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• W2130271420 abstract "The α-helix of the designed amphipathic peptide antibiotic LAH4(KKALLALALHHLAHLALHLALALKKA-NH2) strongly interacts with phospholipid membranes. The peptide is oriented parallel to the membrane surface under acidic conditions, but transmembrane at physiological pH (Bechinger, B. (1996) J. Mol. Biol. 263, 768–775). LAH4 exhibits antibiotic activities against Escherichia coli and Bacillus subtilis; the peptide does not, however, lyse human red blood cells at bacteriocidal concentrations. The antibiotic activities of LAH4 are 2 orders of magnitude more pronounced at pH 5 when compared with pH 7.5. Although peptide association at low pH is reduced when compared with pH 7.5, the release of the fluorophore calcein from large unilamellar 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles is more pronounced at pH values where LAH4 adopts an orientation along the membrane surface. The calcein release experiments thereby parallel the results obtained in antibiotic assays. Despite a much higher degree of association, calcein release activity of LAH4 is significantly decreased for negatively charged membranes. Pronounced differences in the interactions of LAH4 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine membranes also become apparent when the mechanisms of dye release are investigated. The results presented in this paper support models in which antibiotic activity is caused by detergent-like membrane destabilization, rather than pore formation by helical peptides in transmembrane alignments. The α-helix of the designed amphipathic peptide antibiotic LAH4(KKALLALALHHLAHLALHLALALKKA-NH2) strongly interacts with phospholipid membranes. The peptide is oriented parallel to the membrane surface under acidic conditions, but transmembrane at physiological pH (Bechinger, B. (1996) J. Mol. Biol. 263, 768–775). LAH4 exhibits antibiotic activities against Escherichia coli and Bacillus subtilis; the peptide does not, however, lyse human red blood cells at bacteriocidal concentrations. The antibiotic activities of LAH4 are 2 orders of magnitude more pronounced at pH 5 when compared with pH 7.5. Although peptide association at low pH is reduced when compared with pH 7.5, the release of the fluorophore calcein from large unilamellar 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles is more pronounced at pH values where LAH4 adopts an orientation along the membrane surface. The calcein release experiments thereby parallel the results obtained in antibiotic assays. Despite a much higher degree of association, calcein release activity of LAH4 is significantly decreased for negatively charged membranes. Pronounced differences in the interactions of LAH4 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine membranes also become apparent when the mechanisms of dye release are investigated. The results presented in this paper support models in which antibiotic activity is caused by detergent-like membrane destabilization, rather than pore formation by helical peptides in transmembrane alignments. KKALLALALHHLAHLALHLALALKKA-NH2 attenuated total reflection Fourier transform infrared spectroscopy large unilamellar vesicle 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol As pathogenic bacteria and fungi turn resistant against many commonly used antibiotics, considerable efforts are undertaken to develop novel ways to fight infections. Host defensive polypeptides are an integral part of the innate immune system and have been discovered in a wide variety of species, including insects, vertebrates, and humans (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1526) Google Scholar). Some of these peptides are stored in intracellular compartments and their release allows for an immediate response when infections occur. Amphipathic peptides exhibit a strong activity against a wide range of bacteria, fungi, and viruses (2Soravia E. Martini G. Zasloff M. FEBS Lett. 1988; 228: 337-340Crossref PubMed Scopus (179) Google Scholar). Examples include the naturally occurring linear polypeptides PGLa (2Soravia E. Martini G. Zasloff M. FEBS Lett. 1988; 228: 337-340Crossref PubMed Scopus (179) Google Scholar, 3Hoffmann W. Richter K. Kreil G. EMBO J. 1983; 2: 711-714Crossref PubMed Scopus (94) Google Scholar), magainins (4Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Crossref PubMed Scopus (2042) Google Scholar, 5Maloy W. Kari U. Biopolymers. 1995; 37: 105-122Crossref PubMed Scopus (463) Google Scholar), cecropins (6Steiner H. Hultmark D. Engstrom A. Bennich H. Boman H.G. Nature. 1981; 292: 246-248Crossref PubMed Scopus (1122) Google Scholar), and defensins (7Lehrer R.I. Lichtenstein A.K. Ganz T. Annu. Rev. Immunol. 1993; 11: 105-128Crossref PubMed Scopus (906) Google Scholar, 8Martin E. Ganz T. Lehrer R.I. J. Leukocyte Biol. 1995; 58: 128-136Crossref PubMed Scopus (300) Google Scholar), as well as derivatives thereof (5Maloy W. Kari U. Biopolymers. 1995; 37: 105-122Crossref PubMed Scopus (463) Google Scholar, 9Wade D. Andreu D. Mitchell S.A. Silveira A.M. Boman A. Boman H.G. Merrifield R.B. Int. J. Pept. Protein Res. 1992; 40: 429-436Crossref PubMed Scopus (159) Google Scholar, 10Wieprecht T. Dathe M. Schumann M. Krause E. Beyermann M. Bienert M. Biochemistry. 1996; 35: 10844-10853Crossref PubMed Scopus (104) Google Scholar, 11Akiyo I. Yukiko H. Ruriko S. Sanae M. Naganori N. Biol. Pharm. Bull. 1998; 20: 267-270Google Scholar). More recently interest in these peptides has further increased when their tumoricidal activity was demonstrated (12Cruciani R.A. Barker J.L. Zasloff M. Chen H.C. Colamonici O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3792-3796Crossref PubMed Scopus (361) Google Scholar, 13Jacob L. Zasloff M. Ciba Found. Symp. 1994; 186: 197-216PubMed Google Scholar, 14Haimovich B. Tanaka J.C. Biochim. Biophys. Acta. 1995; 1240: 149-158Crossref PubMed Scopus (35) Google Scholar, 15Ohsaki Y. Gazdar A.F. Chen H.C. Johnson B.E. Cancer Res. 1992; 52: 3534-3538PubMed Google Scholar, 16Soballe P.W. Maloy W.L. Myrga M.L. Jacob L.S. Herlyn M. Int. J. Cancer. 1995; 60: 280-284Crossref PubMed Scopus (68) Google Scholar). Linear peptide antibiotics, such as magainins and cecropins, are thought to express their biological activity by related mechanisms (17Segrest J.P. De L. Dohlman J.G. Brouillette C.G. Anantharamaiah G.M. Proteins. 1990; 8: 103-117Crossref PubMed Scopus (600) Google Scholar). Although they show no primary sequence homology, they are all positively charged and form amphipathic α-helices in the presence of lipid membranes. Experimental evidence suggests that direct peptide-lipid interactions are important for the expression of antibiotic activity of these substances (18Wade D. Boman A. Wahlin B. Drain C.M. Andreu D. Boman H.G. Merrifield R.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4761-4765Crossref PubMed Scopus (656) Google Scholar), rather than specific association with a chiral receptor. In particular, the first recognition and membrane association is strongly governed by electrostatic interactions with the negative surface charges of the bacterial cell wall and/or the plasma membrane (19Matsuzaki K. Harada M. Funakoshi S. Fujii N. Miyajima K. Biochim. Biophys. Acta. 1991; 1063: 162-170Crossref PubMed Scopus (207) Google Scholar, 20Wenk M.R. Seelig J. Biochemistry. 1998; 37: 3909-3916Crossref PubMed Scopus (180) Google Scholar). There is good agreement that this class of polypeptides exerts its antibiotic activity by permeabilizing the membranes of sensitive cells, which results in decoupling of the transmembrane ionic gradients and consecutively cell death (21Juretic D. Hendler R.W. Kamp F. Caughey W.S. Zasloff M. Westerhoff H.V. Biochemistry. 1994; 33: 4562-4570Crossref PubMed Scopus (41) Google Scholar, 22Westerhoff H.V. Juretic D. Hendler R.W. Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6597-6601Crossref PubMed Scopus (262) Google Scholar). The mechanisms of membrane permeabilization are, however, unknown and several models have been proposed to explain the interaction of polypeptides with lipid bilayers (23Bechinger B. J. Membr. Biol. 1997; 156: 197-211Crossref PubMed Scopus (553) Google Scholar). Electrophysiological single channel recordings are difficult to perform because amphipathic peptide antibiotics exhibit a strong propensity to destabilize lipid bilayers. In a few cases, however, stepwise fluctuations in conductivity have been observed, the step-size ranging over 3 orders of magnitude (24Cruciani R.A. Barker J.L. Durell S.R. Raghunathan G. Guy H.R. Zasloff M. Stanley E.F. Eur. J. Pharmacol. 1992; 226: 287-296Crossref PubMed Scopus (170) Google Scholar, 25Duclohier H. Molle G. Spach G. Biophys. J. 1989; 56: 1017-1021Abstract Full Text PDF PubMed Scopus (197) Google Scholar). Based on these observations a transmembrane helical bundle, with a water-filled pore lined by basic and polar residues, has been suggested to be the active configuration (26Sansom M.S. Eur. Biophys. J. 1993; 22: 105-124Crossref PubMed Scopus (188) Google Scholar). The formation of a similar peptide aggregate has been suggested to explain the pores formed by the dodecamer alamethicin (27Boheim G. J. Membr. Biol. 1974; 19: 277-303Crossref PubMed Scopus (309) Google Scholar, 28Rizzo V. Stankowski S. Schwarz G. Biochemistry. 1987; 26: 2751-2759Crossref PubMed Scopus (158) Google Scholar). These latter polypeptides lack the high charge density and the electrophysiological properties are defined much more reproducibly when compared with amphipathic peptides. The observed small cation specificity of the channels formed by positively charged peptides resulted in an extension of this first model, in which the basic transmembrane peptides together with negatively charged lipids form the pore (23Bechinger B. J. Membr. Biol. 1997; 156: 197-211Crossref PubMed Scopus (553) Google Scholar). Structural studies indicate that the large majority of charged amphipathic peptides are oriented along the bilayer surface at physiological peptide-to-lipid ratios (29Bechinger B. Zasloff M. Opella S.J. Protein Sci. 1993; 2: 2077-2084Crossref PubMed Scopus (344) Google Scholar). In addition, association of several peptides in transmembrane helical bundles with many basic lysine residues accumulating in the pore lumen seems energetically unfavorable (23Bechinger B. J. Membr. Biol. 1997; 156: 197-211Crossref PubMed Scopus (553) Google Scholar). The possibility of a detergent-like membrane disruption that is based on the bilayer destabilizing properties of amphipathic peptides has, therefore, also been taken into consideration (23Bechinger B. J. Membr. Biol. 1997; 156: 197-211Crossref PubMed Scopus (553) Google Scholar). Our present understanding of the energies involved in peptide-lipid and peptide-peptide interactions is insufficient to safely test for all possible macromolecular aggregation states that could be involved in pore formation by a merely theoretical analysis. Experiments are, therefore, required that allow one to test the validity of the suggested models. In order to gain further insight into the mechanisms of antibiotic activity and channel formation we designed synthetic model peptides and investigated their functional and biophysical properties (30Bechinger B. J. Mol. Biol. 1996; 263: 768-775Crossref PubMed Scopus (175) Google Scholar). One of them, LAH41 with the sequence KKALLALALHHLAHLALHLALALKKA-NH2, forms an amphipathic α-helix (Fig. 1). Whereas four terminal lysines serve as membrane anchors and improve the solubility of the peptide, four histidines in the central part of the peptide allow one to alter the charge of the peptide in a pH-dependent manner without altering its chemical composition. The presence of an amidated carboxyl group is a modification also found in nature and ensures that the COOH terminus is uncharged. Alanines and leucines were chosen to form a hydrophobic surface as they show a high propensity to form α-helical structures. Oriented solid-state NMR and ATR-FTIR spectroscopies indicate that the topology of LAH4 depends on the pH of the external medium (30Bechinger B. J. Mol. Biol. 1996; 263: 768-775Crossref PubMed Scopus (175) Google Scholar, 44Bechinger B. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 1999; 76: 552-563Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The protonation states of the histidines are pH-dependent, and, as a result LAH4 switches reversibly from a transmembrane alignment at neutral pH into an orientation along the bilayer surface when the surrounding medium is acidified. The midpoint of the transition between the two states is governed mainly by hydrophobic, electrostatic, and polar interactions. In case of models in which transmembrane helical aggregates form the active configuration one would, therefore, expect that this peptide is by 2 to 3 orders less active in functional assays at acidic pH when compared with neutral proton activity. We will show that this designed sequence exhibits antibiotic activity against Gram-negative and Gram-positive bacteria in a similar concentration range, as do naturally occurring polypeptides. Under the same experimental conditions, however, this family of antibiotic peptides does not induce release of hemoglobin from red blood cells. The characterization of this peptide is, therefore, not only interesting because of its potential pharmaceutical use, but the analysis of its structural and functional properties as a function of pH also allows one to obtain a more general knowledge of the mechanisms of channel formation and antibiotic activity of amphipathic peptides. The release of the fluorescent dye calcein from POPC and POPG large unilamellar vesicles is used to quantitatively characterize the pore forming activities of LAH4 in well defined model systems. We will show that the LAH4-induced release of fluorescent dye exhibits considerable quantitative and qualitative differences when added to zwitterionic or acidic model membranes. Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL), calcein and fluorescein labeled dextran (M r 3,000 and 10,000) from Molecular Probes (Leiden, The Netherlands). The peptides LAH4(M r 2,777) and LAH4-Trp12 (M r 2,849) with the sequence KKALLALALHHLAHLALHLALALKKA-NH2 and KKALLALALHHWAHLALHLALALKKA-NH2, respectively, were synthesized by solid-phase peptide synthesis on a Millipore 9060 automatic peptide synthesizer using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry. The synthetic product was purified using reversed phase high performance liquid chromatography. The identity and purity was confirmed by mass spectrometry. Escherichia coli andBacillus subtilis with catalogue numbers DSM-3948 and DSM-675, respectively, were from the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). A bacterial pre-culture in Luria broth was started from a single colony and grown overnight at 37 °C. 100 μl of this culture was used to inoculate 10 ml and grown to mid-logarithmic phase. The cells were diluted with LB to OD600 = 0.05 (approximately 4 × 107cells/ml). 30 μl of 2-fold peptide dilutions in LB medium were added to 200-μl aliquots of the bacterial suspension. This mixture was grown at 37 °C until the controls reached an OD600 of 5 (approximately 4 h). 4 ml of fresh human blood, collected in tubes containing 6 mg of EDTA, was washed three times with a buffer containing 100 mm NaCl, 2 mm EDTA, 10 mm Tris, pH 7.3, and resuspended in the same buffer to obtain a 2% suspension. 30 μl of a peptide solution, prepared as 2-fold dilutions, was added to 200 μl of the red blood cell suspension. After incubation at 37 °C for 4 h the solutions were centrifuged. The OD550 in the supernatant provides a reliable indicator of the release of hemoglobin from red blood cells. CD spectra were recorded on an auto-dichrograph mark IV (Jibon-Yvon) in the range 190–260 nm using quartz cuvettes with a path length of 0.2 mm. 10 scans were averaged and corrected for the contributions of vesicles and buffer. Small unilamellar vesicles were prepared by sonication to reduce the influence of scattering at low wavelength. The peptide/lipid ratio was 1/150 (mol/mol) at a peptide concentration of 0.23 mg/ml. The molar ellipticity was calculated usingd 10-camphorsulfonic acid (Θ290.5 = 7783 deg cm2 dmol−1, OD285 = 34.5m−1 cm−1) as in Ref. 31Chen G.C. Yang J.T. Anal. Lett. 1977; 10: 1195-1207Crossref Scopus (397) Google Scholar. For quantitative studies of peptide membrane association the tryptophan analogue of LAH4 was investigated. The tryptophan in this peptide is located at position 12 (LAH4-Trp12), therefore, it resides on the hydrophobic face approximately in the middle of the amphipatic helix (Fig. 1). Tryptophan fluorescence emission spectra were recorded in the range 290–400 nm with an excitation wavelength of 280 nm using a Perkin-Elmer 50b spectrofluorometer. Phospholipid small unilamellar vesicles, prepared by extrusion through 50-nm filters, were injected into a solution of 6 μm LAH4-Trp12 at 37 °C. The measured intensities were corrected for the contribution of light scattering in the presence of vesicles. A membrane insertion model was used to analyze the experimental data, which results in the following sigmoidal function, I=LKd+L(Imax−Io)+IoEquation 1 where I 0 and I are the fluorescent intensities at 330 nm before and after injection of the lipid vesicles, respectively, I max is the maximum intensity, L the lipid concentration, andK d the dissociation constant. Least square line fit analysis was used to extract the dissociation constants from experimental measurements of the fluorescence intensity. Dried films of phospholipids (30 μmol) were hydrated with 1 ml of a solution containing 60 mm calcein or 10 mmfluorescein-labeled dextrans, respectively, 10 mm EDTA, 100 mm NaCl, and 50 mm buffer. The buffers used were: ethanolamine, pH 8.5; Tris-HCl, pH 7.5; and acetate, pH 5.5 or pH 4.5. The suspensions were exposed to 10 freeze-thaw cycles and extruded through 200-nm filters (LipoFast, Avestin, Inc., Ottawa, Canada) (32MacDonald R.C. MacDonald R.I. Menco B. Takeshita K. Subbarao N.K. Hu L.R. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1389) Google Scholar). Fluorescent dye not enclosed within the vesicles was removed by gel filtration (Sephacryl-S300HR, Amersham Pharmacia Biotech). The final concentration of phospholipid was determined by phosphorus analysis (33Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar). The release of calcein from vesicles was followed with fluorescence spectroscopy using an emission wavelength of 517 nm. The excitation wavelength was chosen in the range 430–490 nm to obtain a quantum yield well within the optimal work range of the photo detector. When entrapped within lipid vesicles, calcein self-quenches to approximately 80% at concentrations >20 mm. Under our experimental conditions at the highest lipid concentration (30 μm) the calcein concentration in the cuvette after addition of Triton X-100 is 5 μm, which is well within the lower linear region. The release of fluorescent dye was initiated by injecting 10 μl of peptide solution into 800 μl of buffer containing the appropriate amount of large unilamellar vesicles (≤30 μm) loaded with fluorescent dye. The release of fluorescent dye was calculated according to, Rf=(Ft−F0)/(FT−F0)Equation 2 where R f is the fraction of dye released,F 0, F t, andF T are the fluorescence intensities at timest = 0, t, or after addition of 10 μl 10% reduced Triton X-100, respectively. The ratioF 0/F T ranged from 0.1 to 0.3 and is a function of the pH and the vesicular phospholipid composition. In the absence of peptide or detergent, POPC and POPG large unilamellar vesicles retained the enclosed dye for a time period of several weeks. The experiments were performed at 37 °C, well above the phase transition temperatures of POPC and POPG (both at −2 °C). Control experiments using proton-decoupled 31P solid-state NMR spectroscopy (50Bechinger B. Kinder R. Helmle M. Vogt T.C. Harzer U. Schinzel S. Biopolymers. 1999; 51Crossref PubMed Scopus (73) Google Scholar) show no evidence for major changes in the macroscopic phase properties of these phospholipid membranes due to the presence of LAH4. In order to test for the antibiotic activity of LAH4the growth of bacterial cells was determined as a function of peptide concentration (Fig. 2). At pH 5.5 and in the presence of ≥0.1 mg/ml (∼36 μm) LAH4or magainin 2 the growth rate of the Gram-negative bacterium E. coli is considerably reduced when compared with peptide-free media. At pH 7.5, however, the same LAH4 concentration barely influences the growth of E. coli cells. At this pH the histidines of LAH4 are uncharged and growth inhibition is only observed at much higher peptide concentrations (1 mg/ml). The antibiotic activity of magainin 2 is also shown in Fig. 2 and independent of pH. The minimal concentration at which magainin 2 inhibits growth of E. coli is found to be 42 μg/ml (17 μm) in agreement with previously published results (34Matsuzaki K. Sugishita K. Harada M. Fujii N. Miyajima K. Biochim. Biophys. Acta. 1997; 1327: 119-130Crossref PubMed Scopus (318) Google Scholar). To determine whether LAH4 is bacteriotoxic or bacteriostatic the number of surviving cells at the lowest peptide concentration at which full growth inhibition is observed (28 μg/ml (10 μm) at pH 5.5) was measured by counting the number of surviving cells in a dilution series on agar plates. Out of a starting density of 4 × 107 cells/ml only 103cells/ml survived. A similar result was obtained in the presence of 50 μm magainin 2. Together with the observation that the optical density at 600 nm decreases immediately after the addition of peptide, these results indicate that LAH4 is bacteriotoxic. The antibiotic activity of LAH4 is even more pronounced when added to cultures of the Gram-positive B. subtilis. Full growth inhibition is observed even at the lowest concentrations tested (0.2 μm at pH 7.5 and pH 5.5). The lowest concentration at which full growth inhibition is observed for magainin under the same experimental conditions is 26 μm. As a test for the cytotoxic activity of LAH4 against eukaryotic organisms we studied the release of hemoglobin from human red blood cells. The open symbols in Fig. 2 show the absence of hemolysis at concentrations where LAH4 or magainin exhibit antibiotic activities. The secondary structure of LAH4 was investigated under the experimental conditions that are applied during the studies presented in this paper using circular dichroism. Fig.3 A shows CD spectra of 100 μm LAH4 as a function of pH and in the absence of membranes. At acidic pH, when the histidines are charged, the peptide exhibits random coil structures. At basic pH the increased ellipticity at 222 nm indicates that helical conformations are induced. The helical content at pH 4.5, 5.5, 6.5, 7.5, and 8.5 was calculated from the ellipticity at 222 nm (35Wu C.S. Ikeda K. Yang J.T. Biochemistry. 1981; 20: 566-570Crossref PubMed Scopus (251) Google Scholar) to be 13, 26, 30, 39, and 43%, respectively. The peptide LAH4 was designed to form amphipathic or hydrophobic α-helical structures in the presence of membranes at high or low proton activity. CD spectroscopy indicates that addition of phospholipid membranes (Fig. 3 B) induces helical conformations in LAH4. The apparent helix content of LAH4 at pH 5.5 increases from 26% in the absence of membranes to 55% and 70% in the presence of POPC or POPG, respectively. This observation is also in accordance with previous results obtained by solution NMR spectroscopy in the presence of detergent micelles (30Bechinger B. J. Mol. Biol. 1996; 263: 768-775Crossref PubMed Scopus (175) Google Scholar). At pH 5.5 the formation of helical conformations is more pronounced in the presence of the negatively charged phospholipid POPG when compared with the zwitterionic POPC (Fig. 3 B). The formation of helical structures due to association with membrane interfaces and/or peptide aggregation has also been observed for magainins (36Matsuzaki K. Murase O. Tokuda H. Funakoshi S. Fujii N. Miyajima K. Biochemistry. 1994; 33: 3342-3349Crossref PubMed Scopus (291) Google Scholar), melittin (37Bello J. Bello H.R. Granados E. Biochemistry. 1982; 21: 461-465Crossref PubMed Scopus (165) Google Scholar), and other amphipathic polypeptides (38Sansom M.S.P. Progr. Biophys. Mol. Biol. 1991; 55: 139-235Crossref PubMed Scopus (436) Google Scholar). The association of LAH4 was quantitatively studied with the help of a tryptophan-labeled analogue and fluorescence spectroscopy. Fig. 4 indicates that the addition of phospholipid model membranes to a solution of LAH4-Trp12 results in a blue shift of the emission maximum wavelength (from 350 to 330 nm) and a concomitant increase in intensity at 330 nm. This result suggests that the tryptophan residue is located in the hydrophobic interior of the phospholipid membrane. Association of LAH4-Trp12 reaches equilibrium within 30 s after injection of the vesicle dispersion into the peptide solution. The spectral changes were used to determine the amount of bound peptide as a function of lipid concentration as well as the apparent dissociation constant of LAH4-Trp12 (TableI). The positively charged peptide exhibits a 2 orders of magnitude lower apparent dissociation constant in the presence of negatively charged POPG when compared with the zwitterionic phospholipid POPC. Dissociation constants with liquid crystalline phospholipid membranes in the 10−6m range are also observed for other amphipathic peptides such as magainin (39Matsuzaki K. Harada M. Handa T. Funakoshi S. Fujii N. Yajima H. Miyajima K. Biochim. Biophys. Acta. 1989; 981: 130-134Crossref PubMed Scopus (192) Google Scholar), melittin (40Beschiaschvili G. Seelig J. Biochemistry. 1990; 29: 52-58Crossref PubMed Scopus (295) Google Scholar), or synthetic model polypeptides (41Polozov I.V. Polozova A.I. Mishra V.K. Anantharamaiah G.M. Segrest J.P. Epand R.M. Biochim. Biophys. Acta. 1998; 1368: 343-354Crossref PubMed Scopus (31) Google Scholar). The POPC dissociation constant of LAH4 at pH 7.5 has been determined to be 239 μm, and corresponds to an association energy of −20 to −30 kJ/mol. The higher dissociation constant (356 μm) observed at acidic pH can be explained by the increased positive charge density of LAH4 due to histidines which augments the peptide solubility in aqueous environments.Table IDissociation (Kd) and activity constants (KA) as well as the relative activity of LAH4-Trp12 at pH 5.5 and 7.5, respectivelypH 5.5pH 7.5K dK ARelative activityK dK ARelative activityPOPC356 × 10−6310 × 10−68578239 × 10−6100 × 10−61857POPG4.7 × 10−618 × 10−663.9 × 10−63.3 × 10−61The dissociation constants were determined experimentally in the presence of 6 μm peptide, the activity constants in the presence of 0.14 μm LAH4-Trp12.K d and K A are used to calculate the relative activities of membrane-associated peptide (normalized to the value obtained in the presence of POPG at pH 7.5). The estimated errors from two to four experiments are within 10%. Open table in a new tab The dissociation constants were determined experimentally in the presence of 6 μm peptide, the activity constants in the presence of 0.14 μm LAH4-Trp12.K d and K A are used to calculate the relative activities of membrane-associated peptide (normalized to the value obtained in the presence of POPG at pH 7.5). The estimated errors from two to four experiments are within 10%. Whereas association of LAH4 with zwitterionic membranes is well described by models where the peptide is in exchange between the aqueous and the membrane phase, such a mechanistic model is insufficient when its association to POPG membranes is analyzed. The enhanced association of this basic peptide with this negatively charged membrane can, however, be accounted for by its accumulation within the Helmholtz double layer along the bilayer surface due to electrostatic attraction and subsequent intercalation into the membrane. It is noteworthy that in the absence of lipid the fluorescence maximum of 6 μm LAH4 solutions shifts to a lower wavelength when the pH values of the LAH4 solutions are increased. At the same time the fluorescence intensity measured at 330 nm is 3-fold higher at pH 7.5 as compared with pH 5.5 (Fig. 4). This shift of the fluorescence maximum is also present when the peptide is diluted 10-fold. In the presence of phospholipids the maximal fluorescence intensity and the fluorescence emission maximum are independent of the pH value. These observations suggest that at basic pH small peptide aggregates form in which the hydrophobic residues are buried. This result is in accordance with the pH-dependent increase in helicity observed during CD measurements (Fig. 3 A). The reversible pH and salt-dependent formation of aggregates (probably tetramers) has also been observed for melittin in aqueous environments (37Bello J. Bello H.R. Granados E. Biochemistry. 1982; 21: 461-465Crossref PubMed Scopus (165) Google Scholar). To characterize the functional mechanisms of membrane permeabilization in quantitative detail, the release of calcein from model membrane vesicles was investigated. Fig.5 A shows a recording of the fluorescence intensity of large unilamellar vesicles loaded with calcein. At the beginning of the recording the calcein fluorescence is significantly reduced due to self-quenching of the concentrated fluorophore (60 mm). A significant increase in fluorescence" @default.
• W2130271420 created "2016-06-24" @default.
• W2130271420 creator A5001033334 @default.
• W2130271420 creator A5012991441 @default.
• W2130271420 date "1999-10-01" @default.
• W2130271420 modified "2023-10-17" @default.
• W2130271420 title "The Interactions of Histidine-containing Amphipathic Helical Peptide Antibiotics with Lipid Bilayers" @default.
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