Báo cáo khoa học: Structures and mode of membrane interaction of a short a helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy pdf

Eur J Biochem 269, 3869–3880 (2002) Ó FEBS 2002 doi:10.1046/j.1432-21033.2002.03080.x Structures and mode of membrane interaction of a short a helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy Ziv Oren1,*, Jagannathan Ramesh2,*, Dorit Avrahami1, N Suryaprakash2,†, Yechiel Shai1 and Raz Jelinek2 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel; 2Department of Chemistry, Ben Gurion University of the Negev, Beersheva, Israel The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as a helices or b sheets We have shown in previous studies that incorporating % 30% D-amino acids into a short a helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished However, the mechanisms underlying the unique structural features induced by incorporating D-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown In this study, we analyze in detail the structures of a model amphipathic a helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides Structural analysis reveals that the peptides have an amphipathic organization within The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of secondary structures Electrostatic interactions between the peptides and the lipids are believed to direct the polypeptides to the membrane surface, whereas completion of the folding process involves hydrophobic interactions between Correspondence to R Jelinek, Department of Chemistry, Ben Gurion University of the Negev, Beersheva 84105, Israel Fax: + 972 6472943, Tel.: + 972 6461747, E-mail: razj@bgumail.bgu.ac.il Abbreviations: ATR-FTIR, attenuated total reflectance Fourier transform infrared; Myr2Gro-PCho, dimyristoylphosphocholine; Myr2Gro-PGro, dimyristoylphosphoglycerol; DPCho, dodecylphosphocholine; egg, phosphatidylcholine; PDA, polydiacetylene; PtdEtn, E coli phosphatidylethanolamine; PtdGro, egg phosphatidylglycerol; SM, sphingomyelin; TPPI, time proportional phase increment *Note: these authors contributed equally to the paper  Note: N Suryaprakash is currently on leave from the Sophisticated Instruments Faculty, Indian Institute of Science, Bangalore, India (Received 26 April 2002, accepted June 2002) both membranes Specifically, the a helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces Further insight into the membranebinding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of a helical or b sheet structures Keywords: cytolytic peptides; membrane permeation; peptide–membrane interactions; polydiacetylene nonpolar residues and the hydrophobic core of the lipid bilayer However, these interactions require that the peptides have a defined amphipathic structure The role of the peptide secondary structure in these interactions has been studied extensively The peptides that have been studied adopted predominantly a helix or b sheet structures [1–8] Incorporation of one or two D-amino acids into the a helical regions of some of these peptides was found to destabilize the a helical structure in solution but the diastereomeric peptides retain most of their helical structure upon membrane binding [8–12] Interestingly, the incorporation of several D-amino acids into the a helical cytolytic peptides pardaxin [13] and melittin [14] clearly disrupted the a helical structure However, the resulting diastereomers retained high antibacterial activity but lost their cytotoxic effects on mammalian cells These results correlated with the ability of the peptides to bind and induce leakage preferentially from negatively charged lipid membranes A melittin diastereomer was used to analyze the role of the random coil to secondary structure transition as a driving force for membrane binding and insertion of diastereomeric peptides into lipid bilayers [7] The energetic constraints of secondary structure formation associated with D-amino acid incorporation appear to play Ó FEBS 2002 3870 Z Oren et al (Eur J Biochem 269) a role in the preferential binding of the diastereomers to the negatively charged outer surface of bacteria The role of secondary structure formation in cell selectivity was further demonstrated by a comparison of leucine–lysine short model peptides composed of either all L-amino acids or the diastereomeric analog [15] Here, we show that the electrostatic interaction between the positively charged diastereomer and negatively charged bacterial membranes might allow the peptides to cross the energy barrier and adopt a stable secondary structure that would enable them to insert into the membrane Furthermore, the results demonstrate the feasibility of a novel approach, based upon incorporation of D-amino acids into the peptide sequence, for regulating cell-selective membrane lysis through modulating the transformation from a random coil into secondary structures This approach differs from the prevalent method of varying the net positive charge or the hydrophobicity of antimicrobial peptides [16–18] The unique structural features induced by incorporating D-amino acids that enable short diastereomeric antimicrobial peptides to retain membrane binding and lytic capabilities have not been determined In this study we analyze in detail the structural properties and interactions of a model amphipathic a helical cytolytic peptide and its diastereomeric analog with zwitterionic and negatively charged membranes Previous studies of the biological functions of these peptides revealed that both peptides have similarly high antibacterial activity against most Gram-positive and Gram-negative bacteria examined, however, only the wildtype peptide exhibits hemolytic activity towards human red blood cells (hRBC) [19] Here, calculations based on highresolution NMR data yield three-dimensional structures of both peptides in membrane models Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid membrane has been obtained through tryptophan quenching experiments and the recently developed lipid/polydiacetylene (PDA) colorimetric assay [20,21] The combined NMR, FTIR, fluorescence, and colorimetric studies point to the significance of segregation between the charged and hydrophobic regions of lytic peptides to membrane binding and its disruption, compared with the formation of a helical or b sheet structures EXPERIMENTAL PROCEDURES Materials 4-Methyl benzhydrylamine resin (BHA) and butyloxycarbonyl (Boc) amino acids were purchased from Calibochem– Novabiochem (La Jolla, CA, USA) Other reagents used for peptide synthesis included trifluoroacetic acid (Sigma), methylene chloride (peptide synthesis grade, Biolab, IL, USA), dimethylformamide (peptide synthesis grade, Biolab, IL, USA), piperidine (Merck, Darmstadt, Germany), and benzotriazolyl-n-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP, Sigma) Egg phosphatidylcholine (PtdCho), egg phosphatidylglycerol (PtdGro), phosphatidylethanolamine (PtdEtn) (type V, from Escherichia coli) and cholesterol were purchased from Sigma Dimyristoylphosphocholine (Myr2Gro-PCho), dimyristoylphosphatydilglycerol (Myr2Gro-PGro), and sphingomyelin (SM) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) The diacetylene monomer 10,12-tricosadiynoic acid was purchased from GFS Chemicals (Powell, OH, USA) All other reagents were of analytical grade Buffers were prepared in double glass-distilled water d38-DPCho and d25-SDS were purchased from Cambridge Isotope Laboratories (Cambridge, MA, USA) Peptide synthesis and purification The peptides KLLLKWLLKLLK-NH2 (K4L7W) and KLllKWLlKlLK-NH2 (K4L3l4W, bold and lowercase letters indicate D-amino acids) were synthesized by a solid phase method on a 4-methyl benzhydrylamine resin (0.05 milliequivalents) [22] The resin-bound peptides were cleaved from the resins by hydrogen fluoride (HF), and after HF evaporation and washing with dry ether, they were extracted with 50% acetonitrile/water HF cleavage of the peptides bound to the 4-methyl benzhydrylamine resin resulted in C-terminus amidated peptides Each crude peptide contained one major peak, as revealed by RPHPLC, which was 50–70% pure by weight The peptides were further purified by RP-HPLC on a C18 reverse phase ˚ Bio-Rad semipreparative column (250 · 10 mm, 300 A pore size, lm particle size) The column was eluted in 40 min, using a linear gradient of 10–60% acetonitrile in water, both containing 0.05% trifluoroacetic acid (v/v), at a flow rate of 1.8 mLỈmin)1 The purified peptides were shown to be homogeneous (> 97%) by analytical HPLC The peptides were further subjected to amino acid analysis and electrospray mass spectroscopy to confirm their composition and molecular mass Preparation of liposomes Small unilamellar vesicles (SUV) were prepared by sonication of phospholipids dispersions as described in detail previously [23] Vesicles were visualized using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan) as follows A drop of vesicles was deposited on a carbon-coated grid and negatively stained with uranyl acetate Examination of the grids revealed that the vesicles were unilamellar with an average diameter of 20–50 nm [24] NMR experiments Peptides were dissolved in 90% H2O/10% 2H2O, to a total concentration of mM with d38-DPCho or d25-SDS at a lipid/peptide molar ratio of 100 : NaCl was added to a total concentration of % 10 mM Short centrifugation was carried out to remove undissolved aggregates The pH was adjusted to 4.0 in all the samples NMR experiments were carried out at 303 K to achieve optimal spectral resolution Under these conditions, there was no doubling of peaks, indicating all peptide is bound to the micelle homogeneously The NMR spectra were recorded on a Bruker DMX-500 spectrometer operating at an 11.7 Tesla magnetic field Two-dimensional (2D) NOESY [25] experiments were carried out using WATERGATE water suppression [26], with 8192 data points acquired for each free induction decay (FID), and 256 points in the indirect dimension The mixing time used, 200 ms, was confirmed to have a negligible contribution from spin-diffusion Hydrogen bonding was Ó FEBS 2002 Mode of interaction of lytic peptides (Eur J Biochem 269) 3871 evaluated using H–D exchange experiments [27] Hydrogen bonds were assigned by recording 1D 1H spectra after dissolution of lyophilized peptide/micelle assemblies in H2O; hydrogen bonds were assigned only to amide protons that still yielded visible 1H signals after more than h 2D TOCSY experiments [28], using 8192 data points acquired for each free induction decay (FID), and 256 points in the indirect dimension, were carried out using a mixing time of 125 ms, applying the MLEV-17 pulse sequence [29] All 2D NMR data were obtained in the phase-sensitive mode using the TPPI method [30] TMS was used as an external chemical shift reference The NMR spectra were processed using FELIX 98 software (MSI, Inc.) Zero-filling and a quadratic sine-bell window function were applied in both dimensions before Fourier transformation Automatic baseline corrections with a fourth order polynomial function were applied to all spectra Structure calculations Assignment of the proton resonances to their respective sites in the peptides was carried out using both TOCSY and NOESY data Cross peaks in the 2D spectra were classified according to their volume, which was referenced to the distance between the internal Trp-6 ring protons (H5-H6/ H4-H5) of the peptide Three categories were defined (strong, medium and weak), which resulted in restraints on ˚ the upper limits of proton distances of 2.7, 3.3 and 5.0 A, respectively [27] With hydrogen bonds, the distance between the amide proton and receptor carbonyl oxygen ˚ was restrained to 1.6–2.3 A, and the distance between the amide nitrogen and the carbonyl oxygen was determined as ˚ 2.3–3.2 A Structure calculations were carried out using XPLOR (version 3.851), applying a distance geometry-simulated annealing protocol [31] Initially, 40 structures were generated using a full-structure distance geometry protocol to scan the conformational space The distance geometry calculation was followed by simulated annealing in which the structures were annealed at 1000 K for 10 ps and cooled to 300 K in 50 K steps over 10 ps The Knoe was scaled at 50 kcalỈmol)1 throughout the calculations The final refinement included energy minimization (4000 steps using POWELL algorithm) The calculated structures were examined visually using the INSIGHTII (version 98.0) molecular graphics program (MSI Inc.) Quality and accuracy of calculated structures were evaluated using the program PROCHECK [32] ATR-FTIR measurements Spectra were obtained with a Bruker equinox 55 FTIR spectrometer equipped with a deuterated triglyceride sulfate (DTGS) detector and coupled with an ATR device For each spectrum, 200 or 300 scans were collected, with a resolution of cm)1 During data acquisition, the spectrometer was continuously purged with dry N2 to eliminate the spectral contribution of atmospheric water Samples were prepared as previously described [33] Briefly, a mixture of PtdCho/cholesterol [10 : (w/w), mg] alone or with peptide (% 30 lg) was deposited on a ZnSe horizontal ATR prism (80 · mm), establishing a : 60 peptide/lipid molar ratio The aperture angle of 45° yielded 25 internal reflections Before preparing the sample, we replaced the trifluoroacetate (CF3COO-) counterions, which strongly associate to the peptide, with chloride ions through several lyophilizations of the peptides in 0.1 M HCl This allowed the elimination of the strong C¼O stretching absorption band near 1673 cm)1 [34] Lipid–peptide mixtures were prepared by dissolving them together in a : MeOH/ CH2Cl2 mixture and drying under a stream of dry nitrogen while moving a Teflon bar back and forth along the ZnSe prism Polarized spectra were recorded and the respective pure lipid in each polarization was subtracted to yield the difference spectra The background for each spectrum was a clean ZnSe prism The sample was hydrated by introducing excess deuterium oxide (2H2O) into a chamber placed on top the ZnSe prism in the ATR casting, and incubating for h before obtaining the spectra H/D exchange was considered complete due to the complete shift of the amide II band Any contribution of 2H2O vapor to the absorbance spectra near the amide I peak region was eliminated by subtracting the spectra of pure lipids equilibrated with 2H2O under the same conditions ATR-FTIR data analysis Prior to curve fitting, a straight base line passing through the ordinates at 1700 and 1600 cm)1 was subtracted To resolve overlapping bands, we processed the spectra using TM PEAKFIT (Jandel Scientific, San Rafael, CA, USA) software Second-derivative spectra accompanied by 13-data-point Savitsky–Golay smoothing were calculated to identify the positions of the components bands in the spectra These wavenumbers were used as initial parameters for curve fitting with Gaussian component peaks Position, bandwidths, and amplitudes of the peaks were varied until: (a) the resulting bands were shifted by no more than cm)1 from the initial parameters; (b) all the peaks had reasonable half-widths ( 20–25 cm)1); and < (c) good agreement was achieved between the calculated sum of all components and the experimental spectra were achieved (r2 > 0.99) The relative contents of different secondary structure elements were estimated by dividing the areas of individual peaks, assigned to a particular secondary structure, by the whole area of the resulting amide I band The results of four independent experiments were averaged Lipid/polydiacetylene vesicle colorimetric assay Preparation of vesicles composed of Myr2Gro-PCho/SM/ cholesterol/polydiacetylene (PDA) (16 : 16 : : 60, w/w) and Myr2Gro-PCho/Myr2Gro-PGro/PDA (20 : 20 : 60, w/w) was carried out in a similar way as described previously [20,21] Briefly, the lipid constituents are dried together in vacuum, followed by adding deionized water and probe-sonication at around 70 °C The vesicle solution is then cooled and kept at °C overnight, and polymerized using irradiation at 254 nm The resulting solution is intense blue Samples for UV/visible measurements were prepared by adding peptides to 0.06 mL vesicle solutions at concentrations of 0.5 mM total lipid, mM Tris The pH of the solutions was 7.8 in all experiments After adding the peptides, the solutions were diluted to 0.2 mL and the spectra were obtained All measurements were carried out at 3872 Z Oren et al (Eur J Biochem 269) Ó FEBS 2002 27 °C on a Hewlett–Packard 8452 A diode-array spectrophotometer, using a cm optical path cell A quantitative value for the extent of blue-red transition is given by the colorimetric response (%CR), which is defined [21]: %CR ẳ ẵPB0 PBi =PB0 ị 100 and PB ẳ Ablue =ẵAblue ỵ Ared ; where A is the absorbance at either the ÔblueÕ component in the UV/visible spectrum (640 nm) or the ÔredÕ component (500 nm) ÔBlueÕ and ÔredÕ refer to the appearance of the material, not its actual absorbance PB0 is the red/blue ratio of the control sample (without peptides), whereas PBI is the value obtained for the vesicle-peptide solutions Fig Schiffer Edmundson wheel projection [36] of K4L7W and K4L3l4W The dotted background indicates hydrophilic amino acids (Lys), the empty background indicates hydrophobic amino acids, and the grey background indicates hydrophobic D-amino acids Tryptophan fluorescence and quenching experiments To determine the environment and the depth of penetration of the peptides, changes in the intrinsic Trp fluorescence were measured in NaCl/Pi and upon membrane binding [35,36] Emission spectra were measured on a SLMAminco, Series Spectrofluorimeter, with excitation set at 280 nm, using a nm slit, recorded in the range of 300–400 nm (4 nm slit) In these studies, SUV were used to minimize differential light-scattering effects [37], and the lipid/peptide molar ratio was kept high (1000 : 1) in order to assure that spectral contributions of free peptides would be negligible Tryptophan emission maxima Peptide (1 lM) was added to NaCl/Pi, or NaCl/Pi containing mM PtdCho/SM/cholesterol (5 : : 1, w/w) SUV The wavelength at the maximum intensity of the tryptophan emission was determined by fitting the emission spectra to a log-normal distribution Nonlinear least-squares (NLLSQ) analyses and data simulations were performed with ORIGIN 6.1 software package (Microcal, Inc., Northampton, MA, USA) Tryptophan Quenching Experiment Peptides, containing one intrinsic tryptophan residue, were added to Br-PtdCho/PtdCho/cholesterol (2.5 : 7.5 : 1, w/w) or Br-PtdCho/PtdEtn/PtdGro (2.5 : 4.5 : 3, w/w) SUV at a lipid/peptide ratio of 1000 : After the emitted fluorescence was stabilized (10–60 incubation at room temperature), an emission spectrum of the tryptophan was recorded SUVs containing either 6,7-Br-PtdCho or 9, 10-Br-PtdCho, were used Three separate experiments were conducted for each peptide In control experiments, PtdCho/cholesterol (10 : 1, w/w) or PtdEtn/PtdGro (7 : 3, w/w) SUV without Br-PtdCho were used RESULTS Figure depicts the Schiffer & Edmundson wheel projections [38] of the peptides studied, KLLLKWLLKLL–NH2 (K4L7W) and KLllKWLlKlLK-NH2 (K4L3l4W), where bold and lowercase letters indicate D-amino acids Both peptides were amidated and display a net positive charge of +5 The peptide containing only L-amino acids (K4L7W) was designed to fold into an ideal amphipathic a helix In the diastereomer, D-amino acids were substituted in positions likely to disrupt formation of a helical structure [19] Resonance assignment of the peptide and secondary structure determination Figure summarizes sequential and medium range NOEs as well as slow-exchanging amide protons for K4L7W and its diastereomer in DPCho and SDS micelles Micellar systems have been widely used as model systems for structural studies of membrane peptides and proteins [39] DPCho, in particular, was selected in this study as it resembles natural zwitterionic phospholipids, while SDS, which has been extensively used in NMR as a model for membrane environments [39,40], was employed here as a mimic for negatively charged membranes The assignment of backbone resonances assignments was carried out using conventional methods [25] based on TOCSY and NOESY spectra, with trypthophan resonances, in particular, used as the starting points in the sequential assignment analysis [25] The NOE patterns shown in Fig 2A,C feature several strong and medium NOE cross peaks The NOE patterns, combined with the appearance of hydrogen bonds, indicate relatively defined folded structures for the K4L7W peptide in both micelle environments The connectivity pattern of the diastereomer on the other hand, (Fig 2B,D), points to less ordered structures, in particular in SDS Significantly, slow-exchange protons associated with hydrogen bond formation were detected only in K4L7W, while no such protons were detected in the diastereomer This observation confirms that the all L-residue peptide contains stable helical domains Further evidence of helical structures can be inferred from the observation of medium-range NOEs, such as dNN(i (r)i + 2), dbN(i(r)i + 1), daN(i (r)i + 3) and daN(i (r)i + 4) [25] Figure superimposes the 15 lowest energy backbone structures calculated from the NMR data using a distance-geometry/simulated annealing protocol The 15 selected structures incurred no NOE violations greater ˚ than 0.5 A The statistical parameters pertaining to the calculated structures, obtained using the software PROCHECK Ó FEBS 2002 Mode of interaction of lytic peptides (Eur J Biochem 269) 3873 Fig Schematic diagrams summarizing the NOE connectivities observed for the peptides in DPCho micelles: (A) K4L7W and (B) K4L3l4W; and in SDS micelles: (C) K4L7W and (D) K4L3l4W The slowly exchanging amide protons are marked with filled circles The intensities of the NOE connectivities are indicated by the widths of the stripes [32] are shown in Table The calculated structures shown in Fig exhibit a relatively good convergence, consistent with the NOE patterns shown in Fig In particular, the superimposed structures of the L-type peptide, Fig 3A,C, clearly show that the peptide exhibits a helical structure, noticeably apparent in DPC micelles as a righthanded a helical conformation between residues 4–10 (Fig 3A) Circular-dichroism (CD) spectroscopy indicated high populations of similar helical structures of the L-peptide in both neutral PtdCho vesicles and PtdEtn/PtdGro vesicles (data not shown) In K4L3l4W, a nascent helical domain within the central region of the peptide was detected when the peptide was reconstituted in DPCho micelles, Fig 3B However, all protons in the peptide were rapidly exchanged in water, indicting that the putative helix is not stabilized More disordered structural features are apparent in the diastereomer recosntituted in SDS micelles, Fig 3D The data presented in Figs and confirm that the helical content is reduced in K4L3l4W compared with the all L-amino acids peptide Amphipathic peptide organization Further insight into the structural properties of the interactions between the peptide and membranes was obtained upon examination of the NMR-calculated average peptide conformations, displaying the relative positions of the two central lysine residues (Lys-5 and Lys-9) and adjacent leucines (Leu-4 and Leu-8), as shown in Fig The structural features previously discussed for the superimposed structures (Fig 3) are apparent in the average conformations An a helix is clearly observed for K4L7W in the DPCho micelles (Fig 4A), and other helical-type structures, albeit less defined, are observed for K4L3l4W in the DPCho micelles (Fig 4B) and K4L7W in the SDS (Fig 4C) A conformation resembling a wide turn is obtained for K4L3l4W in the SDS micelles (Fig 4D) Importantly, the positions of the leucine and lysine side-chains emphasize the amphipathic organization of the peptides The relative orientations of the side-chain clearly 3874 Z Oren et al (Eur J Biochem 269) Ó FEBS 2002 Fig Superposition of backbone atoms of the 15 lowest energy structures of (A) K4L7W in DPCho micelles; (B) K4L3l4W in DPCho micelles; (C) K4L7W in SDS micelles; (D) K4L3l4W in SDS micelles The superposition has been based upon the backbone conformation of residues 4–10 reveal segregation between charged interfaces (lysine sidechains) and hydrophobic domains (leucine side-chains) in the membrane-associated peptides Secondary structures of the peptides in zwitterionic lipid membranes determined by FTIR spectroscopy Figure depicts FTIR analysis of the two peptides in model membranes The FTIR analysis is aided by the NMR results, which allow assigning specific secondary structures to the amide I peaks of the peptides In the FTIR experiments, the peptides were incorporated into a zwitterionic lipid membrane composed of PtdCho/cholesterol (10 : 1, w/w) Helical and disordered structures might contribute to the amide I vibration at almost identical wavenumbers, and it is difficult to determine from the IR spectra alone the exact ratio between the helix and random coil populations However, exchanging hydrogen with deuterium makes it possible, in some instances, to differentiate a helical components from random structures, as the absorption of the random structure undergoes greater shifts compared to the a helical components following deuteration Thus, we examined the IR spectra of the peptides after complete deuteration The amide I region spectra of K4L7W and K4L3l4W bound to PtdCho/cholesterol (10 : 1, w/w) multibilayers are shown in Fig 5A,C, respectively The second-derivative, combined with a 13-datapoint Savitsky–Golay smoothing, was calculated in order to identify the positions of the component bands in the spectra, and are given in the corresponding panels in Fig 5B,D These wavenumbers were used as initial parameters for curve fitting with Gaussian component peaks The assignments, wavenumbers (t), and relative areas of the component peaks are further summarized in Table Assignment of the different secondary structures to the various amide I regions was based on the values taken from 10 0 0 30 50 60 70 60 1.12 1.12 1.41 1.77 0.045 0.045 0.045 0.051 ± ± ± ± 0.520 0.610 0.419 0.550 0.030 0.045 0.027 0.037 ± ± ± ± 0.457 0.657 0.757 0.687 0.0001 0.0003 0.0002 0.0006 ± ± ± ± 0.004 0.006 0.005 0.008 8 D-SDS L-SDS 96 80 90 67 D-DPC L-DPC Angles (deg) ˚ Bonds (A) H-Bonds Peptide Number of restraints 1.58 1.61 1.94 2.54 2.70 2.98 3.16 3.86 50 30 30 10 DA (%) GA (%) AA (%) MF (%) Mean RMSD to mean ˚ structure (A) MP RMSD (backbone) ˚ (A) Impropers (deg) Convergence MP RMSD (heavy atoms) ˚ (A) Structural Quality (Derived from Ramachandran map) Mode of interaction of lytic peptides (Eur J Biochem 269) 3875 Deviations from Idealized geometry Table Structral Statistics for the L-peptides and their diastereomers in DPCho and SDS micelles MP, mean pairwise MF, most favored region AA, additional allowed region GA, generously allowed region ˚ DA, disallowed region None of the structures had NOE violation greater than 0.5 A and a dihedral angle violation greater than 5° Ó FEBS 2002 a study by Jackson & Mantsch [41], the results of the NMR analysis, and the CD data obtained for K4L7W in PtdCho/ cholesterol (10 : 1, w/w) vesicles (data not shown) The amide I region between 1610 and 1628 cm)1 is characteristic of an aggregated b sheet structure and the region between 1640 and 1645 cm)1 corresponds to a random coil The amide I region of an a helical structure is located between 1650 and 1655 cm)1, and the amide I region from 1656 to 1670 cm)1 is characteristic of a 310 helix or distorted/ dynamic helix [42] The band at approximately 1624 cm)1, observed in both K4L7W and K4L3l4W, probably corresponds to small populations of peptides forming aggregated b sheets The major amide I band of K4L7W in the PtdCho/cholesterol multibilayers (% 1652 cm)1) is centered in the a helical region, similarly to data reported previously in PtdEtn/ PtdGro membranes [19] These results are in accordance with the results of the NMR data in both membrane environments Incorporation of four D-amino acids in K4L3l4W disrupts the helical structure in PtdCho/cholesterol, as confirmed by both the major amide I band shift to % 1657 cm)1, as well as the peak width, which is similar to previous reports for negatively charged membranes [19] Based on the results of the three-dimensional structure of K4L3l4W, this band might arise from a distorted/dynamic helical structure [42] Peptide interactions with membranes determined by a lipid/polydiacetylene colorimetric assay Application of the newly developed lipid/PDA colorimetric assay [20,21] further illuminates the interactions of the peptides with lipid membranes The colorimetric assay was shown to correlate blue-red transitions of lipid/PDA vesicles with the degree of membrane penetration and lipid disruption by membrane-associated peptides [21] In particular, it has been shown that the colour changes, which are due to structural rearrangements of the PDA matrix, depend upon the depth of peptide insertion into the hydrophobic bilayer lipids incorporated within the PDA vesicle framework [20] Here, the lipid/PDA vesicle assay was used to evaluate the interactions and association of the peptides with the lipid domains Figure features titration curves correlating the colorimetric response (%CR) with peptide concentrations, recorded for K4L7W and K4L3l4W, respectively, interacting with two vesicle compositions An assembly consisting of PDA, Myr2Gro-PCho, SM, and cholesterol (6 : : : mol/mol/mol/mol) resembles a zwitterionic membrane surface, while Myr2Gro-PGro/Myr2Gro-PCho/PDA aggregates have been employed to mimic negatively-charged membranes [34] The %CR is a quantitative parameter that measures the blue/red changes from the UV/vis spectra of the vesicle solution [20] Different chromatic responses are recorded following peptide interactions with the two vesicle systems In the case of Myr2Gro-PCho/SM/cholesterol/PDA, for example, the colorimetric titration curves show similar blue-red transitions induced by both peptides These results indicate that the peptides penetrate and disrupt the membrane to a similar extent However in the negatively-charged membrane model [Myr2Gro-PCho/Myr2Gro-PGro/PDA], K4L3l4W seems to induce more pronounced blue-red Ó FEBS 2002 3876 Z Oren et al (Eur J Biochem 269) tryptophan emission of K4L7W (338 ± nm) and K4L3l4W (342 ± nm) were observed, reflecting their relocation to more hydrophobic environments [45] Blue shifts in the tryptophan emission were similarly recorded for both peptides within PtdEtn/PtdGro vesicles [19] Depth of peptide penetration determined by tryptophan-quenching Fig Calculated average structures of the peptides (based on the 15 lowest energy structures presented in Fig 3), showing the orientation of residues Leu-4, Leu-8, Lys-5 and Lys-9 (A) K4L7W in DPCho micelles; (B) K4L3l4W in DPCho micelles (C) K4L7W in SDS micelles; (D) K4L3l4W in SDS micelles transitions, i.e higher %CR, compared to the L-peptide These data suggest that K4L7W penetrates deeper into the lipid bilayer compared to the diastereomer, a result that is consistent with the FTIR data discussed above Characterization of the tryptophan environment using fluorescence spectroscopy More information upon the membrane environment of the peptides was obtained by recording the fluorescence emission spectra of tryptophan [43,44] in NaCl/Pi at pH 7.4, and in the presence of vesicles composed of PtdCho/SM/ cholesterol (5 : : 1, w/w/w) In these studies, SUVs were used in order to minimize light-scattering effects [37], and a high lipid/peptide molar ratio was maintained (1000 : 1) to assure that spectral contributions of the free peptide would be negligible In buffer the tryptophan within both peptides gave rise to a fluorescence peak at around 351 nm When PtdCho/SM/cholesterol vesicles were added to the aqueous solutions containing the peptides, blue shifts in the In this set of experiments the depth of penetration of the peptides into lipid membranes was estimated through quenching of the tryptophan fluorescence by bromine, a quencher with an r6 dependence and an apparent R0 of ˚ A [35] Tryptophan, which is sensitive to its environment, has been utilized previously in combination with brominated lipids (PtdCho brominated at various positions within the alkyl chains, denoted Br-PtdCho) to evaluate peptide localization within membranes [35,36] Br-PtdCho quenchers of tryptophan fluorescence are suitable for probing membrane insertion of peptides, as they act over a short distance and not drastically perturb the lipid bilayers Significant quenching (Fig 7) was observed for both peptides with 6,7-Br-PtdCho/PtdCho and 6,7-Br-PtdCho/ PtdEtn/PtdGro (% 30–35% reduction of fluorescence signal), while less quenching was detected in the case of 9,10Br-PtdCho/PtdCho and 9,10-Br-PtdCho/PtdEtn/PtdGro (20–25% reduction in zwitterionic membranes and 5–15% in negatively charged environments) These results are consistent with the blue shifts observed in the tryptophan emission spectra decribed above, and suggest that the peptides not penetrate deeply into the membrane, but are rather located closer to the membrane interface Furthermore, the results indicate that the peptides penetrate less into negatively – charged membranes compared to zwitterionic membrane environments This might be related to the electrostatic attraction between the positive interface of the peptides and the negative headgroups of the phospholipids, which is expected to position the peptides closer to the membrane surface DISCUSSION Membrane binding and lysis by cytolytic peptides are influenced by their secondary structure In previous studies we have used FTIR spectroscopy to examine the membrane-bound structures of a group of newly designed short diastereomeric antimicrobial peptides [10,15] The data showed increased flexibility of the secondary structure of the diastereomers, compared with their all L-amino acids analogs However, FTIR spectroscopy can only provide an average and approximate measure of the secondary structure content Furthermore, assignment of secondary structures to the amide I peaks in the diastereomeric peptides has been ambiguous due to the lack of correlation with other structure determination methods In the present study we have carried out a detailed structural and functional analysis for the native [all L-amino acid] model peptide K4L7W and its diastereomeric analog The data shed new light on the structural features of the membrane-bound peptides and their organization, and point to possible permeating mechanisms of diastereomeric antimicrobial peptides Ó FEBS 2002 Mode of interaction of lytic peptides (Eur J Biochem 269) 3877 Fig FTIR spectra deconvolution of the fully deuterated amide I band (1600–1700 cm)1) of K4L7W (A) and K4L3l4W (C) in PtdCho/cholesterol (10 : 1, w/w) multibilayers The second derivatives, calculated to identify the positions of the component bands in the spectra, are shown in (B) for K4L7W and in (D) for K4L3l4W The component peaks are the result of curve-fitting using a Gaussian line shape The sums of the fitted components superimpose on the experimental amide I region spectra In (A) and (C), the continuous lines represent the experimental FTIR spectra after Savitzky–Golay smoothing; the broken lines represent the fitted components A 60 : lipid/ peptide molar ratio was used Table Assignment, wavenumbers (m), and relative areas of the component peaks determined from the deconvolution of the amide I bands of the peptides incorporated into PtdCho/cholesterol (10 : 1, w/w) multibilayers A : 60 peptide/lipid molar ratio was used The results are the average of four independent experiments All values are given as mean ± SEM Aggregated b sheet and a sheet Peptide designation PtdCho/cholesterol K4L7W K4L3l4W PtdEtn/PtdGroa K4L7W K4L3l4W a Random coil m (cm– 1) area (%) m (cm– 1) area (%) 1624 ± 1624 ± 15 ± 13 ± 1640 ± 27 ± 1621 ± 1634 ± 15 ± 25 ± a Helix Distorted/dynamic helix m (cm– 1) area (%) 1652 ± 85 ± area (%) 1657 ± 60 ± 1658 ± 1651 ± m (cm– 1) 75 ± 85 ± The results were obtain from D Avrahami [19] Incorporation of D-amino acids into a short, amphipathic a helical model peptide results in reorganization of the backbone and side-chains The NMR-calculated structures of K4L7W indicate that the peptide exhibits a right-handed a helical conformation between residues 4–10 in zwitterionic environments (Fig 3A), and a less-defined helical structure in negativelycharged micelles (Fig 3C) In the case of K4L3l4W, extended conformation that might include a nascent helical domain spanning the central region was detected (Figs 3B,D) Previous NMR studies on the effect of incorporating D-amino acids on a helical structures were conducted on a 26-residue diastereomer analog of melittin, and on an 18-residue model amphipathic a helical peptide The NMR structure of a melittin diastereomer (four acids replaced by their D-enantiomers) revealed an amphipathic a helix at its C-terminal region in trifluoroethanol/water, methanol, and DPCho/Myr2Gro-PGro micelles, similar to native melittin [11] However, double D-amino acid replacement in the middle of a model amphipathic a helical peptide resulted in the formation of two separate helices [46] Although in the above cases significant sections of the diastereomers retain their a helical structure, incorporation of four D-amino acids into K4L7W has a substantial effect upon the secondary structure, resulting in a distinct organization of the peptide backbone and side-chains that, although not a helical, still maintains its ability to disrupt membranes L-amino 3878 Z Oren et al (Eur J Biochem 269) Fig Colorimetric data for (A) Myr2Gro-PCho/SM/cholesterol/ PDA vesicles; and (B) Myr2Gro-PCho/Myr2Gro-PGro/PDA vesicles following the addition of K4L7W (solid line) and K4L3l4W (broken line) The graph depicts the change of colorimetric response (%CR, see Experimental procedures) of the vesicle solution as a function of peptide concentration Fig The quenching of tryptophan fluorescence by brominated phospholipids The experiment was conducted with two types of liposomes PtdEtn/PtdGro (7 : 3, w/w) and PtdCho/cholesterol (10 : 1, w/w) each contains 25% of either Br-PtdCho6,7 (light grey) or Br-PtdCho9,10 (dark grey) The results of the FTIR study in PtdCho/cholesterol and PtdEtn/PtdGro membranes [19] have revealed that the major amide I band of K4L3l4W is located at % 1657 cm)1, as compared to % 1652 cm)1 within the a helical peptide K4L7W (Fig and Table 2) Previous studies that examined structural changes in phospholipase A2 [42], bacteriorhodopsin, and other proteins [47,48] have correlated increased amide I frequencies in this region with distorted/ dynamic a helical structures Combined with the threedimensional structural analysis of K4L3l4W presented here, we suggest assigning the band at % 1657 cm)1 to a distorted/ dynamic helical structure Contribution of amphipathic organization and interface location to membrane disruption by the peptides One of the most intriguing observations addressed by this work was the similar high antimicrobial activities of the peptide and its diastereomer [19] The data obtained using the lipid/PDA colorimetric assay further confirm those results, and reveal that both peptides disrupt lipid mem- Ó FEBS 2002 branes to a similar degree (Fig 6) The major structural differences between the peptides point to two properties that may underlie their similar membrane-permeating activities, namely amphipathic organization and interface location The structural analysis presented here reveals that both peptides adopt amphipathic organization within the membrane In a previous study it was shown that the antimicrobial peptide tritrpticin exhibits an amphipathic organization that maximizes both electrostatic and hydrophobic interactions with the membrane, although the peptides does not display either a helical or b sheet structures [49] In the case of K4L7W the a helical structure orients the hydrophobic and polar amino acids onto separate surfaces, thus allowing simultaneous interactions of the peptides with both the hydrophobic core of the membrane and the polar headgroup region Importantly, despite the absence of a a helical structure, similar segregation between the polar and hydrophobic surfaces was observed for the diastereomer K4L3l4W (Figs and 4) The conformation of membrane-bound K4L3l4W is most likely affected by competing factors – ones that favor disordered structures on the one hand, while inducing more defined conformations, on the other hand Specifically, occurrence of disordered structures would likely result from an electrostatic repulsion between positively charged amino acids [50,51] and the destabilizing effect of D-amino acids incorporated into a helical structures In contrast, hydrophobic interactions between nonpolar amino acids and the lipid hydrocarbon core combined with the electrostatic interactions between the charged amino acids and the polar headgroup region are expected to induce formation of organized structures Our results indicate that the capability of K4L3l4W to adopt a defined amphipathic secondary structure in the membrane, despite the incorporation of D-amino acids, is most likely attributed to such hydrophobic and electrostatic interactions Numerous studies have demonstrated the important roles of the a helical structures in binding and incorporation of peptides within membranes [9,12,52,53] However, the results obtained here for K4L3l4W suggest that a stable a helical conformation might not be the essential requirement for membrane association and permeation processes The free energy of the hydrophobic stretches within amino acids is probably a major driving force for membrane binding, compensating for the reduced a helical structure Furthermore, both the blue shift in tryptophan fluorescence observed following membrane binding, and the quenching of Trp fluorescence by brominated lipids, indicate that the all-L-residue peptide and its diastereomer are located at the membrane interface Taken together, these results strongly suggest that the a helical structure is not a prerequisite for maintaining an interface localization of a peptide In summary, the structural and functional 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