Báo cáo khoa học: Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3 pptx

Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3 Hueih Min Chen 1 , King Wong Leung 1 , Nagendra N. Thakur 1 , Anmin Tan 1, * and Ralph W. Jack 2 1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan; 2 Institut fu ¨ r Organische Chemie, Universita ¨ tTu ¨ bingen, Germany Different pathways of bilayer disruption by the structurally related antimicrobial peptides cecropin B, B1 and B3, revealed by surface plasma resonance analysis of immobi- lized liposomes, differential scanning calorimetry of peptide– large unilamellar vesicle interactions, and light microscopic analysis of peptide-treated giant unilamellar vesicles, have been identified in this study. Natural cecropin B (CB) has one amphipathic and one hydrophobic a-helix, whereas cecro- pins B1 (CB1) and B3 (CB3), which are custom-designed, chimaeric analogues of CB, possess either two amphipathic or two hydrophobic a-helices, respectively. Surface plasma resonance analysis of unilamellar vesicles immobilized through a biotin–avidin interaction showed that both CB and CB1 bind to the lipid bilayers at high concentration (>10 l M ); in contrast, CB3 induces disintegration of the vesicles at all concentrations tested. Differential scanning calorimetry showed the concentration-dependent effect of bilayer disruption, based on the different thermotrophic phase behaviours and the shapes of the thermal phase- transition curves obtained. The kinetics of the lysis of giant unilamellar vesicles observed by microscopy demonstrated that both CB and CB1 effect a continuous process involving loss of integrity followed by coalescence and resolution into smaller vesicles, whereas CB3 induces rapid formation of irregular-shaped, nonlamellar structures which rapidly dis- integrate into twisted, microtubule-containing debris before being completely destroyed. On the basis of these observa- tions, models by which CB, CB1 and CB3 induce lysis of lipid bilayers are discussed. Keywords: differential scanning calorimetry; lysis mechan- ism; lytic peptides; microscopic analysis; surface plasma resonance. Cationic antimicrobial peptides have now been isolated from a wide variety of sources including bacteria, inverte- brates, vertebrates and plants [1–7]. Although their structure and chemical nature differ markedly, their function appears to involve protection of the producing organism from competing or pathogenic micro-organisms, and their acti- vity may be directed towards a variety of bacteria, protozoa, fungi and/or viruses. In bacteria, the production of cationic antimicrobial peptides is probably a survival strategy to obtain an ecological advantage over competitors [8–10]. In invertebrates and plants, which lack the adaptive immune system of higher animals, these peptides represent a major component of ÔinnateÕ immune defence [11–13]. Even vertebrates rely heavily on the protection against infection offered by the innate immune system, in which the defence peptides play a pivotal role [14,15]. The general mechanism by which cationic antimicrobial peptides bring about cell death appears to involve perme- abilization of the phospholipid bilayer membranes border- ing their targets. However, closer inspection of this activity reveals significant differences between various peptides. Several cationic antimicrobial peptides from bacteria, including the lantibiotics nisin and epidermin and the bacteriocin pediocin PA-1, are most effective against specific target organisms and utilize docking molecules present in the cell membrane in a mechanism that stabilizes the formation of ion-permeable pores [16–19]. As a result, these peptides are active at nanomolar concentrations and show limited target range with little or no observable activity against membranes of eukaryotic organisms; they may be active in the absence of the docking molecules (e.g. artificial bilayers), but at significantly higher peptide concentrations. Conversely, many of the cationic defence peptides of eukaryotic origin are active at micromolar concentrations, but show little target specificity and may act on many membrane types including those of erythrocytes (haemolytic activity). For example, the synthesis of a variety of analogues of the 13-amino acid, tryptophan-rich bovine antimicrobial peptide indolicidin has suggested that chiral and/or sequence-specific determinants are not required for either antibacterial or haemolytic activity, although target specificity is strongly influenced by the overall physico- chemical nature of the analogue [20], and similar results Correspondence to H. M. Chen, Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan 115. Fax: + 886 2 2788 8401, Tel.: + 886 2 2785 5696 ext. 8030, E-mail: robell@gate.sinica.edu.tw Abbreviations: CB, cecropin B; CB1, cecropin B1; CB3, cecropin B3; ESR, electron spin resonance; DSC, differential scanning calorimetry; GUV, giant unilamellar vesicle; LUV, large unilamellar vesicle; PA, phosphatidic acid; PtdEtn, phosphatidylethanolamine; RU, resonance units; SPR, surface plasma resonance. *Present address: Department of Biophysical Chemistry, Biocenter, University of Basel, Basel, Switzerland. (Received 30 July 2002, revised 28 October 2002, accepted 7 January 2003) Eur. J. Biochem. 270, 911–920 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03451.x have been obtained with analogues of the insect defence peptide cecropin B [21]. Thus, the cationic defence peptides, which are active against a broad range of targets, seem to offer the best opportunity for the study of the molecular mechanisms involved in generalized pore formation in the absence of specific stabilizing structures. Because of their relatively broad spectrum of activity, it has been suggested that defence peptides may offer an alternative source of antimicrobial chemotherapeutics [22,23]. Moreover, it has also been shown that certain magainins (isolated from frog skin) and cecropins (insect- derived peptides) also possess antitumour cell activities, making these peptides of special interest as molecular models for the study of pore formation in biological membranes [24–27]. Structure–function analysis of the various subclasses of defence peptides, combined with dissection of the molecular events involved in pore forma- tion, offers the possibility to develop customized peptides with defined activities for anti-infective and antitumour applications. In general, two models for disruption of membrane integrity have generally been adopted: pore formation by the Ôbarrel-staveÕ model, for which alamethicin and melittin are considered prime examples, and the Ôflip-flopÕ action (so-called Ôcarpet-likeÕ action) of peptides such as cecropin P1 [28–32]. However, it is not currently possible to assess the likely mechanism of membrane disruption of a given peptide simply on the basis of its structure. Thus, we have used specific peptides with high homology, but which contain specific structural variations, to investigate the relationship between these two cell-killing models [33,34]. We have previously reported the synthesis and biological activities of three model peptides: native cecropin B (CB), which contains both a hydrophobic and an amphipathic helix, and the custom-designed, synthetic analogues cecro- pin B1 (CB1) and cecropin B3 (CB3), which contain two amphiphilic and two hydrophobic helices, respectively [27,35–42]. Recently, we explored the kinetics of liposome lysis using fluorescent quenching and used surface plasma resonance (BIACore) and oriented circular dichroism [43] to investigate the two different modes of membrane disruption and to detect the orientation of the peptides with respect to the membrane surface [42]. Kinetic analysis suggests that bilayer disruption occurs in two distinct steps, whereas oriented circular dichroism provides evidence that the peptides exist in at least two different membrane-associated states, depending on the orientation of the helical segments with respect to the bilayer surface. Similar transitions of peptide orientation in membrane multilayers have also been shown for melittin, alamethicin and magainin [44,45]. In this study, we examined and compared the disruptive mechanisms of the natural peptide (CB) and the custom peptide analogues (CB1) and (CB3) against liposomes, large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs) using surface plasma resonance (SPR), differential scanning calorimetry (DSC) and real-time light microscopy, respectively. The results indicate that the different mecha- nisms of disruption of lipid bilayers observed are dependent on the particular physicochemical characteristics of the different peptides. Investigation of such differences will provide useful information for the future custom design of peptidyl and mimetic drugs. Experimental procedures Materials Phosphatidylethanolamine (PtdEtn, an egg derivative con- taining fatty acids of C 16 ,C 18 and C 20 ). 1,2-Diacyl- sn-glycero-3-phosphatidic acid (PA) is a monosodium salt synthetic lipid (98% purity). The symmetric fatty acid of PA contains 18-carbon double chains. Both PtdEtn and PA were obtained from Sigma-Aldrich (St Louis, MO, USA) and were used without further purification. CM5 Sensor Chips, amine coupling kits, and BIAevaluation software were all purchased from BIAcore AB (Uppsala, Sweden). Analytical grades of Triton X-100 and SDS were obtained from Sigma-Aldrich, and Biotin-X-DHPE (N-{[6-(bioti- noyl)amino]hexanoyl)}-1,2-dihexadecanoyl-sn-glycero- 3-phosphoethanolamine) was purchased from Molecular Probes (Eugene, OR, USA). Avidin obtained from Sigma- Aldrich is made from egg white and is chromatographically purified and a lyophilized powder. One unit of avidin [12.3 UÆ(mg solid) )1 ] will bind 1.0 lg biotin. All water used in these experiments was deionized and distilled. Peptide sequences, preparation and solutions The preparation of the 35-amino-acid peptide amides CB ( H) KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGE AKAL )CONH 2 ), CB1 ( H) KWKVFKKIEKMGRNIRNGI VKAGPKWKVFKKIEK )CONH 2 )andCB3( H) AIAVLGE AKALMGRNIRNGIVKAGPAIAVLGEAKAL )CONH 2 ) used throughout this study has been previously described [35]; CB contains a 10-amino-acid amphiphilic (italicized) and a 10-amino-acid hydrophobic (underlined) helix, and CB1 contains two 10-amino-acid amphiphilic helices (itali- cized) and CB3 contains two 10-amino-acid hydrophobic helices (underlined), each derived from the parent, CB. The peptides were all > 95% pure, as determined by RP-HPLC, and were stored lyophilized at )20 °C until use. The concentration of peptide stock solutions was determined from the net weight of peptides (the weight of the associated counter ions was not taken into consideration) and their molecular masses. Concentrations measured by the above method were confirmed with a bicinchoninic acid assay (Micro BCA protein assay; Pierce Chemical Co.). A negligible deviation between these two methods was observed. Analysis of liposome–peptide interactions by SPR Peptides were dissolved in HBS-EP buffer [10 m M Hepes, 0.15 M NaCl, 3 m M EDTA and 0.005% surfactant P20 (or Tween 20), pH 7.4] to give a stock solution with a concentration of 500 l M , and these were further diluted as necessary in the same buffer. Surfactant P20 is a 10% aqueous solution of the nonionic surfactant Polysorbate 20. The purpose of the surfactant is to reduce sample loss caused by the adsorption of hydrophobic molecules to the surfaces of the flow system of the BIAcore instrument. Peptides dissolved in HBS-EP buffer are not lysed by the liposomes. Avidin was prepared as a stock at 50 lgÆmL )1 dissolved in 10 m M sodium acetate (pH 4.8). To prepare biotin-containing liposomes, PtdEtn (7 mg) and PA (3 mg) 912 H. M. Chen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 were dissolved in 1 mL chloroform, and 50 lgbiotin- X-DHPE was added before removal of the chloroform in an argon stream. Instead of our previous use of phosphatidyl- choline/PA (7 : 3) [27,33–42], PtdEtn used in these experi- ments is for the comparison with a microbial lipid. The lipid film was rehydrated in 2 mL HBS-EP buffer, sonicated for 30 s, and liposomes were produced after 30 passes through a LipoFast extruder fitted with two 100-nm polycarbonate filter stacks. SPR experiments were conducted using a BIAcore 2000 biosensor (Biacore). Avidin was immobilized on the sensor surface first using EDC [1-ethyl-3-(3-dimethylaminopro- pyl)carbodiimide] and NHS (succinimide), and the activated esters that had not bound ligand were capped with ethanolamine. The maximum avidin loading was achieved by optimizing the flow rate used (1 lLÆmin )1 ). Anionic liposomes (1 mgÆmL )1 ) were then immobilized on the surface of the chip using various flow rates. Peptides dissolved in HBS-EP buffer were applied at the concentra- tions and flow rates indicated in order to assess their effect on the immobilized liposomes (1 lLÆmin )1 is the optimum). HBS-EP was used a running buffer. An avidin-free chip surface, liposomes without biotin, or applications of BSA served as controls to assess the specificity of the responses observed. Peptide-induced responses were measured by SPR, and results were collected and analysed using the manufacturer’s software and protocols. A diagram of the construction of the biosensor surface is shown in Fig. 1. Binding of peptides to the immobilized liposomes causes an increase in resonance units (RU), whereas the disruption of liposomes results in a decrease in RU. The sensor chip surface was regenerated by using 10 m M HCl. DSC LUVs for use in DSC experiments were prepared from PtdEtn and PA by dissolving 3.5 mg PtdEtn and 1.5 mg PA (PtdEtn/PA ¼ 7 : 3, w/w) in 1 mL chloroform. After removal of the solvent at room temperature under an argon gas stream, the lipids were rehydrated in 1 mL NaCl/P i and then sonicated for 30 s. LUVs were subsequently formed by extrusion through a LiposoFast extruder fitted with two 100-nm polycarbonate filters for 25 repetitions. The thermotrophic phase behaviour of the LUVs was measured in a nano differential scanning calorimeter (Calorimetry Sciences Corp., Provo, UT, USA), with temperature scans performed at a rate of 1 °CÆmin )1 . The DSC cells were pressurizedto0.31kgÆcm )2 (0.30 MPa) throughout the experiment, and liposomes were used undiluted (i.e. at 5mgÆmL )1 ). Peptides, dissolved in NaCl/P i were added to give final concentrations in the range 1–100 l M ,and controls consisted of DSC analysis of each peptide in the absence of lipid and the liposomes without peptide. Preparation of GUVs GUVs were used for light microscopic observation of the membrane disruptive effects of the peptides (see section below). To obtain GUVs, we used the method of Akashi et al. [46] with some modification. Briefly, PtdEtn and PA (7 : 3) were dissolved together in chloroform/methanol (2 : 1, v/v) to a final concentration of 10 mgÆmL )1 and stored under argon at )20 °C. Subsequently, 10-lL aliquots were placed in the bottom of a 1.5-cm diameter, 10-mL glass tube, and the solvent was dried at room temperature in a stream of argon gas, to provide a thin lipid film. After removal of residual solvent under high vacuum for at least 10 h, the film was prehydrated with a stream of water- saturated argon gas for 30 min at 43–45 °C and then fully rehydrated by addition of 2 mL argon-saturated 1 m M MgCl 2 solution for 48 h at 37 °C. MgCl 2 concentration dependence experiments on GUV lysis induced by peptides were conducted and it was found that the electrostatic interactions between peptides and lipids are little influenced by the addition of MgCl 2 . The liposomes, which appear as an almost transparent, milky-white cloud in the middle of the solution, were collected and stored at 4 °C in plastic tubes. Microscopic observation of GUVs The stored GUVs were typically diluted 10-fold to prevent liposome fusion, and 40-lL aliquots were observed on an inverted microscope (Diaphot TMD; Nikon) under phase contrast, and images were captured with a charge-coupled- device camera (microflex UFX-DX; Nikon). The unilamel- lar nature of liposomes was assessed optically; those with an appropriately thin contour were judged unilamellar and were used for further study. Peptide solutions (20 lLofa 100-l M stock, final concentration  33.3 l M ) were added after selection of a single GUV (diameter generally 40–120 lm). Results SPR analysis of peptide–liposome interactions: effect of peptides on immobilized liposomes Analysis of chips carrying immobilized PtdEtn/PA lipo- somes containing biotin-X-DHPE revealed that the SPR response varies greatly with both the type and concentration of the peptide applied. At higher concentrations (> 10 l M ) of either CB or CB1 (Fig. 2), an increase in relative response was observed, indicating a time-dependent increase in mass and suggesting that the peptides were interacting intimately Fig. 1. Diagram detailing the liposome immobilization procedure. Liposomes were immobilized by binding of embedded biotin with avidin-EDC/NHS, which in turn was first immobilized on the surface of the sensor chip. Peptides passing over the immobilized liposome either bind to the liposome or disrupt it. Ó FEBS 2003 Identifying the disruptive mechanisms of cecropins B, B1 and B3 (Eur. J. Biochem. 270) 913 with the liposome membranes. Moreover, this effect was slightly higher for CB1, as indicated by the higher relative response (Fig. 2A,C). However, at lower peptide concen- trations (< 2.5 l M ), the relative response was negative. These ÔnegativeÕ observations may be due to the lower flow rates used in this experiment. Further experiments will be performed to clarify this. Figure 2B,D shows the non- specific binding of CB and CB1 (respectively) to the chip surface in the absence of liposomes; the level of nonspecific binding between peptides and sensor chip surface should, however, be significantly lower when immobilized liposomes mostly occupy the chip surface. By contrast, SPR analysis of the disruptive effects of CB3 generates negative RU values for all concentrations tested (Fig. 2E). About 80% of the liposome RU is lost at 100 l M CB3. Moreover, the amount of nonspecific binding of CB3 to the sensor chip surface in the absence of immobilized liposome was not significant (Fig. 2F). The negative RU values observed with CB3 at all concentrations when passing through the immobilized liposomes were consistent compared with the positive RU values of peptides with nonspecific bindings. Plots of peptide concentration against the relative reduction in mass estimated from the SPR response (data not shown) suggested that CB3 caused a time-dependent disruption of membrane integrity (Fig. 3C); the effect was more pronounced when higher concentrations of CB3 were applied. DSC analysis of the effect of peptides on PtdEtn/PA LUV thermotrophic phase behaviour The effect of CB, CB1 and CB3 on the thermotrophic phase behaviour of LUVs is shown in Fig. 4. The phase-transition temperature (T m ) for the PtdEtn/PA (7 : 3) liposome varied considerably as a function of peptide concentration (data not shown). Typical examples were shown at two peptide concentrations (lower concentration at 1 l M and 20 l M for CB/CB1 and CB3, respectively; higher concentration at 20 l M and 50 l M for CB/CB1 and CB3, respectively). In the case of the parent peptide (CB) with liposomes, low concentrations of peptide (1 l M ) lowered T m by  1.7 °C and broadened the DSC endotherm as compared with liposome alone. Higher concentrations of peptide (20 l M ) resulted in DSC endotherms that split to yield multiple peaks, one of which was considerably lower than the T m of the liposomes alone, and one of which yielded a T m even higher than that for LUVs in the absence of peptide. These two distinguishable outcomes of concentration-dependence are comparable to those observed by SPR (Fig. 2A). Similar results were obtained for the analogue CB1, although the peak splitting at higher peptide concentrations was less pronounced than for the parent peptide. However, a slightly different situation could be discerned for the analogue CB3; considerably higher concentrations (20 l M ) were required to obtain a decrease in the DSC endotherm similar to that observedwith1l M CB or CB1. At even higher peptide Fig. 2. Effect of peptides CB, CB1 and CB3 on biotin-loaded PtdEtn/PA (7 : 3, w/w) liposomes bound to the surface of a sensor chip and comparison with nonspecific binding to surfaces devoid of liposomes. (A) and (B) Application of various concentrations (0.25, 0.5, 0.75, 2.5, 10, 50 and 100 l M )of peptide CB to the surface of a sensor with and without (respectively) biotin-loaded PtdEtn/PA liposomes. (C) and (D) Application of various concentrations (0.25, 0.5, 0.75, 2.5, 10, 50 and 100 l M ) of peptide CB1 to the surface of a sensor with and without (respectively) biotin-loaded PtdEtn/PA liposomes. (E) and (F) Application of various concentrations (0.25, 0.5, 0.75, 2.5, 10, 50 and 100 l M )ofpeptideCB3tothesurfaceofa sensor with and without (respectively) biotin-loaded PtdEtn/PA liposomes. 914 H. M. Chen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 concentrations (50 l M ), more peak splitting was observed, although it was not as pronounced as that measured with the other two peptides. Control experiments (flat lines shown in each figure) using samples of each peptide at concentrations of 20 l M (CB and CB1) and 50 l M (CB3) in the absence of liposomes suggested that endothermic variations observed in the presence of liposomes arise exclusively from phase transi- tions in the liposomes, which are effected by antimicrobial peptides. Microscopic investigation of peptide-induced disruption of GUVs Our initial optimization studies showed that the most stable GUVs were formed in low salt concentrations, in particular MgCl 2 (1 m M ), and that liposome stability increased with increasing incubation time, although the relative size decreased with increasing stability (data not shown). With the optimized procedure given in Materials and Methods, we routinely produced stable GUVs with diameters ranging between 40 and 120 lm. To study the step-by-step disrup- tion of a GUV, we added the peptide solution and allowed it to reach the target by diffusion. When CB was added in this way (Fig. 5A), no observable alteration to the GUV occurred within the first 1–2 min, but, after this initial lag phase, the liposome began to decrease in size without evidence of significant micelle or small liposome formation (Fig 5B–D). Finally, during the terminal stages of liposome disintegration induced by CB, the GUV transformed rapidly into a series of very small vesicles which eventually disappeared (Fig. 5D). Similar analysis of the effect of the analogues CB1 and CB3 revealed several differences. When a GUV was exposed to CB1 (Fig. 5E), the lag phase was markedly shorter, at  30–45 s. Moreover, disintegration of the GUV was hastened in comparison with that observed with CB, with small vesicles rapidly becoming visible. Subsequently, 4–5 min after addition of the peptide, the liposome crum- pled to leave multivesicular and debris-like structures (Fig. 5F–H). The presence of CB1 leads to a propensity to form mutlilamellar vesicles and other fused structures, not observed with the parent peptide (data not shown). By contrast, a markedly different disintegration pattern was observed when the GUV was exposed to CB3 (Fig. 5I–L). After an initial lag phase of  2–3 min, the vesicles began to rapidly fluctuate in size and shape, deviating strongly from their normally spherical appearance. Moreover, hair-like microtubules appeared with increasing frequency, and liposomes that had deviated significantly from their spheri- cal form either burst to leave only the microtubules or rearranged into substantially smaller, microtubule-covered spherical structures. Discussion Cationic antimicrobial defence peptides such as cecropins [1] disrupt cell membranes, and thereby kill their targets, by associating with the membrane lipids [21]. The mechanism of bilayer disruption by cecropin A was recently shown to occur via an ion channel (pore-forming model), and this system has been extensively analysed and characterized using solid-state NMR [47–49]. In addition, experiments using synthetic enantio analogues of a variety of defence peptides, including cecropins, magainins and melittin or melittin-cecropin chimaeras, have shown that these peptides do not function via enzymatic processing or a chiral-specific receptor, but by formation of ion-conducting pores com- posed of self-aggregates [21,50]. However, an alternative model to the barrel-stave pore formation, involving a ÔparallelÕ arrangement of peptides on the membrane surface (Ôcarpet-likeÕ model) has also been suggested for the antimicrobial peptide dermaseptin [51]. This alternative Fig. 3. Comparison of the change in relative response (DRU) measured by SPR for biotin-loaded PtdEtn/PA (7 : 3, w/w) liposomes coupled to the surface of a sensor chip and treated with various concentrations of (A) CB, (B) CB1, or (C) CB3. DRU, Difference between RU at the time of association and the time after the dissociation: i.e. the total capacity of peptides bound to the immobilized liposomes. Ó FEBS 2003 Identifying the disruptive mechanisms of cecropins B, B1 and B3 (Eur. J. Biochem. 270) 915 model further suggests that, as a result of this mechanism of disruption, the membrane decomposes into fragments after a threshold concentration of peptide has been reached [52]. In this study, we have demonstrated the distinction between these two models of membrane disruption, using closely related but structurally different chimaeric peptides from within the same general family by microscopic, thermo- dynamic and biosensor analysis of specific peptide–lipid interactions. Microscopic analysis of the effects of each of the peptides on LUVs reveals differences in their respective activities in real-time. Interestingly, despite their differences in structure, the peptides CB (one amphiphilic helix and one hydrophobic helix) and CB1 (two amphiphilic helices) appeared to have a similar overall effect on the liposome integrity, except that a longer lag time preceded visible effects in the case of CB and the vesicles were more rapidly disrupted by CB1 than by the parent peptide. These observations may suggest that the more hydrophilic and cationic CB1 is more rapidly attracted to the anionic bilayer and that the amphiphilic helix is the predominant cause of membrane disruption when it is present. Overall, the observable effect of these peptides resembles that of phospholipase A 2 from cobra (Naja naja) venom [53]. In contrast, the peptide CB3 (two hydrophobic helices) showed a markedly different disruptive effect on the liposome bilayer, particularly evidenced by loss of the spherical liposome structure and the formation of many hair-like microtubules. Overall, these microscopic observa- tions are consistent with previous suggestions that the peptides CB and CB1 may act by pore formation, whereas CB3 may cause gross membrane destabilization. DSC is a thermodynamic technique for probing the nature, stoichiometry, location, and aggregation state of peptides in their lipid-bound state by analysis of the thermal transitions that these peptide–lipid interactions generate and has been shown to provide valuable information for the analysis of materials that destabilize membrane structure [54,55]. DSC analysis of the thermotrophic phase behaviour of membranes exposed to CB and CB1 revealed that their effect is strongly concentration-dependent; low concentra- tions (1 l M ) lowered and broadened the DSC endotherm, whereas higher concentrations (> 20 l M )resultedin endotherms with two shoulders, one lower and one higher than that observed for untreated liposomes. Multiple peaks in phase-transition endotherms obtained for model mem- branes treated with membrane disrupters such as human defensin, magainin, viral fusion peptides, staphylococcal d-lysin and gramicidin S have been reported previously [55–59]. However, in each case, both peaks were at lower temperatures than for untreated lipids. The observation of endotherm peaks at higher temperatures in this study suggests that aggregation (or pore formation) of the peptides (CB or CB1 at higher concentration, 20 l M )may occur within the lipid bilayers of liposome. The stronger interactions between peptide pore and the lipids causes the higher binding-endothermic effect. However, at lower concentration (1 l M ), the peptides (CB or CB1) disrupt liposomes into smaller species (without pore formation) which causes smaller effects of phase transition than that of liposome alone (see light microscopic results). By contrast, CB3 had significantly less effect on the endotherm, and higher concentrations (50 l M ) were required to obtain multiple peaks similar to the endotherms obtained with CB or CB1, suggesting that CB3 binds less efficiently to the liposomes and has a lower tendency to aggregate at the membrane. SPR has been used to study a variety of biological interactions; here we applied the technique to study peptide–lipid interactions in greater detail by immobilizing liposomes through an avidin–biotin interaction. To reduce any non-specific interactions between the applied peptides and the chip surface, we optimized the derivatization of the chip surface and tested liposome-free chips for Fig. 4. Microcalorimetric determination of the effects of (A) CB, (B) CB1, and (C) CB3 on the phase-transition temperature of PtdEtn/PA (7 : 3, w/w) liposomes. Thick solid lines indicate the DSC curves for liposomes alone (PtdEtn/PA ¼ 7:3,5mgÆmL )1 ), and dashed lines show the DSC curves for peptides CB and CB1 at 20 l M and CB3 at 50 l M in the absence of liposomes. Dotted and thin solid lines represent the thermal curves for CB and CB1 with liposomes (PtdEtn/PA ¼ 7:3, 5mgÆmL )1 )at1l M and 20 l M (respectively) and CB3 with liposomes (PtdEtn/PA ¼ 7:3, 5mgÆmL )1 )at20l M and 50 l M (respectively). 916 H. M. Chen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 nonspecific binding; in each case non-specific interactions were concentration-dependent and minimal, suggesting that the effects observed in the presence of liposomes result primarily from specific peptide–liposome inter- action(s). SPR analysis with higher concentrations of the peptides, CB and CB1, reveals a net increase in mass. One Fig. 5. Microscopic observations of the effect of CB, CB1 and CB3 on GUVs (PtdEtn/PA at 7 : 3). A single, isolated vesicle was treated with a 50 l M concentration of the indicated peptide by application to the side of the vesicle-containing drop and was followed microscopically for the period indicated. For CB, A–D were obtained at time (min) 0, 6, 20 and 24, respectively. For CB1, E–H were obtained at time (min) 0, 1, 3 and 4, respectively. For CB3, I–L were obtained at time (min) 0, 5, 8 and 15, respectively. Ó FEBS 2003 Identifying the disruptive mechanisms of cecropins B, B1 and B3 (Eur. J. Biochem. 270) 917 explanation for these observations could be that higher peptide concentrations favour peptide aggregation and that these aggregates then bind to the liposome surface without causing their destruction. Alternatively, as observed in the DSC analyses, two populations of peptide may exist at higher concentrations: a predominant proportion that is aggregated and interacts directly with the liposome surface, and a second minor population of free peptides that may interact with liposomes in a disruptive manner. Thus, the mass increase observed at higher peptide concentrations would be the sum total of the positive RU values generated by the major popula- tion. By contrast, treatment of surface-bound liposomes with the peptide CB3 had a markedly different effect: at each concentration tested, the peptide disrupted liposome integrity (seen as a loss of mass). Moreover, liposome disruption was even more pronounced at higher peptide concentrations, and there was a short lag in CB3-induced liposome disruption. Initially the peptide appeared to bind to the liposome, as indicated by the short period of mass increase, and binding was followed by mass loss, indica- ting liposome destruction. This lag phase probably indicates that CB3 accumulates on the membrane until a critical concentration is reached, after which the peptides may coalesce to co-operatively cause membrane destruc- tion. Together these observations suggest that CB3 functions by a markedly different mechanism from that used by CB and CB1. In summary, on the basis of liposomal lysis as a function of time observed by the change in SPR, the thermotrophic phase transition of the final states of interactions between liposomes and peptides, and the time course of morphological change in giant liposome observed by microscopy, CB and CB1, having at least one amphipathic a-helix, appear to follow the pore- forming lysis model. In contrast, CB3, having no amphipathic a-helix, appears to have a completely different lysis mechanism, following the carpet-like lysis model. These proposed lysis pathways are also supported by our previous studies using spin-label electron spin resonance [39]. The report indicated that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. CB1 was located in the lipid bilayers by measuring the collision rate with chromium oxalate in solution [39]. Results from electron spin resonance power saturation measurements suggested that the N-terminal a-helix of CB1 is located on the surface of the lipid bilayers, whereas the C-terminal a-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/phosphatidylcholine ratios and the amounts of free peptides [39]. An understanding of the modes of action of these peptides should help in the design of more potent and more specific antimicrobial peptides. Acknowledgements This work was partially supported by the National Science Council (Taiwan) (grant NSC 91-2311-B-001-065). References 1. Steiner, H., Hultmark, D., Engstrom, A., Bennich, H. & Boman, H.G. (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature (London) 292, 246–248. 2. Zasloff, M. 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