Distinguishing between different pathways of bilayer disruption by the related antimicrobial...

Distinguishing between different pathways of bilayer disruptionby the related antimicrobial peptides cecropin B, B1 and B3

Hueih Min Chen1, King Wong Leung1, Nagendra N. Thakur1, Anmin Tan1,* and Ralph W. Jack2

1Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan; 2Institut fur Organische Chemie,

Universitat Tubingen, Germany

Different pathways of bilayer disruption by the structurallyrelated 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 microscopicanalysis of peptide-treated giant unilamellar vesicles, havebeen identified in this study.Natural cecropinB (CB)hasoneamphipathic and one hydrophobic a-helix, whereas cecro-pins B1 (CB1) and B3 (CB3), which are custom-designed,chimaeric analogues of CB, possess either two amphipathicor two hydrophobic a-helices, respectively. Surface plasmaresonance analysis of unilamellar vesicles immobilizedthrough a biotin–avidin interaction showed that both CBand CB1 bind to the lipid bilayers at high concentration(>10 lM); in contrast, CB3 induces disintegration of thevesicles at all concentrations tested. Differential scanningcalorimetry showed the concentration-dependent effect of

bilayer disruption, based on the different thermotrophicphase behaviours and the shapes of the thermal phase-transition curves obtained. The kinetics of the lysis of giantunilamellar vesicles observed by microscopy demonstratedthat both CB and CB1 effect a continuous process involvingloss of integrity followed by coalescence and resolution intosmaller vesicles, whereas CB3 induces rapid formation ofirregular-shaped, nonlamellar structures which rapidly dis-integrate into twisted, microtubule-containing debris beforebeing completely destroyed. On the basis of these observa-tions, models by which CB, CB1 and CB3 induce lysis oflipid bilayers are discussed.

Keywords: differential scanning calorimetry; lysis mechan-ism; lytic peptides; microscopic analysis; surface plasmaresonance.

Cationic antimicrobial peptides have now been isolatedfrom a wide variety of sources including bacteria, inverte-brates, vertebrates and plants [1–7]. Although their structureand chemical nature differ markedly, their function appearsto involve protection of the producing organism fromcompeting 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 cationicantimicrobial peptides is probably a survival strategy toobtain an ecological advantage over competitors [8–10]. Ininvertebrates and plants, which lack the adaptive immunesystem of higher animals, these peptides represent a majorcomponent of �innate� immune defence [11–13]. Evenvertebrates rely heavily on the protection against infection

offered by the innate immune system, in which the defencepeptides 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 activityreveals significant differences between various peptides.Several cationic antimicrobial peptides from bacteria,including the lantibiotics nisin and epidermin and thebacteriocin pediocin PA-1, aremost effective against specifictarget organisms and utilize docking molecules present inthe cell membrane in a mechanism that stabilizes theformation of ion-permeable pores [16–19]. As a result, thesepeptides are active at nanomolar concentrations and showlimited target range with little or no observable activityagainst membranes of eukaryotic organisms; they may beactive in the absence of the docking molecules (e.g. artificialbilayers), but at significantly higher peptide concentrations.Conversely, many of the cationic defence peptides ofeukaryotic origin are active at micromolar concentrations,but show little target specificity and may act on manymembrane types including those of erythrocytes (haemolyticactivity). For example, the synthesis of a variety ofanalogues of the 13-amino acid, tryptophan-rich bovineantimicrobial peptide indolicidin has suggested that chiraland/or sequence-specific determinants are not required foreither antibacterial or haemolytic activity, although targetspecificity 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: [email protected]

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) � FEBS 2003 doi:10.1046/j.1432-1033.2003.03451.x

have been obtained with analogues of the insect defencepeptide cecropin B [21]. Thus, the cationic defence peptides,which are active against a broad range of targets, seem tooffer the best opportunity for the study of the molecularmechanisms involved in generalized pore formation in theabsence of specific stabilizing structures.Because of their relatively broad spectrum of activity, it

has been suggested that defence peptides may offer analternative source of antimicrobial chemotherapeutics[22,23]. Moreover, it has also been shown that certainmagainins (isolated from frog skin) and cecropins (insect-derived peptides) also possess antitumour cell activities,making these peptides of special interest as molecularmodels for the study of pore formation in biologicalmembranes [24–27]. Structure–function analysis of thevarious subclasses of defence peptides, combined withdissection of the molecular events involved in pore forma-tion, offers the possibility to develop customized peptideswith defined activities for anti-infective and antitumourapplications.In general, two models for disruption of membrane

integrity have generally been adopted: pore formation bythe �barrel-stave� model, for which alamethicin and melittinare considered prime examples, and the �flip-flop� action(so-called �carpet-like� action) of peptides such as cecropinP1 [28–32]. However, it is not currently possible to assess thelikely mechanism of membrane disruption of a givenpeptide simply on the basis of its structure. Thus, we haveused specific peptides with high homology, but whichcontain specific structural variations, to investigate therelationship between these two cell-killing models [33,34].We have previously reported the synthesis and biologicalactivities of three model peptides: native cecropin B (CB),which contains both a hydrophobic and an amphipathichelix, and the custom-designed, synthetic analogues cecro-pin B1 (CB1) and cecropin B3 (CB3), which contain twoamphiphilic and two hydrophobic helices, respectively[27,35–42]. Recently, we explored the kinetics of liposomelysis using fluorescent quenching and used surface plasmaresonance (BIACore) and oriented circular dichroism [43] toinvestigate the two different modes of membrane disruptionand to detect the orientation of the peptides with respect tothe membrane surface [42]. Kinetic analysis suggests thatbilayer disruption occurs in two distinct steps, whereasoriented circular dichroism provides evidence that thepeptides exist in at least two different membrane-associatedstates, depending on the orientation of the helical segmentswith respect to the bilayer surface. Similar transitions ofpeptide orientation in membrane multilayers have also beenshown for melittin, alamethicin and magainin [44,45].In this study, we examined and compared the disruptive

mechanisms of the natural peptide (CB) and the custompeptide analogues (CB1) and (CB3) against liposomes, largeunilamellar vesicles (LUVs) and giant unilamellar vesicles(GUVs) using surface plasma resonance (SPR), differentialscanning calorimetry (DSC) and real-time light microscopy,respectively. The results indicate that the different mecha-nisms of disruption of lipid bilayers observed are dependenton the particular physicochemical characteristics of thedifferent peptides. Investigation of such differences willprovide useful information for the future custom design ofpeptidyl and mimetic drugs.

Experimental procedures

Materials

Phosphatidylethanolamine (PtdEtn, an egg derivative con-taining fatty acids of C16, C18 and C20). 1,2-Diacyl-sn-glycero-3-phosphatidic acid (PA) is a monosodium saltsynthetic lipid (98%purity). The symmetric fatty acid of PAcontains 18-carbon double chains. Both PtdEtn and PAwere obtained from Sigma-Aldrich (St Louis, MO, USA)and were used without further purification. CM5 SensorChips, amine coupling kits, and BIAevaluation softwarewere all purchased from BIAcore AB (Uppsala, Sweden).Analytical grades of Triton X-100 and SDS were obtainedfrom Sigma-Aldrich, and Biotin-X-DHPE (N-{[6-(bioti-noyl)amino]hexanoyl)}-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) was purchased from MolecularProbes (Eugene, OR, USA). Avidin obtained from Sigma-Aldrich is made from egg white and is chromatographicallypurified and a lyophilized powder. One unit of avidin[12.3 UÆ(mg solid))1] will bind 1.0 lg biotin. All water usedin these experiments was deionized and distilled.

Peptide sequences, preparation and solutions

The preparation of the 35-amino-acid peptide amidesCB (H)KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL)CONH

2), CB1 (H)KWKVFKKIEKMGRNIRNGIVKAGPKWKVFKKIEK)CONH

2) and CB3 (H)AIAVLGEAKALMGRNIRNGIVKAGPAIAVLGEAKAL)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, andCB1 contains two 10-amino-acid amphiphilic helices (itali-cized) and CB3 contains two 10-amino-acid hydrophobichelices (underlined), each derived from the parent, CB. Thepeptides were all>95%pure, as determined byRP-HPLC,and were stored lyophilized at )20 �C until use. Theconcentration of peptide stock solutions was determinedfrom the net weight of peptides (the weight of the associatedcounter ions was not taken into consideration) and theirmolecular masses. Concentrations measured by the abovemethod were confirmed with a bicinchoninic acid assay(Micro BCA protein assay; Pierce Chemical Co.). Anegligible deviation between these two methods wasobserved.

Analysis of liposome–peptide interactions by SPR

Peptides were dissolved in HBS-EP buffer [10 mM Hepes,0.15 M NaCl, 3 mM EDTA and 0.005% surfactant P20 (orTween 20), pH 7.4] to give a stock solution with aconcentration of 500 lM, and these were further diluted asnecessary 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 losscaused by the adsorption of hydrophobic molecules to thesurfaces of the flow system of the BIAcore instrument.Peptides dissolved in HBS-EP buffer are not lysed by theliposomes. Avidin was prepared as a stock at 50 lgÆmL)1

dissolved in 10 mM sodium acetate (pH 4.8). To preparebiotin-containing liposomes, PtdEtn (7 mg) and PA (3 mg)

912 H. M. Chen et al. (Eur. J. Biochem. 270) � FEBS 2003

were dissolved in 1 mL chloroform, and 50 lg biotin-X-DHPEwas added before removal of the chloroform in anargon 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 30passes through a LipoFast extruder fitted with two 100-nmpolycarbonate filter stacks.SPR experiments were conducted using a BIAcore 2000

biosensor (Biacore). Avidin was immobilized on the sensorsurface first using EDC [1-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide] andNHS (succinimide), and the activatedesters that had not bound ligand were capped withethanolamine. The maximum avidin loading was achievedby optimizing the flow rate used (1 lLÆmin)1). Anionicliposomes (1 mgÆmL)1) were then immobilized on thesurface of the chip using various flow rates. Peptidesdissolved in HBS-EP buffer were applied at the concentra-tions and flow rates indicated in order to assess their effecton the immobilized liposomes (1 lLÆmin)1 is the optimum).HBS-EP was used a running buffer. An avidin-free chipsurface, liposomes without biotin, or applications of BSAserved as controls to assess the specificity of the responsesobserved. Peptide-induced responses were measured bySPR, and results were collected and analysed using themanufacturer’s software and protocols. A diagram of theconstruction of the biosensor surface is shown in Fig. 1.Binding of peptides to the immobilized liposomes causes anincrease in resonance units (RU), whereas the disruption ofliposomes results in a decrease in RU. The sensor chipsurface was regenerated by using 10 mM HCl.

DSC

LUVs for use in DSC experiments were prepared fromPtdEtn and PA by dissolving 3.5 mg PtdEtn and 1.5 mg PA(PtdEtn/PA ¼ 7 : 3, w/w) in 1 mL chloroform. Afterremoval of the solvent at room temperature under an argongas stream, the lipids were rehydrated in 1 mLNaCl/Pi andthen sonicated for 30 s. LUVs were subsequently formedby extrusion through a LiposoFast extruder fitted withtwo 100-nm polycarbonate filters for 25 repetitions. The

thermotrophic phase behaviour of the LUVs was measuredin a nano differential scanning calorimeter (CalorimetrySciences Corp., Provo, UT, USA), with temperature scansperformed at a rate of 1 �CÆmin)1. The DSC cells werepressurized to 0.31 kgÆcm)2 (0.30 MPa) throughout theexperiment, and liposomes were used undiluted (i.e. at5 mgÆmL)1). Peptides, dissolved in NaCl/Pi were added togive final concentrations in the range 1–100 lM, andcontrols consisted of DSC analysis of each peptide in theabsence of lipid and the liposomes without peptide.

Preparation of GUVs

GUVs were used for light microscopic observation of themembrane disruptive effects of the peptides (see sectionbelow). To obtain GUVs, we used the method of Akashiet 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 andstored under argon at )20 �C. Subsequently, 10-lL aliquotswere placed in the bottom of a 1.5-cm diameter, 10-mL glasstube, and the solvent was dried at room temperature in astream of argon gas, to provide a thin lipid film. Afterremoval of residual solvent under high vacuum for at least10 h, the film was prehydrated with a stream of water-saturated argon gas for 30 min at 43–45 �C and then fullyrehydrated by addition of 2 mL argon-saturated 1 mM

MgCl2 solution for 48 h at 37 �C. MgCl2 concentrationdependence experiments on GUV lysis induced by peptideswere conducted and it was found that the electrostaticinteractions between peptides and lipids are little influencedby the addition of MgCl2. The liposomes, which appear asan almost transparent, milky-white cloud in the middle ofthe solution, were collected and stored at 4 �C in plastictubes.

Microscopic observation of GUVs

The stored GUVs were typically diluted 10-fold to preventliposome fusion, and 40-lL aliquots were observed on aninverted microscope (Diaphot TMD; Nikon) under phasecontrast, 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 anappropriately thin contour were judged unilamellar andwere used for further study. Peptide solutions (20 lL of a100-lM stock, final concentration � 33.3 lM) were addedafter selection of a single GUV (diameter generally40–120 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 SPRresponse varies greatly with both the type and concentrationof the peptide applied. At higher concentrations (> 10 lM)of either CB or CB1 (Fig. 2), an increase in relative responsewas observed, indicating a time-dependent increase in massand 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.

� FEBS 2003 Identifying the disruptive mechanisms of cecropins B, B1 and B3 (Eur. J. Biochem. 270) 913

with the liposome membranes. Moreover, this effect wasslightly higher for CB1, as indicated by the higher relativeresponse (Fig. 2A,C). However, at lower peptide concen-trations (< 2.5 lM), the relative response was negative.These �negative� observations may be due to the lower flowrates used in this experiment. Further experiments will beperformed to clarify this. Figure 2B,D shows the non-specific binding of CB and CB1 (respectively) to the chipsurface in the absence of liposomes; the level of nonspecificbinding between peptides and sensor chip surface should,however, be significantly lowerwhen immobilized liposomesmostly occupy the chip surface. By contrast, SPR analysis ofthe disruptive effects of CB3 generates negative RU valuesfor all concentrations tested (Fig. 2E). About 80% of theliposome RU is lost at 100 lM CB3. Moreover, the amountof nonspecific binding of CB3 to the sensor chip surface inthe absence of immobilized liposome was not significant(Fig. 2F).The negative RU values observed with CB3 at all

concentrations when passing through the immobilizedliposomes were consistent compared with the positive RUvalues of peptides with nonspecific bindings. Plots ofpeptide concentration against the relative reduction inmass estimated from the SPR response (data not shown)suggested that CB3 caused a time-dependent disruption ofmembrane integrity (Fig. 3C); the effect was morepronounced when higher concentrations of CB3 wereapplied.

DSC analysis of the effect of peptideson PtdEtn/PA LUV thermotrophic phase behaviour

The effect of CB, CB1 and CB3 on the thermotrophic phasebehaviour of LUVs is shown in Fig. 4. The phase-transitiontemperature (Tm) for the PtdEtn/PA (7 : 3) liposome variedconsiderably as a function of peptide concentration (datanot shown). Typical examples were shown at two peptideconcentrations (lower concentration at 1 lM and 20 lM forCB/CB1 and CB3, respectively; higher concentration at20 lM and 50 lM for CB/CB1 andCB3, respectively). In thecase of the parent peptide (CB) with liposomes, lowconcentrations of peptide (1 lM) lowered Tm by � 1.7 �Cand broadened the DSC endotherm as compared withliposome alone. Higher concentrations of peptide (20 lM)resulted in DSC endotherms that split to yield multiplepeaks, one of which was considerably lower than the Tm ofthe liposomes alone, and one of which yielded a Tm evenhigher than that for LUVs in the absence of peptide. Thesetwo distinguishable outcomes of concentration-dependenceare comparable to those observed by SPR (Fig. 2A). Similarresults were obtained for the analogue CB1, although thepeak splitting at higher peptide concentrations was lesspronounced than for the parent peptide. However, a slightlydifferent situation could be discerned for the analogue CB3;considerably higher concentrations (20 lM) were requiredto obtain a decrease in the DSC endotherm similar to thatobserved with 1 lM 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 lM) ofpeptide 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 lM) 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 lM) of peptide CB3 to the surface of asensor with and without (respectively) biotin-loaded PtdEtn/PA liposomes.


concentrations (50 lM), more peak splitting was observed,although it was not as pronounced as that measured withthe other two peptides.Control experiments (flat lines shown in each figure)

using samples of each peptide at concentrations of 20 lM(CB and CB1) and 50 lM (CB3) in the absence of liposomessuggested that endothermic variations observed in thepresence of liposomes arise exclusively from phase transi-tions in the liposomes, which are effected by antimicrobialpeptides.

Microscopic investigation of peptide-induceddisruption of GUVs

Our initial optimization studies showed that the most stableGUVs were formed in low salt concentrations, in particularMgCl2 (1 mM), and that liposome stability increased withincreasing incubation time, although the relative sizedecreased with increasing stability (data not shown). Withthe optimized procedure given in Materials and Methods,we routinely produced stable GUVs with diameters rangingbetween 40 and 120 lm. To study the step-by-step disrup-tion of aGUV,we added the peptide solution and allowed itto reach the target by diffusion. When CB was added in thisway (Fig. 5A), no observable alteration to the GUVoccurred within the first 1–2 min, but, after this initial lagphase, the liposome began to decrease in size withoutevidence of significant micelle or small liposome formation(Fig 5B–D). Finally, during the terminal stages of liposomedisintegration induced by CB, the GUV transformedrapidly into a series of very small vesicles which eventuallydisappeared (Fig. 5D).Similar analysis of the effect of the analogues CB1 and

CB3 revealed several differences.When aGUVwas exposedto CB1 (Fig. 5E), the lag phase was markedly shorter, at� 30–45 s. Moreover, disintegration of the GUV washastened in comparison with that observed with CB, withsmall 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 toform mutlilamellar vesicles and other fused structures, notobserved with the parent peptide (data not shown). Bycontrast, a markedly different disintegration pattern wasobserved when the GUV was exposed to CB3 (Fig. 5I–L).After an initial lag phase of� 2–3 min, the vesicles began torapidly fluctuate in size and shape, deviating strongly fromtheir normally spherical appearance. Moreover, hair-likemicrotubules appeared with increasing frequency, andliposomes that had deviated significantly from their spheri-cal form either burst to leave only the microtubules orrearranged into substantially smaller, microtubule-coveredspherical structures.

Discussion

Cationic antimicrobial defence peptides such as cecropins [1]disrupt cell membranes, and thereby kill their targets, byassociating with the membrane lipids [21]. The mechanismof bilayer disruption by cecropin A was recently shown tooccur via an ion channel (pore-forming model), and thissystem has been extensively analysed and characterizedusing solid-state NMR [47–49]. In addition, experimentsusing synthetic enantio analogues of a variety of defencepeptides, including cecropins, magainins and melittin ormelittin-cecropin chimaeras, have shown that these peptidesdo not function via enzymatic processing or a chiral-specificreceptor, but by formation of ion-conducting pores com-posed of self-aggregates [21,50]. However, an alternativemodel to the barrel-stave pore formation, involving a�parallel� arrangement of peptides on the membrane surface(�carpet-like� model) has also been suggested for theantimicrobial peptide dermaseptin [51]. This alternative

Fig. 3. Comparison of the change in relative response (DRU) measuredby 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.


model further suggests that, as a result of this mechanism ofdisruption, the membrane decomposes into fragments aftera threshold concentration of peptide has been reached [52].In this study, we have demonstrated the distinction betweenthese two models of membrane disruption, using closelyrelated but structurally different chimaeric peptides fromwithin the same general family by microscopic, thermo-dynamic and biosensor analysis of specific peptide–lipidinteractions.Microscopic analysis of the effects of each of the

peptides on LUVs reveals differences in their respectiveactivities in real-time. Interestingly, despite their differencesin structure, the peptides CB (one amphiphilic helix andone hydrophobic helix) and CB1 (two amphiphilic helices)appeared to have a similar overall effect on the liposomeintegrity, except that a longer lag time preceded visibleeffects in the case of CB and the vesicles were more rapidlydisrupted by CB1 than by the parent peptide. Theseobservations may suggest that the more hydrophilic andcationic CB1 is more rapidly attracted to the anionicbilayer and that the amphiphilic helix is the predominantcause of membrane disruption when it is present. Overall,the observable effect of these peptides resembles that ofphospholipase A2 from cobra (Naja naja) venom [53]. Incontrast, the peptide CB3 (two hydrophobic helices)showed a markedly different disruptive effect on theliposome bilayer, particularly evidenced by loss of thespherical liposome structure and the formation of manyhair-like microtubules. Overall, these microscopic observa-tions are consistent with previous suggestions that thepeptides CB and CB1 may act by pore formation, whereasCB3 may cause gross membrane destabilization.DSC is a thermodynamic technique for probing the

nature, stoichiometry, location, and aggregation state ofpeptides in their lipid-bound state by analysis of the thermaltransitions that these peptide–lipid interactions generate andhas been shown to provide valuable information for the

analysis of materials that destabilize membrane structure[54,55]. DSC analysis of the thermotrophic phase behaviourof membranes exposed to CB and CB1 revealed that theireffect is strongly concentration-dependent; low concentra-tions (1 lM) lowered and broadened the DSC endotherm,whereas higher concentrations (> 20 lM) resulted inendotherms with two shoulders, one lower and one higherthan that observed for untreated liposomes. Multiple peaksin phase-transition endotherms obtained for model mem-branes treated with membrane disrupters such as humandefensin, magainin, viral fusion peptides, staphylococcald-lysin and gramicidin S have been reported previously[55–59]. However, in each case, both peaks were at lowertemperatures than for untreated lipids. The observation ofendotherm peaks at higher temperatures in this studysuggests that aggregation (or pore formation) of thepeptides (CB or CB1 at higher concentration, 20 lM) mayoccur within the lipid bilayers of liposome. The strongerinteractions between peptide pore and the lipids causes thehigher binding-endothermic effect. However, at lowerconcentration (1 lM), the peptides (CB or CB1) disruptliposomes into smaller species (without pore formation)which causes smaller effects of phase transition than that ofliposome alone (see light microscopic results). By contrast,CB3 had significantly less effect on the endotherm, andhigher concentrations (50 lM) were required to obtainmultiple peaks similar to the endotherms obtained with CBor CB1, suggesting that CB3 binds less efficiently to theliposomes and has a lower tendency to aggregate at themembrane.SPR has been used to study a variety of biological

interactions; here we applied the technique to studypeptide–lipid interactions in greater detail by immobilizingliposomes through an avidin–biotin interaction. To reduceany non-specific interactions between the applied peptidesand the chip surface, we optimized the derivatization ofthe 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, 5 mgÆmL)1), and dashed lines show the DSC curves

for peptides CB and CB1 at 20 lM and CB3 at 50 lM 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, 5 mgÆmL)1) at 1 lM and 20 lM (respectively) and CB3 with liposomes (PtdEtn/PA ¼ 7 : 3,

5 mgÆmL)1) at 20 lM and 50 lM (respectively).


nonspecific binding; in each case non-specific interactionswere concentration-dependent and minimal, suggestingthat the effects observed in the presence of liposomes

result primarily from specific peptide–liposome inter-action(s). SPR analysis with higher concentrations of thepeptides, 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 lMconcentration 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.


explanation for these observations could be that higherpeptide concentrations favour peptide aggregation andthat these aggregates then bind to the liposome surfacewithout causing their destruction. Alternatively, asobserved in the DSC analyses, two populations of peptidemay exist at higher concentrations: a predominantproportion that is aggregated and interacts directly withthe liposome surface, and a second minor population offree peptides that may interact with liposomes in adisruptive manner. Thus, the mass increase observed athigher peptide concentrations would be the sum total ofthe positive RU values generated by the major popula-tion. By contrast, treatment of surface-bound liposomeswith the peptide CB3 had a markedly different effect: ateach concentration tested, the peptide disrupted liposomeintegrity (seen as a loss of mass). Moreover, liposomedisruption was even more pronounced at higher peptideconcentrations, and there was a short lag in CB3-inducedliposome disruption. Initially the peptide appeared to bindto the liposome, as indicated by the short period of massincrease, and binding was followed by mass loss, indica-ting liposome destruction. This lag phase probablyindicates that CB3 accumulates on the membrane untila critical concentration is reached, after which the peptidesmay coalesce to co-operatively cause membrane destruc-tion. Together these observations suggest that CB3functions by a markedly different mechanism from thatused by CB and CB1.In summary, on the basis of liposomal lysis as a

function of time observed by the change in SPR, thethermotrophic phase transition of the final states ofinteractions between liposomes and peptides, and the timecourse of morphological change in giant liposomeobserved by microscopy, CB and CB1, having at leastone amphipathic a-helix, appear to follow the pore-forming lysis model. In contrast, CB3, having noamphipathic a-helix, appears to have a completelydifferent lysis mechanism, following the carpet-like lysismodel. These proposed lysis pathways are also supportedby our previous studies using spin-label electron spinresonance [39]. The report indicated that the lysis actionof CB1 is related to its capacity to bind to the lipidbilayers. In contrast, there is no evidence of binding forCB3. CB1 was located in the lipid bilayers by measuringthe collision rate with chromium oxalate in solution [39].Results from electron spin resonance power saturationmeasurements suggested that the N-terminal a-helix ofCB1 is located on the surface of the lipid bilayers,whereas the C-terminal a-helix of CB1 is embeddedbelow the surface of the lipid bilayers. These conclusionswere further supported by the observed relationshipbetween the partition distribution of peptides bound toliposomes at different PA/phosphatidylcholine ratios andthe amounts of free peptides [39].An understanding of the modes of action of these

peptides should help in the design of more potent and morespecific antimicrobial peptides.

Acknowledgements

This work was partially supported by the National Science Council

(Taiwan) (grant NSC 91-2311-B-001-065).

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