide synthesis like 1-hydroxybenzotriazole (HOBT), di-isopropylcarbodiimide (DIC), 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and N,N′-diisopropylethylamine (DIPEA) were purchased from Sigma. Dichloromethane, N,N′ dimethylformamide (DMF) and piperidine were of standard grades and procured from reputed local companies. Acetonitrile (HPLC grade) was procured from Merck, India while trifluoroacetic acid, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) were purchased from Sigma. Egg phosphatidylcholine (PC) and egg phosphatidylglycerol (PG) were obtained from Northern Lipids Inc., Canada while cholesterol (Chol) was purchased from Sigma. 3,3′-dipropylthiadicarbocyanine iodide (diS-C3-5), NBD-fluoride (4-fluoro-7-nitrobenz-2-oxa-1, 3-diazole) and tetramethylrhodamine succinimidyl ester were procured from Molecular Probes (Eugene, OR). The rest of the reagents were of analytical grade and procured locally; buffers were prepared in milli Q water. Peptide Synthesis, Fluorescent Labeling, and Purification—All the peptides were synthesized manually on solid phase. Stepwise solid phase syntheses were carried out on rink amide MBHA resin (0.15 mmol) utilizing the standard Fmoc chemistry, employing DIC/HOBT or TBTU/HOBT/DIPEA coupling procedure (16Fields G.B. Noble R.L. Int. J. Pep. Prot. Res. 1990; 35: 161-214Crossref PubMed Scopus (2333) Google Scholar, 17Wild C. Oas T. Mcdanal C. Bolognesi D. Matthews T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10537-10541Crossref PubMed Scopus (482) Google Scholar, 18Bouchayer E. Stassinopoulou C.I. Ch Tzougraki Marion D. Gans P. J. Pep. Res. 2001; 57: 39-47Crossref PubMed Scopus (12) Google Scholar). De-protection of α-amino group and the coupling of amino acids were checked by Kaiser test (19Kaiser E. Colescott R.L. Bossinger C.D. Cook P. Anal. Biochem. 1970; 34: 595Crossref PubMed Scopus (3510) Google Scholar) for primary amines and chloranil test for secondary amine (20Vojkovsky T. Pept. Res. 1995; 8: 236PubMed Google Scholar). After the synthesis was over, each peptide was cleaved from the resin with simultaneous de-protection of side chains by treatment with a mixture of trifluoroacetic acid/phenol/thioanisole/1,2-ethanedithiol/water (82.5:5:5:2.5:5 v/v) for 6-7 h. Labeling at the N terminus of a peptide was achieved by a standard procedure reported earlier (21Rapaport D. Shai Y. J. Biol. Chem. 1992; 267: 6502-6509Abstract Full Text PDF PubMed Google Scholar, 22Ghosh J.K. Ovadia M. Shai Y. Biochemistry. 1997; 36: 15451-15462Crossref PubMed Scopus (48) Google Scholar). In brief, 15-20 mg of resin-bound peptide was treated with 25% piperidine (in DMF) to remove the Fmoc group from the N-terminal amino group. The resin was washed and dried. Then Fmoc de-protected resin-bound peptides were incubated with tetramethylrhodamine succinimidyl ester (2-3 equiv.) in dimethylformamide in the presence of 5% diisopropylethylamine for 48-72 h, which ultimately resulted in the formation of Nα-Rho-peptides. Similarly, resin-bound peptides were treated with NBD-fluoride (2-3 equiv.) to obtain Nα-NBD-peptides. After sufficient labeling, the resins were washed with DMF and DCM in order to remove the unreacted probe. The peptides were cleaved from the resin as above and precipitated with dry ether. All the peptides were purified by RP-HPLC on an analytical Vydac C4 column using a linear gradient of 0-80% acetonitrile in 45 min with a flow rate of 0.6 ml/min. Both acetonitrile and water contained 0.05% trifluoroacetic acid. The purified peptides were ∼90% homogeneous as shown by HPLC. Each peptide was subjected to ES-MS analysis for the detection of molecular mass. Preparation of Small Unilamellar Vesicles (SUVs)—SUVs were prepared by a standard procedure (23Shai Y. Hadari Y.R. Finkels A. J. Biol. Chem. 1991; 266: 22346-22354Abstract Full Text PDF PubMed Google Scholar, 24Ghosh J.K. Shai Y. J. Biol. Chem. 1998; 273: 7252-7259Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) as follows. Dry lipids (either PC/cholesterol (8:1 w/w) or PC/PG/cholesterol (4:4:1 w/w)) of required amounts were dissolved in CHCl3/MeOH (2:1 v/v) in small glass vial. Solvents were evaporated under a stream of nitrogen, which resulted in the formation of a thin film on the wall of glass vessel. The thin film was resuspended in buffer at a concentration of 8.2 mg/ml by vortex mixing. The lipid dispersions were then sonicated in a bath-type sonicator (Laboratory Supplies Company, New York) for 10-20 min until it became transparent. The lipid concentration was determined by phosphorus estimation (25Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Circular Dichroism (CD) Experiments—The CD spectra of peptides were recorded in phosphate-buffered saline (PBS, pH 7.4), 40% TFE, and 1% SDS by utilizing a Jasco J-710 spectropolarimeter. The spectropolarimeter was calibrated routinely with 10-camphor sulfonic acid. The samples were scanned at room temperature (∼30 °C) with the help of a capped quartz cuvette of 0.2-cm path length in the wavelength range of 250-195 nm. An average of 4-6 scans were taken for each sample with a scan speed of 20 nm/min and data interval of 0.5 nm for peptide concentration of 10-20 μm. The fractional helicities were calculated by Equation 1 (26Greenfield N. Fasman G.D. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3322) Google Scholar, 27Wu C.S.C. Ikeda K. Yang J.T. Biochemistry. 1981; 20: 566-570Crossref PubMed Scopus (251) Google Scholar),Fh=[θ]222-[θ]2220[θ]222100-[θ]2220(Eq. 1) where [θ]222 was the experimentally observed mean residue ellipticity at 222 nm. The values for [θ]222100 and [θ]2220 that correspond to 100 and 0% helix contents were considered to have mean residue ellipticity values of -32,000 and -2,000, respectively, at 222 nm (27Wu C.S.C. Ikeda K. Yang J.T. Biochemistry. 1981; 20: 566-570Crossref PubMed Scopus (251) Google Scholar). Membrane Binding Experiments—The affinities of the peptides for phospholipid vesicles were determined by binding experiments as reported earlier (28Schwarz G. Gerke H. Rizzo V. Stankowski S. Biophys. J. 1987; 52: 685-692Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 29Beschiaschvili G. Seelig J. Biochemistry. 1990; 29: 52-58Crossref PubMed Scopus (294) Google Scholar, 30Rapaport D. Shai Y. J. Biol. Chem. 1991; 266: 23769-23775Abstract Full Text PDF PubMed Google Scholar,22Ghosh J.K. Ovadia M. Shai Y. Biochemistry. 1997; 36: 15451-15462Crossref PubMed Scopus (48) Google Scholar). In brief, small unilamellar vesicles were added gradually to 0.2-0.3 μm of NBD-labeled peptide at room temperature. Fluorescence intensities of NBD-labeled peptides alone and after each addition of lipid vesicles were recorded on a PerkinElmer spectrofluorimeter LS-50B, with the excitation set at 467 nm and emission at 528 nm. The excitation and emission slits were fixed at 8 and 6 nm, respectively. The contributions of lipid to any of the recorded signal were measured by titrating the unlabeled peptide (concentration same as NBD-labeled peptide) with the same amount of lipid vesicles and were subtracted from the original signal. The binding isotherms were analyzed by Equation 2,Xb*=Kp*Cf(Eq. 2) where Xb* is defined as the molar ratio of bound peptide per 60% of the total lipid, assuming that the peptides were initially partitioned only over the outer leaflet of the SUV as suggested by Beschiaschvili and Seelig (29Beschiaschvili G. Seelig J. Biochemistry. 1990; 29: 52-58Crossref PubMed Scopus (294) Google Scholar). Kp* represents the partition coefficient and Cf indicates the concentration of the free peptide at equilibrium. Xb was calculated by extrapolating the fluorescence signal Finfinity (fluorescence signal when all the peptides were bound to lipid) from a double-reciprocal plot of F (peptide fluorescence in the presence of lipid) versus CL (lipid concentration). Fraction of peptide bound (fb) was determined by Equation 3,fb=(F-F0)/(Finfinity-F0)(Eq. 3) where F is the fluorescence of the peptide when it is bound to lipid and F0 is the fluorescence of the peptide in its unbound state. If fb is known, Cf can easily be calculated for each concentration of the lipid. Kp* can easily be determined from the slope of the plot of Xb* and Cf. Enzymatic Cleavage Experiments—In order to detect the location of a NBD-labeled peptide in its membrane-bound state, enzymatic cleavage experiments were performed as reported earlier (31Gazit E. Shai Y. Biochemistry. 1993; 32: 3429-3436Crossref PubMed Scopus (70) Google Scholar, 22Ghosh J.K. Ovadia M. Shai Y. Biochemistry. 1997; 36: 15451-15462Crossref PubMed Scopus (48) Google Scholar). In brief, lipid vesicles made of either PC/Chol or PC/PG/Chol were first added to the NBD-labeled peptide. When major portion of the peptide was bound to the lipid vesicles as detected by the saturation of the fluorescence level, proteinase-k (final concentration, 10.0 μg/ml) was added. In this experiment fluorescence of NBD-labeled peptide was recorded at 528 nm with respect to time (in s) with excitation wavelength set at 467 nm. In the control experiment proteinase-k was first added to NBD-labeled peptide, and then lipid vesicles were added. Fluorescence Resonance Energy Transfer Experiment—Fluorescence energy transfer experiments were performed with excitation wavelength set at 467 nm and emission range of 500-600 nm. Desired amount of the NBD-labeled peptide was taken in a fluorimeter cuvette. Sufficient amounts of the phospholipid vesicles were added to the NBD-labeled peptide to ensure that the peptides were bound to the membrane. Now Rho-labeled acceptor peptide was added to the donor peptide-lipid complex. Energy transfer from donor to acceptor was determined by subtracting the acceptor fluorescence in the presence of lipid and unlabeled donor from the fluorescence signal obtained in the presence of donor, acceptor and lipid vesicles. The efficiency of energy transfer (E) was determined by the decrease in donor fluorescence in the presence of the acceptor as reported earlier (32Gazit E. Shai Y. J. Biol. Chem. 1995; 270: 2571-2578Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Ghosh J.K. Ovadia M. Shai Y. Biochemistry. 1997; 36: 15451-15462Crossref PubMed Scopus (48) Google Scholar). The percentage of energy transfer was calculated by Equation 4,E={(ID0-IDA)/ID0}×100(Eq. 4) where ID0 and IDA are the fluorescence intensities of the NBD-labeled donor peptide in the absence and presence of the Rho-labeled acceptor peptide respectively at the emission maxima of the donor after correcting the light scattering of the lipid vesicles and emission of the acceptor. Membrane Permeability Assays: Dissipation of Diffusion Potential from the Small Unilamellar Lipid Vesicles—The ability of the peptides to destabilize the phospholipid bilayer was detected by their efficacy to dissipate the diffusion potential across the membrane. The experiments were performed as follows (33Sims P.J. Waggoner A.S. Wang C.H. Hoffmann J.R. Biochemistry. 1974; 13: 3315-3330Crossref PubMed Scopus (764) Google Scholar, 34Loew L.M. Rosenberg I. Bridge M. Gitler C. Biochemistry. 1983; 22: 837-844Crossref PubMed Scopus (63) Google Scholar). Lipid vesicles were prepared in K+ buffer (50 mm K2SO4/25 mm HEPES sulfate, pH 6.8). Required amounts of the lipid vesicles were mixed with isotonic (K+-free) Na+ buffer (50 mm Na2SO4/25 mm HEPES sulfate, pH 6.8) followed by the addition of the potential sensitive dye diS-C3-5. Addition of valinomycin created a negative potential inside the lipid vesicles by the selective efflux of K+ ions from the lipid vesicles. As a result of that a quenching of the fluorescence of the dye occurred. When the dye exhibited a steady fluorescence level, peptides were added. Membrane permeability of the peptide was detected by the increase in fluorescence, which resulted from the dissipation of diffusion potential. Fluorescence was monitored at 670 nm with respect to time (s) with excitation wavelength of 620 nm. The peptide-induced dissipation of diffusion potential was measured in terms of percentage of fluorescence recovery (Ft) as defined by Refs. 21Rapaport D. Shai Y. J. Biol. Chem. 1992; 267: 6502-6509Abstract Full Text PDF PubMed Google Scholar and 22Ghosh J.K. Ovadia M. Shai Y. Biochemistry. 1997; 36: 15451-15462Crossref PubMed Scopus (48) Google Scholar in Equation 5,Ft=[(It-I0)/(If-I0)]×100%(Eq. 5) where It = the observed fluorescence after the addition of a peptide at time t (10 min after the addition of the peptide), I0 = the fluorescence after the addition valinomycin and If = the total fluorescence observed before the addition of valinomycin. Calcein Release from Calcein-entrapped Lipid Vesicles—Peptide induced release of calcein from calcein-entrapped vesicles is often employed to detect the pore-forming activity of proteins and peptides. Calcein-entrapped lipid vesicles were prepared with a self-quenching concentration (60 mm) of the dye in 10 mm HEPES at pH 7.4 as reported earlier (35Allen T.M. Cleland L.G. Biochim. Biophys. Acta. 1980; 597: 418-426Crossref PubMed Scopus (433) Google Scholar, 36Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Biochemistry. 1992; 31: 12416-12423Crossref PubMed Scopus (571) Google Scholar). Briefly, thin film of lipid (either PC/PG/Chol or PC/Chol) was re-suspended in calcein solution, vortexed for 1-2 min and then sonicated in a bath-type sonicator. The non-encapsulated calcein was removed from the liposome suspension by gel filtration using a Sephadex G-50 column. Usually lipid vesicles are diluted to ∼10-fold after passing through a G-50 column. The eluted calcein-entrapped vesicles were diluted further in the same buffer to a final lipid concentration of 3.0 μm for the experiment. Peptide-induced release of calcein from the lipid vesicles was monitored by the increase in fluorescence due to the dilution of the dye from its self-quenched concentration. Fluorescence was monitored at room temperature with excitation and emission wavelengths fixed at 490 and 520 nm, respectively. Calcein release as measured by the fluorescence recovery is defined by the same equation as used to determine the dissipation of diffusion potential. However, in this case If, the total fluorescence, was determined after the addition of Triton X-100 (0.1% final concentration) to the dye-entrapped vesicle suspension. Hemolysin E is a recently identified toxin from a pathogenic strain of E. coli. Since it is a pore-forming toxin, membrane-interaction and assembly therein are the key steps, associated with its mechanism of action. Recently membrane interaction of several protein and peptide toxins like pneumolysin (37Bonev B.B. Gilbert R.J.C. Andrew P.W. Byron O. Watts A. J. Biol. Chem. 2001; 276: 5714-5719Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), Vibrio cholerae cytolysin (38Zitzer A. Zitzer O. Bhakdi S. Palmer M. J. Biol. Chem. 1999; 274: 1375-1380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), equinatoxin (39Malovrh P. Viero G. Serra M.D. Podlesek Z. Lakey J.H. Macek P Menestrina G. Anderluh G. J. Biol. Chem. 2003; 278: 22678-22685Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), E. coli α-hemolysin (40Schindel C. Zitzer A. Schulte B. Gerhards A. Stanley P. Hughes C. Koronakis V. Bhakdi S. Palmer M. Eur. J. Biochem. 2001; 268: 800-808Crossref PubMed Scopus (48) Google Scholar, 41Hyland C. Vuillard L. Hughes C. Koronakis V. J. Bacteriol. 2001; 183: 5364-5370Crossref PubMed Scopus (45) Google Scholar) B. thuringiensis δ-toxin (31Gazit E. Shai Y. Biochemistry. 1993; 32: 3429-3436Crossref PubMed Scopus (70) Google Scholar, 42Gerber D. Shai Y. J. Biol. Chem. 2000; 275: 23602-23607Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and pardaxin (21Rapaport D. Shai Y. J. Biol. Chem. 1992; 267: 6502-6509Abstract Full Text PDF PubMed Google Scholar) have been studied in detail in order to understand their mechanism of action. Although, pore-formation has been demonstrated by electrophysiological experiments (4Ludwig A. Bauer S. Benz R. Bergmann B. Goebel W. Mol. Microbiol. 1999; 31: 557-567Crossref PubMed Scopus (96) Google Scholar, 7Ludwig A. Tengel C. Bauer S. Bubert A. Benz R. Mollenkopf H.J. Goebel W. Mol. Gen. Genet. 1995; 249: 474-486Crossref PubMed Scopus (99) Google Scholar) and visualized by electron microscopy (10Wallace A.J Stillman T.J. Atkins A. Jamieson S.J. Bullough P.A. Green J. Artymiuk P.J. Cell. 2000; 100: 265-276Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), membrane-interacting segments of hemolysin E are not yet known. In order to understand the possible contribution of different parts of hemolysin E to its membrane interaction and assembly, we have identified an amphipathic leucine zipper-like motif H-205 (amino acid 205-234) in this toxin. Sequence alignment, shown in panel A of Fig. 1, clearly describes that this particular segment and the heptadic amino acids are conserved in the homologous proteins of the same family. Panel B of Fig. 1 depicts all the sequences of the peptides including their NBD- and Rho-labeled analogues, used in this study. H-205 and two mutants were synthesized by standard solid phase synthesis. Experimental mass of each of the peptides as detected by ES-MS was within 0.5 Da of the respective desired mass. Panel C of Fig. 1 shows the helical-wheel projections of H-205 and Mu1-H-205. Panel D of Fig. 1 represents the Schiffer Edmundson wheel projections of the wild-type H-205, which shows the segregation of hydrophobic and hydrophilic amino acids in opposite sides, indicating amphipathic nature of the peptide. Both the Wild Type and Mutant Peptides Derived from the Leucine Zipper-like Motif Bind to the Phospholipid Membrane—In order to detect the ability of the peptides to bind to phospholipid vesicles, they were labeled by fluorescent probe NBD. The sensitivity of the NBD probe to the dielectric constant of the mediu" @default.
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• W2023756559 date "2003-12-01" @default.
• W2023756559 modified "2023-09-30" @default.
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