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:
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).
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