Báo cáo khoa học: Contribution of a central proline in model amphipathic a-helical peptides to self-association, interaction with phospholipids, and antimicrobial mode of action ppt

Contribution of a central proline in model amphipathic
a-helical peptides to self-association, interaction with
phospholipids, and antimicrobial mode of action
Sung-Tae Yang
1
, Ju Yeon Lee
1
, Hyun-Jin Kim
1
, Young-Jae Eu
1
, Song Yub Shin
2
,
Kyung-Soo Hahm
2
and Jae Il Kim
1
1 Department of Life Science, Gwangju Institute of Science and Technology, Korea
2 Department of Bio-Materials, Graduate School and Research Center for Proteineous Materials, Chosun University, Gwangju, Korea
Antimicrobial peptides are produced as components of
the innate immune system by a wide variety of insects,
amphibians, and mammals, including humans [1–4]. In
recent decades, the structures and functions of many
antimicrobial peptides have been extensively studied to
elucidate their mode of action. Typically, antimicrobial
Keywords
aggregation; amphipathic helix; antimicrobial
peptides; membrane depolarization; proline
Correspondence
J. Kim, Department of Life Science,

Gwangju Institute of Science and
Technology, Gwangju 500-712, Korea
Fax: +82 62 970 2484
Tel: +82 62 970 2494
E-mail:
(Received 9 February 2006, revised 28 June
2006, accepted 5 July 2006)
doi:10.1111/j.1742-4658.2006.05407.x
Model amphipathic peptides have been widely used as a tool to determine
the structural and biological properties that control the interaction of pep-
tides with membranes. Here, we have focused on the role of a central Pro
in membrane-active peptides. To determine the role of Pro in structure,
antibiotic activity, and interaction with phospholipids, we generated a ser-
ies of model amphipathic a-helical peptides with different chain lengths
and containing or lacking a single central Pro. CD studies showed that
Pro-free peptides (PFPs) formed stable a-helical structures even in aqueous
buffer through self-association, whereas Pro-containing peptides (PCPs)
had random coil structures. In contrast, in trifluoroethanol or SDS mi-
celles, both PFPs and PCPs adopted highly ordered a-helical structures,
although relatively lower helical contents were observed for the PCPs than
the PFPs. This structural consequence indicates that a central Pro residue
limits the formation of highly helical aggregates in aqueous buffer and cau-
ses a partial distortion of the stable a-helix in membrane-mimetic environ-
ments. With regard to antibiotic activity, PCPs had a 2–8-fold higher
antibacterial activity and significantly reduced hemolytic activity compared
with PFPs. In membrane depolarization assays, PCPs passed rapidly across
the peptidoglycan layer and immediately dissipated the membrane potential
in Staphylococcus aureus, whereas PFPs had a greatly reduced ability.
Fluorescence studies indicated that, although PFPs had strong binding
affinity for both zwitterionic and anionic liposomes, PCPs interacted

weakly with zwitterionic liposomes and strongly with anionic liposomes.
The selective membrane interaction of PCPs with negatively charged
phospholipids may explain their antibacterial selectivity. The difference in
mode of action between PCPs and PFPs was further supported by kinetic
analysis of surface plasmon resonance data. The possible role of the
increased local backbone distortion or flexibility introduced by the proline
residue in the antimicrobial mode of action is discussed.
Abbreviations
DiSC
3
(5), 3,3¢-dipropylthiadicarbocyanine iodide; PamOlePtdCho, 1-palmitoyl-2-oleoylphosphatidylcholine; PamOlePtdGro, 1-palmitoyl-2-
oleoylphosphatidylglycerol; PCPs, proline-containing peptides; PFPs, proline-free peptides; SPR, surface plasmon resonance.
4040 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS
peptides contain multiple basic amino acids and am-
phipathic structures with clusters of hydrophobic and
hydrophilic residues [5–9]. Although their precise
mechanism of action is not yet fully understood, it is
widely accepted that cationic antimicrobial peptides
interact with negatively charged bacterial membranes
by electrostatic interactions and then cause cell death
by permeabilizing cell membranes by forming barrel-
stave or toroidal pores [10–14] or by disrupting the
membrane via a ‘carpet’ mechanism [15–17]. It is also
known that in some cases peptides inhibit the macro-
molecular synthesis by penentrating into the bacterial
cytoplasm followed by DNA ⁄ RNA binding without
causing membrane permeabilization [18–21]. Some
antimicrobial peptides can lyse not only microbial but
also eukaryotic cells [22]. This activity against eukary-
otic cells should be eliminated so that the antimicrobial

peptides can be used therapeutically. Thus, consider-
able attention has been focused on the design of new
antimicrobial peptides with good selectivity for bacter-
ial cells.
Structure–function studies of antimicrobial peptides
have shown that a number of variables modulate
antibiotic activity, including chain length, helical pro-
pensity, amphipathicity, net positive charge, hydro-
phobicity, and hydrophobic moment [23–29]. A variety
of model amphipathic a-helical peptides and artificial
membranes have been used to analyze the molecular
structure–function relationships, understand the gen-
eral aspects of peptide–lipid interactions, and deter-
mine the variables that control cell selectivity. For
example, to investigate the effect of hydrophobic–
hydrophilic balance on biological and membrane-lytic
activities, Kiyota et al. [30] synthesized five 18-residue
model peptides composed of nonpolar (Leu) and basic
(Lys) residues of varying hydrophobic–hydrophilic
balance. In addition, to determine the proper chain
length for potent antimicrobial peptides, Blondelle &
Houghten [31] prepared a series of 8–22-residue model
amphipathic peptides comprising Leu and Lys. Also,
Papo et al. [32] generated several short model peptides
and their diastereomeric analogs to study the structural
and functional effects of d-amino acids in amphipathic
a-helices. These systematic analyses have helped to
clarify the characteristics needed for the design of
potent, selective antimicrobial peptides as antibiotics.
The presence of Pro residues in a-helices generally cre-

ates a bend or kink in the peptide backbone because of
the lack of an amide proton, which normally provides a
hydrogen bond donor, and they are commonly found
within the amphipathic a-helices of antimicrobial pep-
tides. Pro residues in amphipathic a-helical peptides
have been the focus of extensive research because they
are functionally important in peptide–lipid interactions.
For example, several recent studies have investigated the
effect of Pro substitutions on the biological activity and
structure of naturally occurring antimicrobial peptides
such as PMAP-23 [33], melittin [34], gaegurin [35], tri-
trpticin [36], and maculatin [37]. These studies revealed
that the replacement of a Pro with an Ala maintained or
decreased the antimicrobial activity but significantly
increased the hemolytic activity. In addition, Oh et al.
[38] reported that a cecropin A–magainin II hybrid pep-
tide and its analog P2, which have amphipathic a-helical
structures with a central hinge region due to the
presence of Gly or Pro, have potent and selective antimi-
crobial activity. We also reported that the replacement
of a Pro with Leu or Ala in the hybrid analog P18
decreases its antibacterial activity and increases its
hemolytic activity [39].
In general, introduction of a Pro near the central
region of a-helical antimicrobial peptides reduces the
a-helical structure. This partial disruption of the struc-
ture appears to contribute to selective cytotoxicity. On
the other hand, Pro residues are also found in ion-
channel-forming peptides. Alamethicin, for example,
has a Pro-kink helical structure which is important for

its insertion into lipid bilayers. Once in the lipid bilay-
ers, they form transmembrane helices that contribute
to transmembrane pores or voltage-induced channels
[40,41]. In addition, statistical analysis of transmem-
brane helices has established the significance of Pro-
containing motifs in transmembrane a-helices [42], and
several studies have investigated the structural and
dynamic role of Pro residues in transmembrane helices
[43]. Although there is growing evidence that the Pro
residues largely contribute to the ability of antimicro-
bial peptides to kill various types of microbial cells
and to form transmembrane helices, the role of the
internal kink induced by Pro in amphipathic a-helices
has not been systematically studied, and the kinetic
significance of this structure remains unknown.
Here, we have systematically examined the role of a
central Pro residue by using model 17–25-residue am-
phipathic a-helical peptides that either contain or lack
a Pro residue. We also applied biosensor technology
to distinguish the kinetics of membrane binding by
Pro-containing peptides (PCPs) and Pro-free peptides
(PFPs). We found that the synthetic PCPs have much
more potent antibacterial activity and significantly
reduced hemolytic activity than the PFPs. In addition,
the PCPs were able to selectively bind and strongly
permeabilize negatively charged liposomes. We further
discuss the role of the helix–bend–helix structure
induced by a central Pro residue in the mechanism of
selective antimicrobial activity.
S T. Yang et al. Central proline in amphipathic a-helix

FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4041
Results
Peptide design
To investigate the influence of a central Pro on the
biological activity, structure, membrane binding, and
membrane-disrupting activity of antimicrobial pep-
tides, we generated amphipathic a-helical peptides with
different chain lengths (17, 21, and 25 residues) and
containing or lacking a Pro residue. The model pep-
tides are composed of repeats of hydrophobic (Leu)
and basic (Lys) residues to create perfect amphipathic
a-helices. A single Trp residue was introduced in posi-
tion 2 of these peptides to allow fluorescent deter-
mination of their concentration and peptide–lipid
interactions. The Pro-free peptides (PFPs) included
M17, M21, and M25, and their counterpart central
Pro-containing peptides (PCPs) were M17P, M21P,
and M25P, respectively (Table 1).
Comparison of antimicrobial and hemolytic
activities of the peptides
The model amphipathic a-helical peptides were studied
for their ability to inhibit the growth of Gram-negative
and Gram-positive bacteria as well as for their cyto-
toxicity against human erythrocytes. The minimal
inhibitory concentrations for the peptides against
bacteria are summarized in Table 2, and the dose–
response relationship of the hemolytic activity is depic-
ted in Fig. 1. As shown in Table 2, the different chain
length of the peptides did not significantly affect their
activity toward both Gram-negative and Gram-positive

bacteria. These results suggest that a long chain length
is not required for improved antibacterial activity.
Interestingly, compared with PFPs, PCPs had % 2–8-
fold greater antibacterial activities. As shown in Fig. 1,
however, PFPs (M17, M21, and M25) were relatively
strongly hemolytic (63%, 65%, and 75% at 50 lm,
respectively), whereas PCPs (M17P, M21P, and M25P)
displayed significantly reduced hemolytic activity (4%,
21%, and 11% at 50 lm, respectively). These data
suggest that introduction of Pro residues at a central
position improved the peptide selectivity for bacterial
versus mammalian cells.
Structural analysis of the peptides
CD spectroscopy was used to monitor the secondary
structure of the peptides. The CD spectra of peptides
were collected in 50 mm sodium phosphate buffer ⁄ 50%
trifluoroethanol ⁄ 30 mm SDS micelles, PamOlePtd-
Cho ⁄ PamOlePtdGro (1 : 1) liposomes or PamOlePtd-
Cho liposomes (Fig. 2). The CD spectra of all of the
synthetic peptides dissolved in water in the absence of
salt showed that they were mainly random coils (data
not shown). In buffer (50 mm sodium phosphate
buffer, pH 7.2), however, PFPs (Fig. 2A, filled
Table 1. Amino-acid sequences and molecular masses of the
model peptides. Observed mass was from Kratos Kompact MALDI
TOF MS.
Peptide Sequence
Mass
Calculated Observed
M25 KWKKLLKKLLKLLKKLLKKLKKLLK-NH

2
3114.3 3115.2
M25P KWKKLLKKLLKLPKKLLKKLKKLLK-NH
2
3098.2 3098.8
M21 KWKKLLKKLLKLLKKLLKKLK-NH
2
2631.6 2631.9
M21P KWKKLLKKLLPLLKKLLKKLK-NH
2
2600.5 2601.4
M17 KWKKLLKKLLKLLKKLL-NH
2
2133.9 2134.3
M17P KWKKLLKKPLKLLKKLL-NH
2
2117.8 2118.6
Table 2. Minimal inhibitory concentration (lM) for the peptides.
Results indicate the range of three independent experiments, each
performed in triplicate.
Bacterial strain
Peptide
M25 M25P M21 M21P M17 M17P
E. coli 16–32 4–8 16 4–8 4–8 2–4
S. typhimurium 16 4 8–16 2–4 4–8 2–4
P. aeuroginosa 16–32 4–8 16–32 8 8–16 4–8
B. subtilis 8–16 4–8 8–16 4–8 8 2–4
S. aureus 8–16 2–4 8 2–4 4–8 2–4
S. epidermidis 8–16 4–8 8–16 2–4 4–8 1–4
Fig. 1. Dose–response curves of hemolytic activity of the peptides

toward human erythrocytes. Hemolysis assays were carried out for
the following peptides: M25 (d), M25P (s), M21 (.), M21P (,),
M17 (n), and M17P (h). Results represent the means of duplicate
measurements from three independent assays.
Central proline in amphipathic a-helix S T. Yang et al.
4042 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS
symbols) exhibited typical a-helical CD spectra, with
minimal mean residue molar ellipticity values at 208
and 222 nm, whereas the CD spectra of PCPs
(Fig. 2A, empty symbols) had a negative band below
200 nm, indicating a lack of ordered structure. This
result supports the idea that proline is an effective
a-helix breaker, as previously reported for globular
proteins [44]. As expected, the CD spectra of all of the
peptides indicated a-helix structures in the presence of
trifluoroethanol (Fig. 2B) or SDS micelles (Fig. 2C),
but there was little difference in the helical contents
between PCPs and PFPs. These results suggest that
PCPs have a partially distorted helix structure with a
kink around the central Pro in membrane-mimetic
environments. Interestingly, the shape of PFP spectra
in the presence of PamOlePtdCho ⁄ PamOlePtdGro
(1 : 1) liposomes is apparently different from that in
the presence of SDS or trifluoroethanol (Fig. 2D). This
may point to strong aggregation in this type of mem-
brane, which may correlate with the increased cytotox-
icity of these peptides. In PamOlePtdCho liposomes
(Fig. 2E), PCPs had no distinct secondary structure,
but there was a weak shoulder in their spectra, com-
pared with aqueous solution, suggesting that some

interaction does occur. In contrast, PFPs adopted
a-helical structures, indicating that PFPs can strongly
interact with zwitterionic liposomes.
Next, to determine in detail the effect of salt on
the conformational transition from a random coil to
an a-helix, the CD spectra were recorded as a func-
tion of the NaCl concentration from 0 to 100 mm at
a constant peptide concentration (Fig. 3). The results
for peptide M21 are shown as an example in
Fig. 3A. The CD spectra of M21 in the presence of
various NaCl concentrations exhibited an isodichroic
point at 203 nm, indicating a two-state equilibrium
between a random coil and an a-helix. In pure
water, M17 and M25 also became more a-helical as
the NaCl concentration was increased (Fig. 3B).
Helix formation by the PFPs appears to be accom-
panied by self-association. In addition, the ratio of
ellipticity values at 222 ⁄ 208 nm is close to 1 in buf-
fer, which this is taken to indicate aggregation [45].
The CD spectra of PCPs (M17P, M21P, and M25P),
however, did not change as the NaCl concentration
was increased. These results suggest that the presence
of a kink induced by a Pro residue in amphipathic
a-helices is essential for maintaining them as mono-
mers in aqueous solution.
Peptide-induced dye leakage from liposomes
We next measured the membrane-disrupting abilities
of the peptides by examining calcein leakage from neg-
atively charged PamOlePtdCho ⁄ PamOlePtdGro (1 : 1)
or zwitterionic PamOlePtdCho liposomes. Upon

addition of the peptides to the liposomes, the
entrapped calcein (70 mm) was released into the buf-
fer by lysis. This relieves self-quenching of the dye
within the liposomes, increasing the fluorescence
intensity. Relative lytic efficiencies were determined
by comparing the effects of the peptides with those
of Triton X-100, which corresponds to the total
fluorescence. Dose responses of peptide-induced calc-
ein release from the PamOlePtdCho ⁄ PamOlePtdGro
(1 : 1) and PamOlePtdCho liposomes are shown in
Fig. 4. Compared with PCPs, PFPs released as much
or slightly more calcein from the PamOlePtdCho ⁄
PamOlePtdGro (1 : 1) liposomes. All of the amphi-
pathic peptides caused an almost total disruption of
Fig. 2. CD spectra of the model peptides under various conditions. CD spectra were obtained at 25 °C in (A) 50 mM sodium phosphate buf-
fer (pH 7.2), (B) 50% trifluoroethanol, (C) 30 m
M SDS micelles, (D) PamOlePtdGro ⁄ PamOlePtdCho (1 : 1) liposomes, or (E) PamOlePtdCho
liposomes and in the presence of the following peptides at 25 l
M concentration: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), and
M17P (h).
S T. Yang et al. Central proline in amphipathic a-helix
FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4043
the PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) liposomes
at 1 : 20 molar ratio of peptide to liposome. In con-
trast, the PFPs (M17, M21, and M25) caused relat-
ively large calcein leakage (57%, 60%, and 66%,
respectively) from PamOlePtdCho liposomes at a
peptide to liposome molar ratio of 1 : 10, whereas
the PCPs showed a relatively reduced ability to
reduce PamOlePtdCho membranes. These results

agree well with those from analysis of hemolysis, and
they indicate that introduction of Pro into amphi-
pathic a-helical peptides confers the ability to selec-
tively disrupt anionic versus zwitterionic liposomes.
Fig. 3. CD spectra of M21 and [.]
222
for the model peptides at various NaCl concentrations. (A) CD spectra were recorded as a function of
the NaCl concentration (from 0 to 50 m
M at increments of 5 mM) for 25 lM peptide M21 at 25 °C. (B) Plot of [h]
222
versus NaCl concentra-
tion (0–100 m
M) for the following peptides at 25 lM concentration: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), and M17P (h).
Fig. 4. Calcein leakage as a function of molar ratio of peptide to lipid. Calcein-containing (A) PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or (B)
PamOlePtdCho liposomes at 25 °C were mixed with the following peptides: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), or M17P
(h). Results represent the means of three independent experiments.
Central proline in amphipathic a-helix S T. Yang et al.
4044 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS
Of the PCPs, M17P and M25P showed negligible cyto-
toxicity against human red blood cells and a relatively
weak ability to disrupt artificial neutral liposomes,
whereas M21P had moderate cytolytic activity. In the
case of M17P and M25P, Pro replaced the central Leu
of the hydrophobic helix face (based on an amphipath-
ic helical wheel diagram), whereas in M21P, it replaced
the central Lys of the hydrophilic helix face. The mod-
erate cytotoxicity of M21P suggests that placement of
Pro in the hydrophobic face of amphipathic a-helical
peptides is more effective than placement in the hydro-
philic region for generating peptides with selectivity for

bacterial versus red blood cells.
Tryptophan fluorescence
To study the interaction of PCPs and PFPs with
membranes, we next examined changes in Trp fluor-
escence in pure water, aqueous buffer, or anionic
PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or zwitterionic
PamOlePtdCho liposomes. As the fluorescence emis-
sion characteristics of the Trp are sensitive to its
immediate environment, it can be used to monitor
the binding of peptides to membranes. All the pep-
tides listed in Table 1 have a single Trp residue at
position 2. The corresponding maximum emission
wavelength (k
max
) is plotted as a function of the
lipid ⁄ peptide molar ratio in Fig. 5, and the k
max
val-
ues of the peptides at lipid ⁄ peptide molar ratio of
50 : 1 are shown in Table 3. In Tris ⁄ HCl buffer, the
k
max
values for PCPs were % 352 nm, indicating that
Trp residues are fully exposed to a hydrophilic envi-
ronment. In contrast, the k
max
value of the Trp resi-
due in PFPs was % 343 nm, indicating that Trp was
surrounded by a hydrophobic environment through
self-association of the peptides in buffer. Addition of

PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) liposomes with
both PFPs and PCPs results in a large blue shift (22–
24 nm) in the k
max
and an increase in the fluores-
cence quantum yield for all the peptides, indicating
that the peptides strongly bind to negatively charged
membranes. With zwitterionic PamOlePtdCho lipo-
somes, there was an almost constant k
max
around
349 nm for M17P and M25P at all lipid ⁄ peptide
ratios and a small blue shift (9 nm) for M21P, indica-
ting a lack of binding to PCPs. In contrast, the three
model PFPs displayed a large blue shift (18–19 nm).
These results indicate that PCPs interact weakly with
zwitterionic phospholipids but strongly with anionic
phospholipids.
Fig. 5. k
max
of tryptophan fluorescence as a function of the lipid ⁄ peptide ratio. Fluorescence spectra were recorded at increasing concentra-
tions of (A) PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or (B) PamOlePtdCho liposomes in Tris ⁄ HCl buffer (pH 7.4) at 25 °C and at 3 l
M of the
following peptides: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), or M17P (h). The excitation wavelength was 280 nm, the excitation
band width was 5 nm, and the emission band width was 3 nm. Results represent the means of three independent experiments.
Table 3. k
max
(nm) of tryptophan fluorescence for the peptides and
K
SV

in the presence of liposomes. Assays were carried out in
Tris ⁄ HCl buffer or in the presence of PamOlePtdCho ⁄ PamOlePtd-
Gro (1 : 1) or PamOlePtdCho liposomes at a lipid ⁄ peptide molar
ratio of 50 : 1.
Peptide
Pure
water
Tris ⁄ HCl
buffer
PtdCho ⁄
PtdGro
PamOle-
PtdCho
K
SV
(M
)1
)
PtdCho ⁄
PtdGro
PamOle-
PtdCho
M25 351 342 328 332 1.96 3.24
M25P 352 351 330 348 2.47 9.14
M21 351 343 329 333 1.83 2.83
M21P 353 351 330 341 2.33 6.29
M17 352 344 330 333 2.20 3.60
M17P 353 352 329 349 2.61 9.88
S T. Yang et al. Central proline in amphipathic a-helix
FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4045

Quenching of the intrinsic fluorescence
by acrylamide
To compare the membrane-integrated state of PCPs
and PFPs following their interaction with negatively
charged PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or
neutral PamOlePtdCho liposomes, we next performed
a fluorescence quenching experiment using the neutral
fluorescence quencher acrylamide. This quencher can
approach Trp more easily when the peptide is free in
solution than when it is bound to model membranes.
Stern-Volmer plots for fluorescence quenching of Trp
by acrylamide in the presence of PamOlePtdCho ⁄
PamOlePtdGro (1 : 1) or PamOlePtdCho liposomes
are depicted in Fig. 6, and the apparent K
SV
values are
shown in Table 3. The Trp fluorescence intensity for
PFPs decreased in a similar concentration-dependent
manner for both types of liposome after the addition
of acrylamide, indicating that PFPs are buried in both
anionic and neutral liposomes. The tendency of PFPs
to self-associate appears to affect their nonselective
interaction. However, quenching of Trp fluorescence of
PCPs is less efficient with PamOlePtdCho ⁄ PamOlePtd-
Gro (1 : 1) than with PamOlePtdCho vesicles, suggest-
ing that the Trp residue of PCPs penetrates more
efficiently into the hydrophobic core of negatively
charged bilayers than zwitterionic bilayers.
Membrane depolarization by PFPs and PCPs
It is widely believed that many membrane-active anti-

microbial peptides pass through the peptidoglycan
layer and then kill the target micro-organism by inter-
acting with and permeabilizing the cytoplasmic mem-
brane. To further study this hypothesis, we examined
the ability of PFPs and PCPs to depolarize the mem-
brane using the membrane-potential-sensitive dye 3,3¢-
dipropylthiadicarbocyanine iodide [DiSC
3
(5)] (Fig. 7).
Upon addition to a suspension of S. aureus, the fluor-
escence of DiSC
3
(5) (first arrow) is strongly quenched
and quickly stabilized. Addition of peptides (second
arrow) increased the fluorescence caused by membrane
depolarization, and subsequent addition of gramicidin
D (third arrow) fully disrupted the membrane poten-
tial. Interestingly, PCPs almost completely dissipated
the membrane potential at 0.3 lm, but self-associated
PFPs showed a largely reduced ability to cause mem-
brane depolarization. In addition, all PCPs caused an
immediate increase in fluorescence intensity, indicating
rapid membrane depolarization, whereas the PFPs
caused a gradual increase in the fluorescence. These
results suggest that the self-association of PFPs, which
have less potent antimicrobial activity, interferes with
their passage across the peptidoglycan layer.
Analysis of binding using an surface plasmon
resonance (SPR) biosensor
Finally, we used SPR to monitor the binding of PFPs

and PCPs to PamOlePtdCho ⁄ PamOlePtdGro (1 : 1)
liposomes immobilized on an L1 sensor chip. Figure 8
shows representative sensorgrams for the binding of
M17 and M17P. The sensorgrams for M25 and M21
were similar to that for M17, whereas the sensorgrams
for M25P and M21P were similar to that for M17P
(data not shown). Examination of the shape of the
sensorgrams for M17 and M17P reveals significantly
different binding kinetics. In particular, the sensor-
grams indicate that the initial association of M17P
with the lipid surface starts as a very fast process
Fig. 6. Stern-Volmer plots for the quenching of Trp fluorescence by
the peptides. Quenching assays were carried out in the presence
of 150 l
M of either (A) PamOlePtdGro ⁄ PamOlePtdGro or (B)
PamOlePtdCho liposomes and the following peptides at 3 l
M con-
centration: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), or
M17P (h). Results represent the means of three independent
experiments.
Central proline in amphipathic a-helix S T. Yang et al.
4046 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS
compared with that of M17. In addition, whereas
M17P exhibited a distinct association and dissociation,
M17 had a very slow dissociation at a low peptide
concentration (less 20 lm) and failed to dissociate
from the liposomes at high concentrations (more
40 lm). When the sensorgrams are fitted using differ-
ent concentrations of M17P, the two-state reaction
model fits better than the simple 1 : 1 Langmuir bind-

ing model, suggesting that a two-step process mediates
the interaction of the peptide with lipid bilayers. How-
ever, M17 had similar C
2
values in both fitting models.
Only peptide sensorgrams obtained at low peptide
concentrations (2.5–20 lm) were used to calculate the
association constants for M17 because the peptide was
bound irreversibly to the lipid bilayers at high concen-
trations. The average values for the rate constants and
affinity constants obtained from the two-state model
analysis are listed in Table 4. There were striking dif-
ferences between PFPs and PCPs in the association
rate (k
a1
) for first step and the dissociation rate (k
d2
)
for second step. The observations with PFPs and PCPs
seem to be in line with those of Zelezetsky et al. [46],
using different types of aggregating ⁄ nonaggregating
model peptides.
Discussion
Membrane-active peptides mediate a wide range of
biological events, including signal transduction, trans-
port through the membrane, membrane fusion and
lysis, ion channel formation, and antimicrobial def-
ense. These peptides exhibit a structural transition
from an extended coil to a well-defined secondary
structure upon binding to membrane surfaces. Interac-

tion of the peptides with membranes plays an import-
ant role in many cellular processes. In particular, Pro
residues often appear in the central region of mem-
brane-active peptides, and they may control the folding
process and affect the membrane translocation or pen-
etration [43,47–49].
Recently, model peptides have been intensively stud-
ied as tools for determining the structural and biologi-
cal properties of antimicrobial peptides. In particular,
model amphipathic a-helical peptides have been stud-
ied extensively to identify general properties related to
peptide–lipid interaction and their relationships with
the biological activity of the peptides [28–32]. In the
present study, we carried out a systematic structure–
activity study on a series of model peptides to deter-
mine the role of a central Pro on the biological activ-
ity, peptide structure, and interaction with membranes.
One interesting finding of this study was that intro-
duction of a Pro in the middle position of the
sequences of nonselective cytolytic peptides confers
high selectivity for bacterial cells. In particular, we
found that the depolarization of bacterial membranes
caused by PCPs is more potent and rapid than that
caused by PFPs. There was a direct correlation
between the ability of the peptides to dissipate the
membrane potential and their antimicrobial activity. In
Fig. 7. Kinetics of membrane depolarization of S. aureus by PFPs
and PCPs. DiSC
3
(5) was added to exponential-phase S. aureus

cells. once the fluorescence was stable, the peptides (0.3 l
M) were
added, and membrane depolarization was measured. Gramicidin D
(0.22 n
M) was used to induce full collapse of the membrane poten-
tial. The results are representative of two independent experi-
ments.
S T. Yang et al. Central proline in amphipathic a-helix
FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4047
addition, Trp fluorescence measurements indicated that
PFPs interacted nonselectively with negatively charged
and zwitterionic liposomes, whereas PCPs bound
strongly and selectively to anionic liposomes. These
results are consistent with the ability of the peptides to
induce dye leakage preferentially from negatively
charged lipid membranes. The selective membrane
interaction of Pro-containing peptides with negatively
charged phospholipids may explain the selective anti-
bacterial activity because zwitterionic phospholipids
are the major constituent of the outer leaflet of red
blood cells.
Understanding the process of peptide folding in
aqueous buffer or in membrane-mimetic environments
is critical for elucidating the mechanism of antimicro-
Fig. 8. Sensorgrams for the binding of peptides to 1 : 1 PamOlePtdCho ⁄ PamOlePtdGro lipid bilayers. Overlay of the experimental (solid line)
and calculated (dotted line) sensorgrams using a two-state model (A and C) or a 1 : 1 Langmuir model (B and D). Lower plot, 5 l
M; upper
plot, 20 l
M. Results are representative of two independent experiments.
Table 4. Kinetic interaction of the peptides with PamOlePtd-

Cho ⁄ PamOlePtdGro (1 : 1) lipid bilayers. Association (k
a1,
k
a2
) and
dissociation (k
d1,
k
d2
) kinetic rate constants for the interaction of
PFPs and PCPs with PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) were
determined by numerical integration using a two-state reaction
model. The affinity constant (K) was determined as (k
a1
⁄ k
d1
)
(k
a2
⁄ k
d2
).
Peptide
k
a1
(1 ⁄ Ms) k
d1
(1 ⁄ s) k
a2
(1 ⁄ s) k

d2
(1 ⁄ s) K (1 ⁄ M)
M25 601 1.41 · 10
)2
3.49 · 10
)2
1.64 · 10
)6
9.07 · 10
8
M25P 4347 5.92 · 10
)2
2.03 · 10
)2
3.17 · 10
)3
4.70 · 10
5
M21 598 1.33 · 10
)2
2.23 · 10
)2
2.55 · 10
)6
3.93 · 10
8
M21P 4822 5.76 · 10
)2
1.56 · 10
)2

3.80 · 10
)3
3.44 · 10
5
M17 634 1.02 · 10
)2
2.95 · 10
)2
1.96 · 10
)6
9.35 · 10
8
M17P 4080 5.92 · 10
)2
1.08 · 10
)2
2.65 · 10
)3
2.81 · 10
5
Central proline in amphipathic a-helix S T. Yang et al.
4048 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS
bial action. CD spectra of the model amphipathic pep-
tides revealed that, in buffer, the central Pro residue
effectively disrupts the a-helical structure, but, in mem-
brane-mimetic environments, the Pro kept a-helical
structures, which means that Pro does not always
behave as a strong helix breaker in certain surround-
ings including membrane-mimetic environments. These
findings with amphipathic a-helical peptides agree with

those reported by Li et al. [50] for model transmem-
brane helical peptides.
The CD and Trp fluorescence spectra of PFPs were
very sensitive to the salt concentration. In buffer, PFPs
are thought to take on an a-helical structure because
of self-association. An increase in ionic strength seems
to lead to a decrease in the electrostatic repulsive for-
ces between the positively charged residues because of
the presence of counterions. In contrast, despite the
reduced electrostatic repulsion in the presence of a
high salt concentration, PCPs had unordered struc-
tures. As suggested by Sansom & Weinstein [51], this
is presumably due to structural dynamics such as twist-
ing and kinking induced by a central Pro residue.
The aggregation of PFPs in buffer correlates with
the ability of the peptides to cause the lysis of human
red blood cells and zwitterionic liposomes. In contrast,
the self association of PFPs appears to interfere with
their ability to cross the peptidoglycan layer and reach
the cytoplasmic membrane. Therefore, PFPs are likely
to show less potent membrane depolarization and
greatly reduced antimicrobial activity. Despite the
cytotoxicity of PFPs, however, it appears that the
structural stability and oligomeric form of PFPs in
the presence of a high NaCl concentration could be
useful for treating cystic fibrosis patients if their anti-
microbial versus hemolytic activity is optimized.
Kinetic analysis of the sensorgram results suggests
that the binding of M17P to the lipid bilayer occurs by
a distinct two-step process: the peptides may first bind

to the lipid head groups via electrostatic interaction
and then insert further into the hydrophobic interior
of the membrane via hydrophobic interactions. The
largest differences between M17 and M17P were
increases in the rate of association (k
a1
; Table 4) in the
first step and dissociation (k
d2
, Table 4) in the second
step. These findings indicate, respectively, that the
central Pro of a-helical peptides is important for fast
electrostatic interaction with PtdCho ⁄ PtdGro mem-
branes and that the Pro is important for effective
translocation across the membrane. In addition, the
values of k
a1
⁄ k
d1
(K
1
; initial binding) and k
a2
⁄ k
d2
(K
2
;
insertion) correspond, respectively, to the affinity con-
stants for electrostatic and hydrophobic interaction of

the peptides with lipid bilayers. As observed in other
amphipathic a-helical peptides such as magainin
[52,53], the initial binding of M17P (K
1
¼
6.8 · 10
4
m
)1
) was much faster than the following
insertion step (K
2
¼ 4.0). This suggested that the elec-
trostatic interaction is a crucial factor for M17P and is
responsible for its selective cytotoxicity. In contrast,
for M17, the rate of the first step (K
1
¼ 6.2 · 10
4
m
)1
)
was similar to that of the second (K
2
¼ 1.5 · 10
4
). The
fact that the K
2
value for M17 is much higher than

that for M17P indicates that the affinity of the pep-
tides for membranes is driven predominantly by hydro-
phobic interactions. This may explain the nonselective
interaction of M17 with both zwitterionic and negat-
ively charged membranes.
Clarifying the structural aspects of the peptides that
confer selective binding to negatively charged lipid
membranes and identification of the driving forces for
membrane partitioning are essential for understanding
the mechanism of permeabilization and improving
antimicrobial selectivity. The interaction of PCPs with
negatively charged membranes is thought to confer
selective antimicrobial function, but the induction of
plasma membrane leakage alone may not be sufficient
to explain the action of these peptides. Our results
indicate that Pro residues of amphipathic a-helical
peptides may promote formation of a bent structure
by inducing the formation of a helix turn in mem-
brane-mimetic environments. The bending of PCPs is
presumably to provide a membrane anchor after their
initial interaction with the membrane surface. The
overall amphipathic helix of PCPs lies approximately
parallel to the bilayer plane, so the bending potential
may be the driving force for penetration of the N-ter-
minus or C-terminus of the peptides into the core of
the bilayer. In other words, partial conformational
flexibility may be a prerequisite for import of the pep-
tides into membranes or the cytosol. For example, a
single Pro residue has been found to be a key struc-
tural factor for the penetration of cells by buforin II

[20]. Also, the Pro residue is thought to promote trans-
location across lipid bilayers [21]. Many signal peptides
also contain a helix-breaking residue and adopt a
dynamic helix–break–helix conformation, and this
structural motif is thought to be important for the effi-
cient initiation of translocation [54–56]. In addition, all
enveloped viruses enter cells by peptide-mediated mem-
brane fusion. The viral fusion peptides involved in this
process interact with and destabilize the target mem-
brane. A common feature of many internal viral fusion
peptides is the presence of a Pro near the center of
their sequence, and it is known that the central Pro
residue in fusion peptides is important for the forma-
tion of their native structure as well as for the
S T. Yang et al. Central proline in amphipathic a-helix
FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4049
membrane interactions that lead to fusion [57,58]. Fur-
thermore, Niidome et al. [59] reported that a peptide
in which in the central double Pro was replaced with
double Ala was less able to promote membrane fusion
and was more lytic. Therefore, it is plausible that the
improved bactericidal activity of PCPs is due to the
promotion of translocation by the higher bending pot-
ential, their ability to cause membrane disruption, and
the existence of an intracellular target for the peptides.
In summary, we have demonstrated that a central
Pro in amphipathic a-helical peptides effectively dis-
rupts their a-helical structures and aggregation in
buffer but that they maintain a-helical structures
in membrane-mimetic environments despite somewhat

reduced a-helical contents. The tendency of PFPs to
self-associate in buffer correlated with their cytotoxici-
ty to human red blood cells and their ability to lyse
artificial zwitterionic liposomes. In addition, biosensor
technology indicated that the interaction of PCPs with
membranes was predominantly influenced by initial
electrostatic interactions, whereas the interaction of
PFPs with membranes was most affected by hydropho-
bic interactions. These properties may lead to the selec-
tivity of PCPs and⁄ or the nonselectivity of PFPs for
bacterial versus red blood cells. It is likely that the
collapse of self-association caused by a central Pro
enhances the ability of the peptide to cross the pepti-
doglycan layer and rapidly reach the cytoplasmic mem-
brane. Although cation-rich and Pro-rich antimicrobial
peptides including PR-39 and bactenecin can penetrate
the cell membrane without membrane disruption
[60,61], further studies are needed to clarify the role of
a single central Pro residue in allowing amphipathic
a-helical peptides to cross lipid bilayers.
Experimental procedures
Materials and micro-organisms
N-a-Fmoc (fluoren-9-yl-methoxycarbonyl) amino acids with
orthogonal side-chain-protecting groups were purchased
from Novabiochem (Laufelfingen, Switzerland). The reagents
and solvents (highest purity commercially available) for
peptide synthesis were obtained from Applied Biosystems
(Foster City, CA, USA). Membrane potential measurements
were performed with a membrane potential-sensitive probe
DiSC

3
(5) (Molecular Probes, Eugene, OR, USA). The
phospholipids PamOlePtdCho and PamOlePtdGro were
purchased from Avanti Polar Lipids (Alabster, AL, USA).
All other regents were of analytical grade. Escherichia coli
KCTC 1682, Salmonella typhimurium KCTC 1926, Pseudo-
monas aeruginosa KCTC 1637, Bacillus subtilis KCTC 3068,
S. aureus KCTC 1621, and Staphylococcus epidermidis
KCTC 1917 were purchased from the Korean Collection for
Type Cultures, Korea Research Institute of Bioscience &
Biotechnology.
Peptide synthesis, purification, and
characterization
Peptides were synthesized using solid-phase methodology
with Fmoc-protected amino acids [62]. Fmoc-protected
peptides were deprotected and cleaved using a mixture of
trifluoroacetic acid, phenol, water, thioanisole, and ethane-
1,2-dithiol (82.5 : 5 : 5 : 5: 2.5, by vol.) for 3 h at room
temperature. HPLC analysis was performed using an LC-
6AD or a LC-10Avp system (Shimadzu, Tokyo, Japan)
with an ODS column (4.6 · 250 mm). Purification by pre-
parative RP-HPLC gave final products that were > 98%
pure as determined by analytical RP-HPLC. Their calcula-
ted average masses were identified by Kratos Kompact
MALDI TOF MS (Shimadzu). Peptides were analysed in
linear mode, and the samples were prepared by the dried-
droplet method. The matrix solution contained saturated
a-cyano-4-hydroxycinnamic acid in 50% acetonitrile ⁄ 0.1%
trifluoroacetic acid.
Determination of antimicrobial activity

The antimicrobial activity of peptides against a range of
micro-organisms was determined by broth microdilution
assay. Briefly, a single colony of bacteria was inoculated
into culture medium (Luria–Bertani broth) and cultured
overnight at 37 °C. An aliquot of this culture was trans-
ferred to 10 mL fresh culture medium and incubated for an
additional 3–5 h at 37 °C to obtain mid-exponential phase
organisms. A twofold dilution series of peptides in 1% pep-
tone was prepared, and 100 lL was added to bacteria
(2 · 10
6
colony-forming unitsÆmL
)1
; 100 lL) in 96-well
microtiter plates (F96 microtiter plates; Nunc, Odense,
Denmark), and the plates were incubated at 37 °C for 16 h.
After incubation, the absorbance in each well at 620 nm
was measured. The lowest peptide concentration that com-
pletely inhibited the growth of the organisms was defined
as the minimal inhibitory concentration. The reported min-
imal inhibitory concentrations are the mean of triplicate
measurements from three independent assays.
Hemolytic activity
Human red blood cells were washed three times with phos-
phate-buffered saline (NaCl ⁄ P
i
;35mm phosphate, pH 7.0,
150 mm NaCl). Then 100 lL4%(v⁄ v) human red blood
cells in NaCl ⁄ P
i

was dispensed into each well of sterile
96-well plates. Next, 100 lL peptide solution was added to
each well. The plates were incubated for 1 h at 37 °C and
then centrifuged at 1000 g for 5 min. Aliquots (100 lL) of
Central proline in amphipathic a-helix S T. Yang et al.
4050 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS
supernatant were transferred to 96-well plates, in which
hemoglobin release was monitored by measuring the
absorbance at 414 nm using an ELISA plate reader
(Molecular Devices, Sunnyvale, CA, USA). No (0%) and
complete (100%) hemolysis were determined in NaCl ⁄ P
i
and 0.1% Triton X-100, respectively. Percentage hemolysis
was calculated using the following formula: % hemoly-
sis ¼ [(A
414
in the presence of peptide solution ) A
414
in
NaCl ⁄ P
i
) ⁄ (A
414
in 0.1% Triton X-100 ) A
414
in
NaCl ⁄ P
i
)] · 100. The % hemolysis recorded was the mean
of duplicate measurements from three independent assays.

CD spectroscopy
The CD spectra of peptides were recorded using a
J-715 CD spectrophotometer (Jasco, Tokyo, Japan) with a
1-mm path-length cell. Wavelengths from 190 to 250 nm
were measured, with a step resolution of 0.1 nm, scan speed
of 50 nmÆmin
)1
, response time of 0.5 s, and bandwidth of
1 nm. CD spectra of peptides were collected and averaged
over four scans at 25 °Cin50mm sodium phosphate buffer
(pH 7.2), 50% trifluoroethanol or 30 mm SDS micelles. The
mean residue ellipticity [h] (in degrees Æ cm
2
Ædmol
)1
) was cal-
culated using [h] ¼ [h]
obs
(MRW ⁄ 10lc), where [h]
obs
is the
ellipticity measured in millidegrees, MRW is the mean resi-
due molecular mass of the peptide, c is the concentration of
the sample in mgÆmL
)1
, and l is the optical path length of
the cell in cm. The spectra were plotted as the molar ellip-
ticity [h] versus wavelength. Measurements were repeated
twice for each condition to ensure reproducibility.
Preparation of liposomes

For SPR experiments, small unilamellar vesicles were pre-
pared by sonication. A 1 : 1 mixture of PamOlePtdCho and
PamOlePtdGro was dissolved in chloroform and dried
under a stream of nitrogen gas to form a thin lipid film on
the wall of a glass tube. The lipid film was dried under
vacuum overnight and then resuspended in 20 mm sodium
phosphate buffer using a vortex mixer. The suspension was
sonicated under nitrogen in an ice bath for % 20 min using
a titanium-tipped sonicator until clear. Large unilamellar
vesicles were prepared for fluorescence experiments by
extrusion. The dried lipid film composed of either Pam-
OlePtdCho or PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) was
hydrated with Tris ⁄ HCl buffer (10 mm Tris ⁄ HCl, pH 7.4,
150 mm NaCl, 0.1 mm EDTA) or 70 mm calcein and resus-
pended with a vortex mixer. The suspension was subjected
to five cycles of freeze–thaw and then successively extruded
20 times through polycarbonate filters (LiposoFast; 100-nm
pore diameter, Avestin Inc., Ottowa, Canada). Calcein-
containing vesicles were separated from free calcein by gel
filtration chromatography in Tris ⁄ HCl buffer using a
Sephadex G-50 column (Pharmacia, Uppsala, Sweden). The
concentration of lipid vesicles used in the various assays is
the lipid concentration initially used for large and small
unilamellar vesicle preparation.
Measurement of peptide-induced dye leakage
The fluorescence intensity of calcein released from lipo-
somes was monitored at 520 nm (excitation at 490 nm) on
a Shimadzu RF-5301 spectrofluorimeter and measured
2 min after the addition of peptides. Fluorescence from
liposomes lysed with Triton X-100 was used as an indicator

of 100% leakage. The percentage of dye leakage caused
by the peptides was calculated according to the following
equation: % leakage ¼ 100 · (F ) F
0
) ⁄ (F
t
–F
0
), where F is
the fluorescence intensity achieved by the peptides, and F
0
and F
t
are the initial fluorescence intensities observed with-
out the peptides and after treatment with Triton X-100,
respectively. All conditions were assayed in triplicate.
Tryptophan fluorescence and quenching
by acrylamide
Tryptophan fluorescence measurements were collected using
a Shimadzu RF-5301 spectrofluorimeter. Liposomes were
added to a fixed amount of peptide (3 lm) dissolved in
10 mm Tris ⁄ HCl buffer. The excitation wavelength was
280 nm, and emission was measured from 300 to 400 nm.
Spectra were recorded as a function of the lipid ⁄ peptide
molar ratio. For fluorescence quenching experiments, Trp
was excited at 295 nm instead of 280 nm to reduce absorb-
ance by acrylamide. Trp fluorescence was quenched by the
titration of acrylamide from a 4 m stock solution to a final
concentration of 0.2 m in the presence of liposomes at a
peptide ⁄ lipid molar ratio of 1 : 50 (3 lm peptide and

150 lm liposomes). Experimental data were plotted accord-
ing to the Stern-Volmer equation, F
0
⁄ F ¼ 1+K
SV
[Q],
where F
0
is the fluorescence of the peptide in the absence
of acrylamide, F is the fluorescence of the peptide in the
presence of acrylamide, K
SV
is the Stern-Volmer quenching
constant, and [Q] is the concentration of acrylamide. All
conditions were assayed in triplicate.
Membrane depolarization
Cytoplasmic membrane depolarization measurements were
performed with the membrane potential-sensitive probe
DiSC
3
(5). Briefly, S. aureus cells were grown at 37 °Cto
mid-exponential phase, centrifuged (3500 r.p.m., 7 min),
washed with 5 mm Hepes, pH 7.2 containing 20 mm glu-
cose, and resuspended in Hepes buffer (5 mm Hepes,
pH 7.2, 20 mm glucose, and 100 mm KCl) to an A
600
of
0.05. Changes in the fluorescence due to the disruption of
the cytoplasmic membrane potential were continuously
monitored at 20 °C using a Shimadzu RF-5301 spectrofluo-

rimeter at an excitation wavelength of 620 nm and an
S T. Yang et al. Central proline in amphipathic a-helix
FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4051
emission wavelength of 670 nm. When dye uptake was
maximal, as indicated by a stable reduction in the fluores-
cence due to quenching of the accumulated dye in the mem-
brane interior, peptides (final concentration 0.3 lm)in
Hepes buffer were added. Full dissipation of the membrane
potential was obtained using 0.22 nm gramicidin D. All
conditions were assayed in duplicate.
SPR analysis of interaction between peptides and
phospholipid bilayers
The interaction of the peptides with the phospholipid bilayers
was analyzed by SPR using a BIACORE 2000 equipped with
an L1 sensor chip as described previously [63]. The running
buffer used for all experiments was 20 mm sodium phosphate
(pH 7.2). The L1 sensor chip was installed and washed with
40 mm N-octyl b-d-glucopyranoside (25 lL) at a flow rate
of 5 lLÆmin
)1
. PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) small
unilamellar vesicles were then immediately applied to the
chip surface for 15 min at a flow rate of 2 lLÆmin
)1
.To
remove any multilamellar structures from the lipid surface or
to regenerate the surface, 10 mm NaOH (50 lL) was injected
at flow rate of 50 lLÆmin
)1
, resulting in a stable baseline.

Complete coverage of the chip surface with lipid was con-
firmed by the absence of nonspecific binding by the negative
control (25 lL 0.1 mgÆmL
)1
BSA in sodium phosphate
buffer). All experiments were carried out at 25 °C, and all
solutions were freshly prepared, degassed, and passed
through 0.22-lm pore filters. Measurements were made twice
for each sample to ensure reproducibility.
The sensorgrams for each peptide ⁄ lipid combination were
analyzed by curve fitting using numerical integration analysis
[64]. The sensorgram data obtained at five different concen-
trations were simultaneously fitted using BIA evaluation
software (version 3.2). Because a poor fit was obtained with
the simple 1 : 1 binding model, the association and dissoci-
ation rate constants were determined using the two-state
reaction model. For peptide–lipid interaction, this may cor-
respond to:
P þ L !
k
a1
k
d1
PL !
k
a2
k
d2
PL
Ã

In the first step, peptide (P) binds to lipids (L) to give the
complex PL as a result of initial electrostatic binding. In
the second step, PL subsequently changes to PL*, which
cannot dissociate directly to P + L and which may corres-
pond to the insertion of the peptide into the hydrophobic
region of the lipid bilayer. The corresponding differential
rate equations for this reaction model are:
dR
1
=dt ¼ k
a1
C
A
ðR
max
À R
1
À R
2
ÞÀk
d1
R
1
À k
a2
R
2
þ k
d2
R

2
dR
2
=dt ¼ k
a2
R
1
À K
d2
R
2
where R
1
and R
2
are the response units for the first and
second steps, respectively, C
A
is the peptide concentration,
and R
max
is the maximal response unit (or equilibrium
binding response). The total affinity constant (K) can then
be determined as (k
a1
⁄ k
d1
)(k
a2
⁄ k

d2
).
Acknowledgements
We are grateful to Dr K. Matsuzaki (Kyoto Univer-
sity, Japan) for assistance in the analysis of peptide–
lipid interactions. This study was supported by the
SRC ⁄ ERC program of MOST ⁄ KOSEF (R11-2000-
083-00000-0), the Molecular and Cellular BioDiscovery
Research Program, the Brain Research Center of
the 21st Century Frontier Research Program
(M103KV010004 03K2201 00430), and the Develop-
ment of Marine Novel Compounds Program of the
Korean Ministry of Maritime Affairs and Fisheries.
J.Y.L. is supported in part by the Research Center for
Biomolecular Nanotechnology at GIST.
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