Investigations into the ability of an oblique a-helical template to provide the basis for design of an antimicrobial anionic amphiphilic peptide Sarah R Dennison1, Leslie H G Morton2, Klaus Brandenburg3, Frederick Harris4 and David A Phoenix1 Faculty of Science, University of Central Lancashire, Preston, UK School of Natural Resources, University of Central Lancashire, Preston, UK Forschungszentrum Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany Department of Forensic and Investigative Science, University of Central Lancashire, Preston, UK Keywords anionic; antimicrobial; a-helical; membrane; peptide Correspondence D A Phoenix, Deans Office, Faculty of Science, University of Central Lancashire, Preston PR1 2HE, UK Fax: +44 1772 892903 Tel: +44 1772 893481 E-mail: daphoenix@uclan.ac.uk (Received 12 January 2006, revised 12 June 2006, accepted 20 June 2006) doi:10.1111/j.1742-4658.2006.05387.x AP1 (GEQGALAQFGEWL) was shown by theoretical analysis to be an anionic oblique-orientated a-helix former The peptide exhibited a monolayer surface area of 1.42 nm2, implying possession of a-helical structure at an air ⁄ water interface, and Fourier transform infrared spectroscopy (FTIR) showed the peptide to be a-helical (100%) in the presence of vesicle mimics of Escherichia coli membranes FTIR lipid-phase transition analysis showed the peptide to induce large decreases in the fluidity of these E coli membrane mimics, and Langmuir–Blodgett trough analysis found the peptide to induce large surface pressure changes in monolayer mimics of E coli membranes (4.6 mNỈm)1) Analysis of compression isotherms based on mixing enthalpy (DH) and the Gibbs free energy of mixing (DGMix) predicted that these monolayers were thermodynamically stable (DH and DGMix each negative) but were destabilized by the presence of the peptide (DH and DGMix each positive) The peptide was found to have a minimum lethal concentration of mm against E coli and was seen to cause lysis of erythrocytes at mm In combination, these data clearly show that AP1 functions as an anionic a-helical antimicrobial peptide and suggest that both its tilted peptide characteristics and the composition of its target membrane are important determinants of its efficacy of action Globally and particularly in developing countries [1], antimicrobial drug resistance has become a major problem, resulting in a decline in the effectiveness of existing antimicrobial agents [2] As a consequence, infections have been rendered more expensive and harder to treat, and epidemics have been made more difficult to control Moreover, many previously treatable infectious diseases such as tuberculosis now have greatly increased rates of morbidity and mortality [3] In response, the pharmaceutical industry has investigated a number of compounds with the potential to act as new and effective antimicrobial agents [4], ranging from photosensitizing dyes [5] to nucleosides [6] A recent focus of these investigations has been a-helical antimicrobial peptides (a-AMPs) which are components of mammalian innate immune systems [7–10] Generally, a-AMPs are cationic [11,12], which facilitates their interaction with the anionic membranes of microbial cells, and they exert their antimicrobial action by the use of nonreceptor-based mechanisms of Abbreviations a-AMP, a-helical antimicrobial peptide; AP1, GEQGALAQFGEWL; FTIR, Fourier transform infrared spectroscopy; Ole2PtdEtn, dioleoylphosphatidylethanolamine; Ole2PtdGro, dioleoylphosphatidylglycerol; SUV, small unilamellar vesicle 3792 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS S R Dennison et al membrane invasion [13,14] The relatively nonspecific nature of these mechanisms renders the development of acquired microbial resistance to a-AMPs unlikely, although several mechanisms of inherent resistance to these peptides have been reported [11,15,16] The most common of these mechanisms is exhibited by both Gram-positive and Gram-negative pathogens and effectively involves the reduction of anionic lipid concentrations in the bacterial cell envelope, thereby inhibiting the membrane-binding ability of cationic a-AMPs [17–19] Most recently, theoretical studies have suggested that many a-AMPs may destabilize bacterial membranes by the use of oblique orientated a-helical structure [20], which has been experimentally demonstrated for the amphibian a-AMPs: aurein 1.2, citropin 1.1 and caerin 1.1 [21] These a-helices have been described in a variety of proteins and peptides, most commonly viral protein segments, and are differentiated from other classes of membrane-interactive a-helices in that they possess a hydrophobicity gradient along the a-helical long axis This structural feature causes an a-helix to penetrate membranes at a shallow angle of 30–60 °, thereby disturbing membrane lipid organization and compromising bilayer integrity [22,23] Among the a-AMPs predicted to form obliqueorientated a-helices [12] are a small number that are negatively charged, such as the amphibian peptide maximin H5 [24] It has been suggested that anionic a-AMPs may have evolved to counter microbe resistance to cationic a-AMPs, which would seem to make these former peptides well suited for development as novel antimicrobial agents directed against such organisms [24,25] There appears to have been little research into the mode of membrane interaction used by anionic a-AMPs, although photodynamic antimicrobial studies have shown nonpeptide anionic molecules to be effective against Gram-negative bacteria because of their ability to penetrate the membranes of these organisms [26,27] Pathogenic Gram-negative bacteria are becoming increasingly problematic in areas ranging from health care to the food industry [28–31], therefore in this study we analysed a novel synthetic peptide, AP1 (GEQGALAQFGEWL), as a potential anionic a-AMP against Escherichia coli The sequence of AP1 was designed to form a membrane-interactive oblique orientated a-helix, shown here by theoretical analysis, and Fourier transform infrared spectroscopy (FTIR) confirmed that the peptide was a-helical in the presence of lipid vesicles that mimicked membranes of E coli A standard toxicity assay showed that AP1 inactivated the organism, and the use of Antimicrobial properties of an anionic a-helical peptide Langmuir-Blodgett troughs showed that the peptide inserted strongly into lipid monolayers that mimicked E coli membranes Compression isotherm analysis indicated that lipid monolayers mimicking E coli membranes were thermodynamically stable but were destabilized by the presence of AP1 FTIR lipid-phase transition analysis showed that the peptide induced changes in the membrane fluidity of E coli membranes, which were consistent with penetration of the hydrophobic core of these membranes AP1 was found to lyse erythrocyte membranes, and, on the basis of these combined data, it is suggested that the peptide functions as an anionic membrane-interactive a-AMP These data also suggest that the antimicrobial activity of AP1 depends on both the structural characteristics of its tilted peptide architecture and the lipid packing of its target membrane Results The influenza HA2 fusion peptide is known to form a membrane-interactive oblique orientated a-helix [32], a secondary-structural motif recently postulated to feature in the action of a range of a-AMPs [20] A segment of the HA2 peptide (GLFGAIAGFIENG), which is key to its structure and underlying hydrophobicity gradient, was used as a basis for the hydrophobicity gradient of the AP1 peptide, thereby giving these peptides 62% sequence homology The sequence of AP1 is predicted to produce an a-helical peptide with structural features that are characteristic of both this oblique orientated a-helix and the established anionic a-AMP, maximin H5 Figure 1A shows that, when the sequences of these three peptides were analysed using extended hydrophobic moment plot methodology, the resulting data points were proximal and, along with those of % 50% of the a-AMPs studied, are candidates to form oblique orientated a-helices Figure 1B shows that, in an a-helical conformation, AP1 would possess a hydrophobic arc size of 90 °, and Fig indicates that this value (Fig 2A) and the mean hydrophobic moment of the peptide (0.35; Fig 2B) are highly comparable to those of maximin H5 However, Fig 2A,B also show that, for both peptides, these values fall in the lower quartile of those observed for the cationic a-AMPs tested Monolayer analysis showed that increasing concentrations of AP1 in the subphase of a LangmuirBlodgett trough led to progressively greater interfacial surface pressures until, at 20 lm peptide, a maximal value of 11.5 mNỈm)1 was observed (Fig 3) Above this peptide concentration, surface pressures were effectively constant, which indicates that 20 lm AP1 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3793 Antimicrobial properties of an anionic a-helical peptide 12 1.5 Surface pressure (mN·m-1) A S R Dennison et al 1.25 0.75 0.5 10 0.25 10 15 20 25 AP1 concentration (M) 30 35 -1 -0.5 0.5 1.5 B L6 G10 Q3 L13 A7 Fig AP1 surface pressure as a function of peptide concentration Increasing concentrations of AP1 were injected into a Tris ⁄ HCl buffer subphase (10 mM, pH 7.5) of a Langmuir-Blodgett system At each AP1 concentration, the peptide was allowed to equilibrate, and the surface pressure determined and plotted, all as described in the text E2 E11 F9 G4 A5 W12 G1 Q8 Fig (A) Extended hydrophobic moment plot analysis of AP1 (m), the known anionic a-AMP, maximin H5 (d), and peptides of the a-AMP dataset (http://www.uclan.ac.uk/biology/bru/amp_data.htm), all as described in the text AP1, maximin H5, and % 50% of the peptides in the dataset are represented by data points that lie in the shaded region, delineating candidacy for oblique-orientated a-helix formation (B) Sequence of AP1 represented as a 2D axial projection This a-helix possesses a hydrophilic face, which is rich in glycine residues and polar residues (circled), and a hydrophobic face formed from bulky apolar residues with a centrally placed glutamate residue A B Hydrophobic Moment (µH) Hydrophobic arc size (º) 350 300 250 200 150 100 50 α-AMP AP1 H5 1.20 C (lM) p (mNỈm)1) 0.5 10 15 20 25 30 0.9 5.15 6.5 7.9 9.32 10.65 11.26 11.32 11.33 A (nm2) G – 4.8 3.1 4.3 6.1 8.3 1.0 1.2 1.3 1.3 · · · · · · · · · 10)8 10)7 10)7 10)7 10)7 10)6 10)6 10)6 10)6 – 48.44 5.44 3.87 2.72 2.00 1.59 1.40 1.27 1.26 1.00 0.80 0.60 0.40 0.20 α-AMP AP1 H5 Fig Box plot for the hydrophobic arc size (A) and mean hydrophobic moment (B) of AP1, maximin H5, which is a known anionic a-AMP, and the a-AMP dataset, http://www.uclan.ac.uk/biology/ bru/amp_data.htm, all determined as described in the text AP1 and maximin H5 show comparable amphiphilic properties, which generally lie in the lower quartile range of the dataset was the minimum bulk concentration required to saturate the air ⁄ water interface with the peptide under these experimental conditions These data were used to 3794 Table Surface excess (G) and interfacial surface area per AP1 molecule (A) for various molar subphase concentrations (C) of the peptide where p is the interfacial pressure increase Values for these parameters were derived using AP1 surface pressure data from Fig with G computed using Eqn (1) and A computed using Eqn (2), all as described in the text determine the corresponding interfacial surface area per AP1 molecule (Table 1), and, for 20 lm peptide, extrapolation provides an estimate of peptide surface of 1.40 nm2, which is comparable to that found for peptides that adopt a-helical structure [33] When spread from chloroform on to the subphase of a Langmuir-Blodgett trough, AP1 formed stable monolayers Under compression, these monolayers showed collapse pressures in the region of 20 mNỈm)1 (Fig 4), indicating the presence of a well-ordered monolayer [34] Compressibility moduli, Cs À1 , were derived from these isotherms (Table 2) and generally decreased with increasing surface pressure, indicating that the monolayer is in the protein phase [35] Figure also shows that the area per AP1 molecule FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS S R Dennison et al Antimicrobial properties of an anionic a-helical peptide Fig A pressure–area isotherm for an AP1 monolayer The peptide was spread from chloroform on to a Tris ⁄ HCl buffer subphase (10 mM, pH 7.5) The variation of surface pressure with area per peptide molecule was monitored as monolayers were compressed and plotted, all as described in the text Table Compressibility moduli (Cs À1 ) of lipid monolayers at varying surface pressure (p) (all values mNỈm)1) Values of Cs À1 were computed using data from compression isotherms (Fig 9) and Eqn (3) Monolayers were formed from either Ole2PtdGro, Ole2PtdEtn, cardiolipin, or lipid mixtures that corresponded to membranes of E coli, all as described in the text Cs À1 (mNỈm)1) Pressure p (mNỈm)1) Ole2PtdGro Ole2PtdEtn Cardiolipin E coli 10 15 17.3 14.5 12.1 29.3 28.8 24.5 11.1 13.8 16.2 26.6 25.3 23.7 corresponding to this collapse pressure was 0.33 nm2 The extrapolated area at p ẳ mNặm)1 for the isotherm provides a measure of the mean monolayer surface area per AP1 molecule [36] This area was 1.42 nm2 per AP1 molecule and is comparable to the value of 1.40 nm2 calculated above for the peptide using Eqns (1) and (2) (Table 1) Although towards the lower end of the expected range, this would approximate to that predicted for AP1 if the peptide was orientated perpendicular to the air ⁄ water interface (1.77 nm2 [37]), but may also indicate the presence of some non-a-helical structure in AP1 FTIR conformational analysis showed that AP1 adopted predominantly b-type structures in solution However, at lipid to peptide ratios of 50 : and above, the peptide adopted % 100% a-helical structure in the presence of lipid assemblies that mimicked membranes of E coli (Fig 5) This conformational behaviour is similar to that shown by most a-AMPs, which are generally non-a-helical in solution but assume a-helical structure at the microbial interface [38–40] Fig FTIR conformational analysis of AP1 in the presence of SUVs with lipid compositions that correspond to those of E coli membranes, all as described in the text The numbers annotating spectra indicate peak band absorbancies For each spectrum, the relative percentages of a-helical structure (1650–1660 cm)1) and b-sheet structures (1625–1640 cm)1) were computed, all as described in the text In aqueous solution, AP1 was predominantly formed from b-type structures (A), but in the presence of E coli membrane mimics (B), the peptide was 100% a-helical A standard toxicity assay established a minimum lethal concentration of mm for AP1 when directed against E coli Analysing the number of colony forming units over time showed that, at this concentration, the peptide took h to induce 100% cell death (Fig 6) Microbial membrane invasion is the primary killing mechanism used by most a-AMPs [41,42] FTIR conformational analysis shows that, in the absence of AP1, small unilamellar vesicles (SUVs) mimetic of E coli membranes underwent transition from the gel phase to liquid crystalline phase over the temperature range 20–70 °C with a concomitant increase in membrane fluidity, as indicated by the rise in wavenumber from % 2851.0 cm)1 to 2852.3 cm)1 The presence of AP1 caused no apparent shift in the temperature range of these phase transitions but, over this temperature range, induced a significant decrease in the membrane fluidity of E coli membranes (Fig 7) AP1 also interacted with lipid monolayers that were mimetic of E coli membranes (Fig 8), inducing FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3795 Antimicrobial properties of an anionic a-helical peptide S R Dennison et al Fig Time course for the viability of E coli represented as percentage death rate in the presence of AP1 (3 mM) At these concentrations, the peptide is bactericidal, achieving a 100% death rate after h The percentage death rate was determined by comparison with identical noninoculated control cultures, all as described above, and error bars represent the standard error on three replicates Fig Time course of interactions between AP1 and monolayers with lipid compositions that correspond to those of E coli membranes, all as described in the text Monolayers were at an initial surface pressure of 30 mNỈm)1, mimetic of naturally occurring membranes, and the peptide was introduced into the subphase to give a final concentration 20 lM, all as described in the text Wavenumber (cm–1) 2854 2853 2852 2851 2850 10 20 30 40 50 60 70 80 Temperature (°C) Fig FTIR lipid-phase transition analyses of SUVs with lipid compositions that correspond to those of E coli membranes, all as described in the text In the absence of AP1 (,) model membranes of E coli underwent a transition from the gel phase to the liquid crystalline phase liquid over the temperature range 30–70 °C with a concomitant increase in membrane fluidity s indicated by the rise in wavenumber from % 2851.0 cm)1 to 2852.3 cm)1 The presence of AP1 caused no apparent shift in this temperature range but induced a significant decrease in the membrane fluidity of E coli membranes, which is consistent with the peptide penetrating the hydrophobic core of these membranes maximal changes in surface pressure of 4.6 mNỈm)1 after 6500 s This was further investigated by thermodynamic analysis of compression isotherms derived from monolayer mimics of E coli membranes in either the absence (Fig 9A) or presence of AP1 (Fig 9B) Cs À1 were derived from these isotherms (Table 2), and Cs À1 is seen to be generally low, indicating that the lipid monolayers analysed were in a liquid expanded phase [35] and, thus, are more fluid and possess high 3796 Fig Compression isotherms of monolayers formed from: lipid compositions that correspond to those of E coli membranes (a), Ole2PtdEtn (b), Ole2PtdGro (c) and cardiolipin (d) The variation of surface pressure with area per lipid molecule was monitored as monolayers were compressed on a Tris ⁄ HCl buffer subphase (10 mM, pH 7.5) either in the absence of AP1 (A) or containing AP1 with a final concentration of 20 lM (B), all as described above FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS S R Dennison et al Antimicrobial properties of an anionic a-helical peptide Table Gibbs free energy of mixing (DGMix), interaction parameter (a) and enthalpy of mixing (DH) at varying surface pressure (p) for lipid mixtures that correspond to membranes of E coli Values for these parameters were computed either in the presence or absence of AP1 using data from compression isotherms (Fig 9) in conjunction with Eqns (4), (5) and (6), respectively, all as described above Pressure p (mNỈm)1) 10 15 DGMix (JỈmol)1) DH (JỈmol)1) a –AP1 +AP1 –AP1 +AP1 –AP1 +AP1 )106.11 )258.02 )387.10 0.60 7.91 12.51 )7.35 )17.88 )26.83 0.04 0.55 0.87 )8986.72 )21852.82 )32785.45 51.00 669.85 1059.37 compressibility Table also shows that AP1 induced a general decrease in Cs À1 with rising monolayer surface pressure, indicating expansion of these bacterial membrane mimics because of peptide interactions Values for the Gibbs free energy of mixing (DGMix) (Table 3) were derived from the compression isotherms shown in Fig It can be seen from Table that DGMix for E coli model membranes varies with surface pressure and according to the presence or absence of AP1 Table shows that values of DGMix for these E coli model membranes are much lower than RT (2444.316 JỈmol)1), indicating that deviations from ideal mixing behaviour are small In the absence of AP1, negative values of DGMix were observed for E coli model membranes (Table 3), indicating a stable monolayer However, in the presence of AP1 (Table 3), positive values of DGMix are observed for E coli model membranes, indicating that, although the lipids forming these monolayers are miscible, repulsive interactions are established in the presence of the peptide, thereby decreasing membrane stability These values of DGMix become progressively more positive as surface pressure increases, showing that, at higher surface pressures, mutual interactions between the component molecules of these membranes are weaker than those occurring in monolayers formed by their pure components [43], becoming increasingly less stable with compression This instability may contribute to the susceptibility of E coli model membranes to the action of AP1 An important determinant of the susceptibility of membranes to a-AMPs is the packing characteristics of the individual membrane lipids [44] To evaluate the nature of interactions between the component lipid molecules in E coli model membranes, the interaction parameter, a, and the mixing enthalpy, DH, were computed (Table 3) It can be seen from Table that, in the absence of AP1, values for a and DH are negative for these model membranes, but, in the presence of the peptide, they are positive These results confirm that E coli membranes are thermodynamically less stable in the presence of AP1, further supporting the suggestion that this instability may contribute to the susceptibility of E coli to the antimicrobial action of the peptide Discussion The biological action of many pore-forming and lytic peptides involves membrane destabilization by the use of lipid-interactive oblique-orientated a-helical structure [22], and such structure also appears to be used by many a-AMPs [20] It has been suggested that anionic a-AMPs and their analogues may serve as complements to their cationic counterparts in some therapeutic contexts [24,25] Here, a synthetic peptide, AP1, was prepared to observe whether anionic peptides with tilted peptide characteristics could be designed to act as potential anionic a-AMPs Theoretical analysis confirmed that the peptide possessed the potential to form an a-helix with a balance between amphiphilicity and hydrophobicity, and structural characteristics that are associated with obliqueorientated a-helices (Figs and 2) It can be seen from Fig 1B that the AP1 a-helix possesses a glycine-rich polar face, and it has previously been shown that similarly located glycine residues are critical for maintaining the hydrophobicity gradients associated with membrane-interactive oblique-orientated a-helices [45] It can also be seen from Fig 1B that the AP1 a-helix possesses a wide hydrophobic face rich in bulky amino-acid residues, and, in combination with a glycine-rich polar face, these structural characteristics give a-helices an effective inverted wedge shape It has been recently shown that a number of a-AMPs, experimentally demonstrated to penetrate membranes in an oblique orientation, appear to possess this inverted wedge shape [12,21] In addition, it can be seen from Fig 1B that a glutamate residue is centrally located in the apolar face of the AP1 a-helix, and previous studies have shown that similarly located glutamate residues are important for the antimicrobial action of other a-AMPs also predicted to form an inverted wedge shape [46] FTIR spectroscopy showed that AP1 was completely a-helical in the presence of model membranes mimetic of those of E coli (Fig 5), although molecular area FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3797 Antimicrobial properties of an anionic a-helical peptide S R Dennison et al determinations showed that the peptide may possess some non-a-helical structure at an air ⁄ water interface It would seem that AP1 specifically requires the amphiphilicity associated with the environment of a membrane or lipid interface to form such structure Monolayer studies confirmed that AP1 was able to partition into model membranes that were mimetic of those of E coli (Fig 8), and toxicity assay showed that AP1 was bactericidal at mm (Fig 6) In combination, these data clearly show that the peptide is able to function as an anionic a-AMP Moreover, these results suggest that interaction with bacterial membranes features in the antibacterial action of AP1, and it is well established that perturbation of the microbial membrane is a primary killing mechanism used by a-AMPs [41,42] This suggestion is strongly reinforced by the observation that AP1 showed haemolytic ability, thereby clearly confirming that the peptide is able to induce cell bilayer disruption AP1 was found to be haemolytic at mm, thereby showing a common characteristic of a-AMPs in that higher concentrations of these peptides are generally required for haemolytic action than for bactericidal action [38] The minimum lethal concentration of AP1 is far in excess of those normally required by cationic a-AMPs to inhibit target micro-organisms (< 20 lm), but is closer to those required by some anionic a-AMPs (80 lm) [12], which are known to generally exhibit lower levels of antimicrobial efficacy than their cationic counterparts Lipid-phase transition analysis clearly suggested that the interactions of the peptide with membranes of E coli induced a significant decrease in the membrane fluidity of E coli membranes (Fig 7), which is consistent with the peptide penetrating deeply into the membranes hydrophobic core To investigate further the mechanism of bacterial membrane interaction used by AP1, thermodynamic analysis of compression isotherms for lipid monolayer mimetics of E coli membranes were undertaken (Table 3, Fig 9) These analyses gave negative values for DGMix, a and DH in the absence of AP1, indicating membrane stability, but, in the presence of AP1, positive values of for DGMix, a and DH were obtained, suggesting that the monolayer had become less stable This shows that the association of AP1 with these model membranes had a destabilizing effect, and, when taken with the FTIR data above, suggests that the peptide may promote toxicity to E coli by a lytic-type mechanism involving disturbance of lipid acyl chains within the membrane core [47] It is well established that the packing characteristics of component lipids is an important factor in determining the stability of membrane bilayers [44] It is interesting to note that E coli membranes possess 3798 high levels of phosphatidylethanolamine (% 85%), which is effectively shaped like an inverted wedge and is known to have a strong preference for the nonlamellar H11 phase [14] Thus, according to the wedge hypothesis of Tytler et al [48], it may be that insertion of the inverted wedge shape formed by the AP1 a-helix into membranes of E coli leads to the formation of nonbilayer structures and thereby membrane destabilization Such a mechanism of membrane perturbation would be consistent with the use of a lytic-type mechanism for antimicrobial action, as predicted by the thermodynamic analyses above and the involvement of oblique-orientated a-helical structure in AP1 The higher concentrations of peptide required for haemolysis would indicate that the membrane composition plays an important role in activity In summary, AP1 was found to function as an anionic a-AMP, indicating that it is possible to design a-AMPs by the use of an oblique-orientated a-helical template It appears from the biophysical data that the peptide uses this structure for the destabilization of membranes of Gram-negative bacteria, thereby promoting the inactivation of these organisms The relatively high concentration required for the minimum lethal concentration indicates though that further lessons with respect to the amino-acid composition are still to be learnt However, as a general lesson, the data presented in this study emphasize that, in development of antimicrobial compounds, both the structural characteristics and composition of their target membrane are important determinants of their efficacy of action Experimental procedures Reagents AP1 (GEQGALAQFGEWL) was supplied by Pepsyn (Liverpool, UK), produced by solid-state synthesis, and purified by HPLC to greater than 95%, which was confirmed by MALDI MS Buffers and solutions for monolayer experiments were prepared from Milli-Q water Nutrient broth was purchased from Amersham Bioscience (GE Healthcare, Chalfont St Giles, UK) Dioleoylphosphatidylglycerol (Ole2PtdGro) and dioleoylphosphatidylethanolamine (Ole2PtdEtn) were purchased from Alexis Corporation (Axxora, Bingham, UK) Cardiolipin, Hepes, Tris and all other reagents were purchased from Sigma (Sigma-Aldrich, Gillingham, UK) Primary structure analyses The sequences of 161 known a-AMPs were obtained from Dennison et al [41] (http://www.uclan.ac.uk/biology/bru/ FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS S R Dennison et al amp_data.htm) and along with those of maximin H5 (ILGPVLGLVSDTLDDVLGIL [24]) and AP1 were analysed according to conventional hydrophobic moment methodology [49] Essentially, this methodology treats the hydrophobicity of successive amino acids in a sequence, as vectors and then sums these vectors in two dimensions, assuming an amino-acid side chain periodicity of 100 ° The resultant of this summation, the hydrophobic moment, provides a measure of a-helix amphiphilicity The analysis of the present study used a moving window of 11 residues, and for each sequence under investigation, the window with the highest hydrophobic moment was identified [49] For these windows, the mean hydrophobic moment, , and the corresponding mean hydrophobicity, , were computed using the online program moment helix prediction (http://www.doe-mbi.ucla.edu/Services/moment/) and the normalized consensus hydrophobicity scale of Eisenberg et al [49] These mean values were plotted on the hydrophobic moment plot diagram of Eisenberg et al [50], as modified by Harris et al [23], to identify candidate obliqueorientated a-helix-forming segments Assuming an idealized a-helix with a residue side chain angular periodicity of 100 °, a 2D axial projection of the peptide was generated [51] The angle subtended by the hydrophobic residue distribution was taken as a measure of hydrophobic arc size Preparation of lipid unilamellar vesicles SUVs with lipid compositions designed to mimic E coli membranes were prepared as described by Keller et al [52] Essentially, chloroform solutions of Ole2PtdGro, Ole2PtdEtn and cardiolipin in molar proportions of : 13.67 : [53] were dried with nitrogen gas and hydrated with Hepes buffer (10 mm, pH 7.5) to give final total lipid concentrations of 150 mm The resulting cloudy suspensions were sonicated at °C with a Soniprep 150 sonicator (amplitude 10 lm) until clear (30 cycles of 30 s), centrifuged (15 min, 3000 g, °C), and the supernatant decanted for immediate use FTIR conformational analysis of AP1 To give final peptide concentration ranging from mm to1 mm, AP1 was solubilized in either Hepes buffer (10 mm, pH 7.5) or suspensions of SUVs formed from Ole2PtdGro, Ole2PtdEtn and cardiolipin as described above These samples were spread individually on a CaF2 crystal, and the free excess water was evaporated at room temperature The single band components of the VAP1 amide I vibrational band (predominantly C¼O stretch) was monitored using an FTIR ‘5-DX’ spectrometer (Nicolet Instruments, Madison, WI, USA), and, for each sample, absorbance spectra were produced For these spectra, water bands were subtracted, and the evaluation of peptide band parameters (peak position, band width and intensity) Antimicrobial properties of an anionic a-helical peptide performed Curve fitting was applied to overlapping bands using a modified version of the curfit procedure written by Dr Moffat, National Research Council, Ottawa, Ontario, Canada The band shapes of the single components are superpositions of Gaussian and Lorentzian band shapes Best fits were obtained by assuming a Gauss fraction of 0.55–0.6 The curfit procedure measures the peak areas of single band components, and, after statistical evaluation, determines the relative percentages of primary structure involved in secondary-structure formation, all as described by Dennison et al [54] FTIR analysis of phospholipid phase-transition properties To give a final peptide concentration of mm, AP1 was solubilized in suspensions of SUVs, which were formed from Ole2PtdGro ⁄ Ole2PtdEtn ⁄ cardiolipin as described above As controls, corresponding lipid SUVs were prepared with no peptide present All samples were then subjected to automatic temperature scans with a heating rate of °C per and within the temperature range 0–60 °C For every °C interval, 50 interferograms were accumulated, apodized, Fourier transformed, and converted into absorbance spectra [55] These spectra monitored changes in the b fi a acyl chain melting behaviour of phospholipids, with these changes determined as shifts in the peak position of the symmetric stretching vibration of the methylene groups, ms(CH2), which is known to be a sensitive marker of lipid order The peak position of ms(CH2) lies at 2850 cm)1 in the gel phase and shifts at a lipid specific temperature Tc to 2852.0– 2852.5 cm)1 in the liquid crystalline state [55] Antimicrobial assay Cultures of the E coli strain W3110, which had been freeze-dried in 20% (v ⁄ v) glycerol and stored at )80 °C, were inoculated into 10 mL nutrient broth After overnight incubation in an orbital shaker (100 r.p.m., 37 °C), 100-lL aliquots of these cultures were used to inoculate 100 mL nutrient broth in 250 mL flasks, which were then incubated with shaking (100 r.p.m., 37 °C) until growth in the midexponential phase was reached (A ¼ 0.6; k ¼ 600 nm) Aliquots (1 mL) of bacterial samples were centrifuged, using a bench top centrifuge (15 000 g, min, 22 °C), and the centrifuged cells washed three times in 1-mL aliquots of Tris ⁄ HCl buffer (10 mm, pH 7.5) These cells were then suspended in mL of this buffer containing AP1 at a final concentration of mm, which corresponds to its minimum inhibitory concentration These culture ⁄ peptide mixtures were incubated at 37 °C, and samples taken at the beginning of the experiment (time zero), and at 15 minute intervals for h and then hourly intervals for h At each time interval, samples were surface-spread on to nutrient agar plates, which were incubated at 37 °C for 12 h As a FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3799 Antimicrobial properties of an anionic a-helical peptide S R Dennison et al control, bacterial cultures were similarly treated but in the absence of peptide Colony counts were expressed as colony forming units (CFU) ml)1 The percentage reduction in colony counts for each time interval was then calculated, and the results were presented graphically against time Monolayer technique All experiments were conducted at 21.0 ± °C using a Langmuir trough measuring · 16 cm, which was fitted with two moveable barriers and was supplied by NIMA Technology (Coventry, UK) Unless otherwise stated, monolayer studies were performed using a Tris ⁄ HCl buffer subphase (10 mm, pH 7.5), which was continuously stirred by a magnetic bar (5 r.p.m.) Surface tension was monitored by the Wilhelmy method using a Whatman’s (Ch1) paper plate in conjunction with a microbalance, as described by Brandenburg et al [56] Changes in monolayer surface pressure ⁄ area were recorded as graphic output on a PC using nima software version 5.16, which interfaces with the Langmuir-Blodgett microbalance Peptide surface activity The barriers of the Langmuir-Blodgett trough were adjusted to their maximum separation (surface area 80 cm2), and this position maintained AP1 was then injected into the buffer subphase to give final concentrations of 1–30 lm, and, at each peptide concentration, changes in surface pressure at the air ⁄ water interface were monitored for h The maximal values of these surface pressure changes were then plotted as a function of the peptide’s final subphase concentration (Fig 3) From these results, the surface excess, G, was calculated by means of the Gibbs’ adsorption isotherm, which is given by Eqn (1) [57]: C¼À Dp  RT D ln C ð1Þ where R is 8.314 Jặmol)1ặK)1, T ẳ 294 K, p is the interfacial pressure increase (mNỈm)1), and c is the molar concentration of peptide in the subphase These values of G were then used to determine values of the interfacial surface area per AP1 molecule (A) according to Eqn (2): Aẳ NC 2ị where N is Avogadro’s number (Table 1) The ability of AP1 to spread on an aqueous surface and to form a stable monolayer was investigated The barriers of the Langmuir-Blodgett trough were adjusted to their maximum separation (surface area 80 cm2) and this position maintained A 10-lL aliquot of AP1 in chloroform (1 mm) was spread on to a buffer subphase and allowed to equilibrate for h The resulting peptide monolayer was compressed using the moveable barriers of the trough to 3800 produce a pressure ⁄ area isotherm, which was converted by nima software in to an output plot of surface pressure vs monolayer surface area per AP1 molecule (Fig 4) Peptide interactions with lipid monolayers The ability of AP1 to penetrate lipid monolayers at constant area was studied Monolayers were formed by spreading on to a buffer subphase, chloroform solutions of Ole2PtdGro, Ole2PtdEtn and cardiolipin in molar proportions of : 13.67 : [53] The solvent was allowed to evaporate off over 30 and then the monolayer compressed at a velocity of cm2Ỉmin)1 to give a surface pressure of 30 mNỈm)1 The barriers were maintained in this position, and peptide was then injected into the subphase to achieve the desired optimum peptide concentration of 20 lm which was determined by analysis of surface activity data described in Fig This subphase concentration of AP1 gave rise to a lipid to peptide ratio of approximately 100 : 1, which was used in all other monolayer studies Interactions of the peptide with lipid monolayers were monitored as changes in monolayer surface pressure vs time The ability of the peptide to interact with lipid monolayers was also investigated using compression isotherms Monolayers were formed by spreading on to a buffer subphase chloroform solutions of either Ole2PtdEtn, Ole2PtdGro, cardiolipin, or these lipids in molar proportions of : 13.67 : [53] The solvent was allowed to evaporate off over 30 min, and monolayers then compressed using a barrier speed of cmỈmin)1 either with AP1 absent from the subphase or included in the subphase at a final peptide concentration of 20 lm Changes in monolayer surface pressure vs changes in area per lipid molecule of the monolayer were monitored and recorded Thermodynamic analysis of compression isotherm data Thermodynamic analysis of compression isotherms was used to investigate the molecular interactions and dynamic behaviour of monolayers The compressibility modulus, Cs À1 , provides a measure of the compressional elasticity of a monolayer and can be used to characterize the phase state of the isotherm, thereby providing information about the compactness and packing of the model membrane [35] Values of Cs À1 (Table 1) were computed according to Eqn (3):   dp 3ị Cs1 ẳ A dA where p is the surface pressure of the monolayer, and A represents the area per molecule in the monolayer The Gibbs free energy of mixing (DGMix) quantifies the stability of monolayer mixtures, thereby providing information on interactions between the components of the monolayers Values of DGMix were computed according to Eqn (4): FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS S R Dennison et al DGMix ¼ Zp Antimicrobial properties of an anionic a-helical peptide ½A1;2 n x1 A1 ỵ x2 A2 ỵ xn An Þdp ð4Þ Frank Grunfeld (NIMA Technology, UK) for his technical advice with the monolayer studies where A1,2, n is the molecular area occupied by the mixed monolayer, A1, A2 .An are the area per molecule in the pure monolayers of component 1, 2, n, x1, x2 xn are the molar fractions of the components and p is the surface pressure These data were then recorded as the variation of DGMix with monolayer surface pressure (Table 3) Numerical data were calculated from compression isotherms using the methodology of Simpson [58] The interaction parameter (a) relates the interaction of each molar fraction of components within a monolayer with the free energy of mixing Values of a were computed (Table 3) according to Eqn (5): a¼ DGMix n n n RTX1 X2 Xn ỵ X1 X2 Xn ỵ X1 X2 Xn ð5Þ where X are the molar fractions of the components, R is 8.314 JỈmol)1ỈK)1, and T is 294 K These data were then used to compute values of monolayer mixing enthalpy (DH) (Table 3) according to Eqn (6): DH ẳ RTa Z 6ị where Z is the packing fraction parameter and calculated using the Quikenden and Tam model [59] Haemolytic assay of AP1 Haemolytic assay was conducted as described by Harris & Phoenix [60] Essentially, packed red blood cells were washed three times in Tris-buffered sucrose (0.25 m sucrose, 10 mm Tris ⁄ HCl, pH 7.5) and resuspended in the same medium to give an initial blood cell concentration of % 0.05% (w ⁄ v) For haemolytic assay, this concentration was adjusted such that incubation with 0.1% (v ⁄ v) Triton X-100 for 30 produced a supernatant with A416 of 1.0, which was taken as 100% haemolysis Aliquots (1 mL) of blood cells at assay concentration were then used to solubilize various amounts of stock AP1 solution, which had been added to a test-tube and dried under nitrogen gas The resulting mixtures were incubated at room temperature with gentle shaking After 30 min, the suspensions were centrifuged at low speed (1500 g, 15 min, 25 °C), and the A416 of the supernatants determined In all cases, levels of haemolysis were determined as the percentage haemolysis relative to that of Triton 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penicillin-binding proteins 4, and to undergo membrane interaction Biochimie 79, 171–174 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3803 ... contribute to the susceptibility of E coli model membranes to the action of AP1 An important determinant of the susceptibility of membranes to a-AMPs is the packing characteristics of the individual... hypothesis of Tytler et al [48], it may be that insertion of the inverted wedge shape formed by the AP1 a-helix into membranes of E coli leads to the formation of nonbilayer structures and thereby... FEBS S R Dennison et al Antimicrobial properties of an anionic a-helical peptide Fig A pressure–area isotherm for an AP1 monolayer The peptide was spread from chloroform on to a Tris ⁄ HCl buffer