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• W1964927873 abstract "Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane. Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane. LPS 2The abbreviations used are: LPSlipopolysaccharideNOEnuclear Overhauser enhancementTr-NOEtransferred NOENOESYnuclear Overhauser effect spectroscopyTOCSYtotal correlation spectroscopyFTIRFourier transform infrared spectroscopyEUendotoxin unitsFITCfluorescein isothiocyanate5-DSA5-doxyl-stearic16-DSA16-doxyl-stearic acidHPLChigh pressure liquid chromatographyLAllimulus amoebocyte lysateDPCdodecylphosphocholineITCisothermal titration calorimetry. or endotoxin, a major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is critically involved in health and diseases of humans (1Raetz C.R. Whitfield C. Ann. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3381) Google Scholar, 2Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1326) Google Scholar). LPS is essential for bacterial survival through establishing an efficient permeability barrier against a variety of antimicrobial compounds including hydrophobic antibiotics, detergents, host-defense proteins, and antimicrobial peptides (3Hancock R.E. Trends Microbiol. 1997; 5: 37-42Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 4Nikaido H. Science. 1994; 264: 382-388Crossref PubMed Scopus (1262) Google Scholar). Several studies have demonstrated that LPS catalyzes folding of outer membrane proteins as a chaperone (5de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5573Crossref PubMed Scopus (65) Google Scholar, 6de, Cock H. Brandenburg K. Wiese A. Holst O. Seydel U. J. Biol. Chem. 1999; 274: 5114-5119Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 7Bulieris P.V. Behrens S. Holst O. Kleinschmidt J.H. J. Biol. Chem. 2003; 278: 9092-9099Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). lipopolysaccharide nuclear Overhauser enhancement transferred NOE nuclear Overhauser effect spectroscopy total correlation spectroscopy Fourier transform infrared spectroscopy endotoxin units fluorescein isothiocyanate 5-doxyl-stearic 16-doxyl-stearic acid high pressure liquid chromatography limulus amoebocyte lysate dodecylphosphocholine isothermal titration calorimetry. LPS, a potent inducer of innate immune systems, hence called endotoxin, is primarily responsible for lethality in sepsis and septic shock syndromes associated with serious Gram-negative infections (8Cohen J. Nature. 2002; 420: 885-891Crossref PubMed Scopus (2163) Google Scholar, 9Fink P.F. Berk J.L. Sampliner J.E. Sepsis Syndrome: Handbook of Critical Care. Little, Brown and Co., Boston1990: 619Google Scholar, 10Hardaway R.M. Am. Surg. 2000; 66: 22-29PubMed Google Scholar). Circulating LPS in bloodstream is intercepted by the phagocytic cells of the innate immune system. Once induced by LPS, these phagocytes produce proinflammatory cytokines, e.g. tumor necrosis factor-α, interleukin-6, and interleukin-1β, through the activation of a Toll-like pattern recognition receptor (11Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar, 12Lee H.K. Dunzendorfer S. Tobias P.S. J. Biol. Chem. 2004; 279: 10564-10574Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The release of cytokines in response to microbial invasion is a natural function of the innate immunity. However, an uncontrolled and overwhelming production of these cytokines may cause “endotoxic shock” or septic shock, typified by endothelial tissue damage, loss of vascular tone, coagulopathy, and multiple organ failure, often resulting in death (9Fink P.F. Berk J.L. Sampliner J.E. Sepsis Syndrome: Handbook of Critical Care. Little, Brown and Co., Boston1990: 619Google Scholar, 10Hardaway R.M. Am. Surg. 2000; 66: 22-29PubMed Google Scholar). Sepsis is the major cause of mortality in the intensive care unit, accounting for 200,000 deaths every year in the United States alone (13Martin G.S. Mannino D.M. Eaton S. Moss M. N. Engl. J. Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4831) Google Scholar). It was demonstrated that release of LPS from antibiotic-treated Gram-negative bacteria can indeed enhance sepsis (14Dofferhoff A.S.M. Buys J. Faist E. Differential Release and Impact of Antibiotic-Induced Endotoxin. Raven Press, New York1995: 11-15Google Scholar). Therefore, an effective antibiotic should not only exert antibacterial activities but also have the ability to sequester LPS and ameliorate its toxicity. Therefore, an amalgamated property of LPS-neutralizing and antimicrobial activity would be highly desirable for antimicrobial agents. Polymyxin B is a prototypical antimicrobial and antiendotoxic antibiotic; however, its neurotoxicity and nephrotoxicity limit its application to topical use (15Rustici A. Velucchi M. Faggioni R. Sironi M. Ghezzi P. Quataert S. Green B. Porro M. Science. 1993; 259: 361-365Crossref PubMed Scopus (143) Google Scholar). The increasing emergence of bacterial strains that are resistant to conventional antibiotics has initiated vital structure/function studies of membrane-perturbing cationic antimicrobial peptides (16Hancock R.E. Scott M.G. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 8856-8861Crossref PubMed Scopus (826) Google Scholar, 17Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 18Shai Y. Biopolymers. 2002; 66: 236-248Crossref PubMed Scopus (1285) Google Scholar, 19Epand R.M. Vogel H.J. Biochim. Biophys. Acta. 1999; 1462: 11-28Crossref PubMed Scopus (1151) Google Scholar, 20Dürr U.H. Sudheendra U.S. Ramamoorthy A. Biochim. Biophys. Acta. 2006; 1758: 1408-1425Crossref PubMed Scopus (754) Google Scholar). More recent studies have been conducted to understand interactions between antimicrobial peptides with LPS to gain insights into the mechanism of outer membrane perturbation, antibacterial activities, and LPS neutralization (21Rosenfeld Y. Papo N. Shai Y. J. Biol. Chem. 2006; 281: 1636-1643Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 22Papo N. Shai Y. J. Biol. Chem. 2005; 280: 10378-10387Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 23Ding L. Yang L. Weiss T.M. Waring A.J. Lehrer R.I. Huang H.W. Biochemistry. 2003; 42: 12251-12259Crossref PubMed Scopus (100) Google Scholar, 24Rosenfeld Y. Barra D. Simmaco M. Shai Y. Mangoni M.L. J. Biol. Chem. 2006; 281: 28565-28574Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 25Chen X. Howe J. Andrä J. Rössle M. Richter W. da Silva A.P. Krensky A.M. Clayberger C. Brandenburg K. Biochim. Biophys. Acta. 2007; 1768: 2421-2431Crossref PubMed Scopus (29) Google Scholar, 26Snyder D.S. McIntosh T.J. Biochemistry. 2000; 39: 11777-11787Crossref PubMed Scopus (117) Google Scholar). These studies have delineated the role of amino acid sequence properties, LPS-peptide interactions by biophysical methods, and global structural parameters, obtained by CD and FTIR. Designing synthetic peptides and elucidation of three-dimensional structures in complex with LPS would be useful for the purpose of rational development of non-toxic antisepsis and antimicrobial therapeutics. Such studies will also be potentially instructive in establishing rules by which folded structures can be stabilized on the LPS surface. Extensive work in the field of peptide design primarily focuses on mimicking secondary structures and tertiary folds of proteins. Usually, short linear peptides are often structurally flexible; however, the functions of these peptides are highly dependent on their ability to adopt folded structures upon complex formation with their cognate receptors. In this regard, designed peptides that would yield high resolution structures in complex with LPS have not been well pursued. LPS, being a negatively charged amphiphilic molecule, interacts with naturally occurring peptides or protein fragments containing basic/polar and hydrophobic amino acids, although there are considerable variations in lengths, sequences, and amino acid compositions among these peptides (27Jerala R. Porro M. Curr. Top. Med. Chem. 2004; 4: 1173-1184Crossref PubMed Scopus (76) Google Scholar, 28Scott M.G. Vreugdenhil A.C. Buurman W.A. Hancock R.E. Gold M.R. J. Immunol. 2000; 164: 549-553Crossref PubMed Scopus (250) Google Scholar). Here, we have determined the three-dimensional structures of a series of 12-residue peptides in the context of LPS. To the best of our knowledge, these results show, for the first time, that atomic resolution structures of designed peptides obtained in LPS could be correlated with their antiendotoxic activities. Furthermore, the LPS-induced structures of active, inactive, and short peptide motif, presented here, may provide building blocks for the designing novel proteins for the outer membrane. LPS of Escherichia coli 0111:B4 and fluorescein isothiocyanate (FITC)-conjugated lipopolysaccharide from E. coli 055:B5 and spin-labeled lipids 5-doxyl-stearic acid (5-DSA) and 16-doxyl-stearic acid (16-DSA) were purchased from Sigma. Peptides were synthesized commercially by GL Biochem (Shanghai, China) and further purified by a reverse-phase HPLC, WatersTM, using a C18 column (300 Å pore size, 5-μm particle size) by a linear gradient of acetonitrile/water mixture. The molecular weight of the peptides was confirmed by mass spectrometry. The ability of the designed peptides to neutralize or inhibit LPS was assessed using a quantitative chromogenic limulus amoebocyte lysate (LAL) with a QCL-1000 (Cambrex) kit. Endotoxin neutralization experiments were carried out following the protocols provided by the vendor and published else where (26Snyder D.S. McIntosh T.J. Biochemistry. 2000; 39: 11777-11787Crossref PubMed Scopus (117) Google Scholar, 27Jerala R. Porro M. Curr. Top. Med. Chem. 2004; 4: 1173-1184Crossref PubMed Scopus (76) Google Scholar). Stock solutions of peptides were prepared in pyrogen-free water provided with the kit. Peptides at concentrations of 0.01, 0.05, 0.1, 5, and 10 μm were incubated with three different endotoxin units (EU) of LPS, namely 1, 3, and 8 EU/ml (1 EU ∼0.1 ng of LPS), in a flat bottom nonpyrogenic 96-well tissue culture plate, at 37 °C for 30 min to allow peptide binding to LPS (26Snyder D.S. McIntosh T.J. Biochemistry. 2000; 39: 11777-11787Crossref PubMed Scopus (117) Google Scholar). A total of 50 μl of this mixture was then added to equal volume of LAL reagent, and the mixture was further incubated for 10 min followed by the addition of 100 μl of chromogenic substrate (Ac-Ile-Ala-Arg-p-nitroaniline). The reaction was terminated by the addition of 25% acetic acid, and the yellow color that developed due to cleavage of the substrate was measured spectrophotometrically at 410 nm using a Benchmark plus microplate spectrophotometer (Bio-Rad). The reduction of A410 as a function of peptide concentrations is directly proportional to the inhibition of LPS by the peptide (29Tack B.F. Sawai M.V. Kearney W.R. Robertson A.D. Sherman M.A. Wang W. Hong T. Boo L.M. Wu H. Waring A.J. Lehrer R.I. Eur. J. Biochem. 2002; 269: 1181-1189Crossref PubMed Scopus (94) Google Scholar, 30Li P. Wohland T. Ho B. Ding J.L. J. Biol. Chem. 2004; 279: 50150-50156Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). All assays were repeated twice, and average values are reported. Antimicrobial activities of the designed peptides were determined following a previously reported method (31Zou G. de Leeuw E. Li C. Pazgier M. Li C. Zeng P. Lu W.Y. Lubkowski J. Lu W. J. Biol. Chem. 2007; 282: 19653-19665Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, bacterial cells used for this assay, e.g. E. coli DH5alpha, Bacillus subtilis, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923, were cultured in Luria-Bertani (LB) media at 37 °C overnight. Cells were centrifuged and washed with the assay buffer (10 mm sodium phosphate buffer, pH 7.4) and diluted to an A600 of 0.2. About 50 μl of these bacterial cell suspensions were incubated, in a sterile 96-well microtiter plate, with the same volume of peptides at various concentrations, ranging from 1 to 200 μm, diluted from a stock solution of 0.3 mm (prepared in the same buffer) at 37 °C for 2 h. The cell suspensions were then plated onto Mueller-Hinton agar plates and incubated overnight. The minimum inhibitory concentration was expressed as the lowest concentration of the peptide where there was no visible growth of the bacteria. All of the fluorescence experiments were performed using a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.). To study the interactions of peptides with FITC-conjugated LPS, 0.5 μm FITC-LPS samples were excited at 480 nm, and change in the emission of FITC at 515 nm was monitored with various concentrations (0.01, 0.02, 0.1, 0.5, 1.0, 5.0, and 10 μm) of peptides. Samples were prepared in 10 mm phosphate buffer, pH 6.0. For the intrinsic Trp fluorescence studies of peptides, 5 μm of each peptide was titrated with varying concentrations of LPS or DPC in a 10 mm sodium phosphate buffer at pH 6.0. The intrinsic tryptophan fluorescence emission spectra of the peptides in their free or lipid-bound forms were acquired by exciting samples at 280 nm using band passes of 5 nm for both the excitation and the emission monochromators in a 0.1-cm path length quartz cuvette. Quenching of tryptophan fluorescence was examined following sequential additions of various concentrations (0.02–3 m) of acrylamide into solutions containing peptide (5 μm) in its free and lipid-bound forms. The results of the quenching reactions were analyzed according to the Stern-Volmer equation, F0/F = 1 + KSV[Q], where F0 and F are the fluorescence intensities at the emission maxima in the absence and presence of quencher, respectively, KSV is the Stern-Volmer quenching constant, and [Q] is the molar quencher concentration. Quenching of Trp fluorescence by spin-labeled lipids (5-DSA and 16-DSA) was used to estimate the depth of insertion of the peptides into LPS bilayer or vesicle by parallax method (32Chattopadhyay A. London E. Biochemistry. 1987; 26: 39-45Crossref PubMed Scopus (596) Google Scholar). LPS bilayer was prepared by dissolving the appropriate amount of LPS in 2:1 chloroform/methanol solution. The organic solvent was evaporated to dryness under vacuum. The lipid film was hydrated with 10 mm phosphate buffer, pH 6.0, at 60 °C and vortexed briefly. This mixture was frozen and thawed five times and extruded through a 0.1-μm membrane with the extruder (Avanti Polar Lipids, Alabaster, AL). Various concentrations of spin-labeled lipids, 5-DSA or 16-DSA, were added, from a stock solution of 1 mm (prepared in methanol) into solutions containing 5 μm peptides and 40 μm LPS vesicle. The location of the tryptophan into LPS bilayer was determined by comparing the extent of quenching observed from shallow (5-DSA) and deep (16-DSA) quenchers following the equation (29Tack B.F. Sawai M.V. Kearney W.R. Robertson A.D. Sherman M.A. Wang W. Hong T. Boo L.M. Wu H. Waring A.J. Lehrer R.I. Eur. J. Biochem. 2002; 269: 1181-1189Crossref PubMed Scopus (94) Google Scholar) Z1F=(1-CInF1F2-L212)/2L21Eq. 1 where Z1F is the difference in depth between the shallow quencher and the tryptophan residue, and F1 and F2 are the difference between the tryptophan fluorescence intensities in the presence and absence of shallow and deep quenchers, respectively. Assuming the usual surface area of the lipid to be 70 Å, C is the quencher mole fraction in unit area. L21 is the difference in depth between the two quenchers. Once Z1F is calculated, the distance of tryptophan from the center of the bilayer was calculated from ZCF = Z1F + Lcl, where Lcl is the distance from the center of the bilayer to the shallow quencher. ITC experiments were performed using a VP-ITC microcalorimeter (MicroCal Inc., Northampton, MA). All samples, dissolved in 10 mm phosphate buffer, pH 6.0, were degassed prior to use. LPS at a concentration of 0.05 mm was loaded into the sample cell (volume 1.4359 ml), and the reference cell was filled with the above mentioned buffer. Peptides, at a concentration of 1 mm, were placed into the injection. A typical titration involved 35 injections of 2.5-μl aliquots of YI12 peptides into the sample cell, at an interval of 4 min, at 25 °C. The reaction cell was stirred continuously at 300 rpm. Raw data were collected and integrated using the MicroCal Origin 5.0 software supplied with the instrument. A single site binding model was fitted to the data by non-linear least square analysis to yield the association constant (Ka) and enthalpy change (ΔH). ΔG and ΔS were calculated using the fundamental equations of thermodynamics: ΔG = −RT ln Ka and ΔS = (ΔH− ΔG)/T, respectively. To obtain information on the ability of designed peptides to dissociate LPS aggregates, dynamic light-scattering measurements were carried out in an BI-9000AT with digital autocorrelator (Brookhaven Instruments Corp., Holtsville, NY). The peptide and buffer solutions were filtered through 0.45-μm filters (Whatman Inc). Measurements were made for 1 μm LPS (without any peptides), and upon incubation with 2 μm peptides, the scattering data were collected at 90°. The data were analyzed through the standard CONTIN method using the dynamic light-scattering software supplied with the instrument. All of the NMR spectra were recorded on a Bruker DRX 600 spectrometer, equipped with cryo-probe and pulse field gradients. Data acquisition and processing were performed with the Topspin software (Bruker) running on a Linux workstation. Sequence-specific resonance assignments of the peptides were achieved from two-dimensional total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra acquired in aqueous solution containing 10% D2O at pH 4.8, 298 K. The peptide concentrations were 0.6 mm, and mixing times were 80 and 400 ms for TOCSY and NOESY, respectively. The interactions of the designed peptides with LPS were examined by recording series of one-dimensional proton NMR spectra whereby 0.6 mm peptides were titrated with various concentrations, 5, 10, and 16 μm LPS. Tr-NOESY spectra were typically obtained either at 10 μm or at 13 μm LPS, which generated a large number of Tr-NOE cross-peaks. The two-dimensional Tr-NOESY spectra were recorded at three different mixing times: 100, 150, and 200 ms with 512 increments in t1 and 2048 data points in t2. Tr-NOESY spectra were also obtained in D2O for unambiguous assignments of aromatic/aromatic or aromatic/aliphatic NOEs. The spectral width was normally 12 ppm in both dimensions. After 16 dummy scans, 72 scans were recorded per t1 increment. NMR data analyses were carried out using the program SPARKY (T. D. Goddard and D. G. Kneller, University of California, San Francisco, CA). 31P NMR spectra of LPS were recorded on a Bruker DRX 400 spectrometer at 298 K. Data acquisition and processing were performed with the Topspin software (Bruker) suite. The interactions of the designed peptides with LPS were examined by recording series of one-dimensional 31P NMR spectra whereby 0.2 mm LPS in water (pH 4.5) was titrated with various concentrations (0.1, 0.2, and 0.4 mm) of designed peptides from a stock solution that was prepared in an unbuffered water (pH 4.5). NMR structures were calculated using the DYANA program, version 1.5 (33Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar). NOE intensities were qualitatively categorized as strong, medium, and weak based on cross-peak intensities in the Tr-NOESY spectra obtained at a mixing time of 150 ms. The NOESY cross-peaks were further translated to upper bound distance limits of 2.5, 3.5, and 5.0 Å, corresponding to strong, medium, and weak intensities, respectively. Only the ϕ dihedral angles were constrained between −30 and −180° to maintain a good stereochemistry of the calculated structures. Out of the 100 structures generated, the 20 lowest energy structures were used for more analysis. FTIR spectra were recorded on a Nicolet Nexus 560 spectrometer (Thermo Fisher Scientific, Inc.) purged with N2 and equipped with a mercury-cadmium-telluride (MCT/A) detector cooled with liquid nitrogen. Attenuated total reflection (ATR) spectra were measured with a 25-reflections ATR accessory from Graseby Specac (Kent, UK) and a wire grid polarizer (0.25 mm, Graseby Specac). Approximately 200 μl of a D2O solution of LPS alone or in the presence of peptide in a 20:1 lipid/peptide molar ratio were applied onto a trapezoidal (50 × 2 × 20 mm) germanium internal reflection element. A dry, or D2O-saturated, N2 stream flowing through the ATR compartment was used to remove bulk water (low hydration) or to fully hydrate the sample (high hydration), respectively. A total of 200 scans were collected at a resolution of 4 cm−1, averaged, and processed with one-point zero filling and Happ-Genzel apodization. In an earlier study, we have determined LPS-bound structure and antiendotoxic activity of a 12-residue synthetic peptide (34Bhattacharjya S. Domadia P.N. Bhunia A. Malladi S. David S.A. Biochemistry. 2007; 46: 5864-5874Crossref PubMed Scopus (42) Google Scholar). This peptide, YVLWKRKRMIFI, was designed using a co-crystal structure of LPS/FhuA (1 QFG) an outer membrane protein. The outer membrane proteins of Gram-negative bacteria are rich in β-sheet, assuming a β-barrel topology (35Schulz G.E. Curr. Opin. Struct. Biol. 2000; 10: 443-447Crossref PubMed Scopus (246) Google Scholar). The folding and stability of these proteins are maintained by a specific environment of LPS (5de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5573Crossref PubMed Scopus (65) Google Scholar, 6de, Cock H. Brandenburg K. Wiese A. Holst O. Seydel U. J. Biol. Chem. 1999; 274: 5114-5119Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 7Bulieris P.V. Behrens S. Holst O. Kleinschmidt J.H. J. Biol. Chem. 2003; 278: 9092-9099Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In complex with LPS, the designed peptide assumed a novel amphipathic structure whereby the cationic and hydrophobic residues were segregated into distinctly different regions (34Bhattacharjya S. Domadia P.N. Bhunia A. Malladi S. David S.A. Biochemistry. 2007; 46: 5864-5874Crossref PubMed Scopus (42) Google Scholar). The C terminus of the peptide showed two consecutive β-turns, whereas the N terminus appeared to be extended. In LPS inhibition assays, the peptide showed relatively weak activity (IC50 ∼ 10 μm). Interestingly, in the LPS-bound state, residues Trp4 and Met9 of the designed peptide showed NOE contacts, indicating that they are within ≤5 Å apart. The Met residue was introduced into the primary sequence as an NMR chemical shift marker (34Bhattacharjya S. Domadia P.N. Bhunia A. Malladi S. David S.A. Biochemistry. 2007; 46: 5864-5874Crossref PubMed Scopus (42) Google Scholar). Here, we have replaced Met9 with aromatic residues, Trp, Phe, and Tyr, to enhance packing interactions with Trp4 in complex with LPS. Such substitution may result in a defined folded structure with a large hydrophobic surface of the peptide in the context of LPS with a plausible enhancement in endotoxin neutralization and antimicrobial activities (Table 1). To underscore the role of presumable aromatic-aromatic interactions, peptides containing Ala4/Ala9 and Leu4/Leu9 were also prepared (Table 1). The Leu residues were particularly introduced to determine correlation of the hydrophobicity and aromatic-aromatic packing to the folding and activities of these peptides. To understand the specific role of Trp in the structure/activity, another peptide containing Phe at positions 4 and 9 has also been made (Table 1). In addition, an octapeptide was prepared to elucidate the role(s) of the hydrophobic residues at the N and C termini (Table 1).TABLE 1Sequences, HPLC retention time, LPS neutralization (in μm) and minimum inhibitory concentration (in μm) of the designed peptidesPeptide NameSequenceRetention timeaA C18 reverse phase semi-preparative column was used. The peptides were eluted using a 60-min linear gradient of acetonitrile (10%) and water (90%) containing 0.1% trifluoroacetic acid (v/v).>80% neutralization of LPS (at 1 and 3 EU/ml)E. coliP. aeruginosa ATCC27853S. aureus ATCC25923B. subtilisminYI12WFYVLWKRKRFIFI26.170.0110301020YI12WWYVLWKRKRWIFI26.070.0160150405YI12WYYVLWKRKRYIFI24.600.0161536YI12FFYVLFKRKRFIFI26.0710bYI12FF peptide shows <40% neutralization of LPS at 10 μm concentration.50>200200>200YI12LLYVLLKRKRLIFI24.82NDcND: no detectable inhibition.50>200>200>200YI12AAYVLAKRKRAIFI21.13NDcND: no detectable inhibition.>200>200>200>200GG8WFGWKRKRFG16.50NDcND: no detectable inhibition.>200>200>200>200a A C18 reverse phase semi-preparative column was used. The peptides were eluted using a 60-min linear gradient of acetonitrile (10%) and water (90%) containing 0.1% trifluoroacetic acid (v/v).b YI12FF peptide shows <40% neutralization of LPS at 10 μm concentration.c ND: no detectable inhibition. Open table in a new tab To determine the ability of the peptides to inhibit or neutralize LPS, sensitive chromogenic LAL assays were conducted (see “Experimental Procedures”). This assay can detect endotoxin at very low concentrations down to ∼1 pm. LAL assays were conducted at three different LPS concentrations, 1, 3, and 8 EU/ml, with six different concentrations of peptides. As can be seen, peptides containing aromatic residues at positions 4 and 9, i.e. YI12WF, YI12WW, YI12WY, and YI12FF (Table 1), demonstrated the inhibition of LPS-mediated activation of LAL enzyme (Fig. 1). Peptides YI12WF, YI12WW, and YI12WY inhibited ≥80% endotoxin even at a concentration of 10 nm at 1 EU/ml (Fig. 1, top) and 3 EU/ml (Fig. 1, middle). At these LPS concentrations, ≥95% inhibition is observed at 100 nm concentrations for YI12WF, YI12WY, and YI12WW peptides (Fig. 1, top and middle). The YI12FF peptide shows a weak inhibitory activity, only ≤40% at 10 μm concentration at 1- and 3-EU/ml doses of LPS (Fig. 1, top and middle). Even at a mu" @default.
• W1964927873 created "2016-06-24" @default.
• W1964927873 creator A5013251288 @default.
• W1964927873 creator A5015514174 @default.
• W1964927873 creator A5031590221 @default.
• W1964927873 creator A5065580640 @default.
• W1964927873 creator A5087297069 @default.
• W1964927873 date "2009-08-01" @default.
• W1964927873 modified "2023-09-27" @default.
• W1964927873 title "Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides" @default.
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• W1964927873 doi "https://doi.org/10.1074/jbc.m109.013573" @default.
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