Báo cáo khoa học: Structures and mode of membrane interaction of a short a helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy pdf

Eur. J. Biochem. 269, 3869–3880 (2002) Ó FEBS 2002

doi:10.1046/j.1432-21033.2002.03080.x

Structures and mode of membrane interaction of a short a helical
lytic peptide and its diastereomer determined by NMR, FTIR,
and fluorescence spectroscopy
Ziv Oren1,*, Jagannathan Ramesh2,*, Dorit Avrahami1, N. Suryaprakash2,†, Yechiel Shai1 and Raz Jelinek2
1

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel; 2Department of Chemistry, Ben Gurion
University of the Negev, Beersheva, Israel

The interaction of many lytic cationic antimicrobial peptides
with their target cells involves electrostatic interactions,
hydrophobic effects, and the formation of amphipathic secondary structures, such as a helices or b sheets. We have
shown in previous studies that incorporating % 30%
D-amino acids into a short a helical lytic peptide composed of
leucine and lysine preserved the antimicrobial activity of the
parent peptide, while the hemolytic activity was abolished.
However, the mechanisms underlying the unique structural
features induced by incorporating D-amino acids that enable
short diastereomeric antimicrobial peptides to preserve
membrane binding and lytic capabilities remain unknown.
In this study, we analyze in detail the structures of a
model amphipathic a helical cytolytic peptide KLLLKWLL
KLLK-NH2 and its diastereomeric analog and their
interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR
experiments in dodecylphosphocholine (DPCho) and
sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals
that the peptides have an amphipathic organization within

The interaction of many lytic cationic antimicrobial peptides
with their target cells involves electrostatic interactions,
hydrophobic effects, and the formation of secondary
structures. Electrostatic interactions between the peptides
and the lipids are believed to direct the polypeptides to
the membrane surface, whereas completion of the folding process involves hydrophobic interactions between

Correspondence to R. Jelinek, Department of Chemistry,
Ben Gurion University of the Negev, Beersheva 84105, Israel.
Fax: + 972 8 6472943, Tel.: + 972 8 6461747,
E-mail:
Abbreviations: ATR-FTIR, attenuated total reflectance Fourier
transform infrared; Myr2Gro-PCho, dimyristoylphosphocholine;
Myr2Gro-PGro, dimyristoylphosphoglycerol; DPCho, dodecylphosphocholine; egg, phosphatidylcholine; PDA, polydiacetylene;
PtdEtn, E. coli phosphatidylethanolamine; PtdGro, egg phosphatidylglycerol; SM, sphingomyelin; TPPI, time proportional phase
increment.
*Note: these authors contributed equally to the paper.
 Note: N. Suryaprakash is currently on leave from the Sophisticated
Instruments Faculty, Indian Institute of Science, Bangalore, India.
(Received 26 April 2002, accepted 6 June 2002)

both membranes. Specifically, the a helical structure of the
L-type peptide causes orientation of the hydrophobic
and polar amino acids onto separate surfaces, allowing
interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite
the absence of helical structures, the diastereomer peptide
analog exhibits similar segregation between the polar and
hydrophobic surfaces. Further insight into the membranebinding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through
tryptophan quenching experiments using brominated

phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR,
fluorescence, and colorimetric studies shed light on the
importance of segregation between the positive charges and
the hydrophobic moieties on opposite surfaces within the
peptides for facilitating membrane binding and disruption,
compared to the formation of a helical or b sheet structures.
Keywords: cytolytic peptides; membrane permeation;
peptide–membrane interactions; polydiacetylene.

nonpolar residues and the hydrophobic core of the lipid
bilayer. However, these interactions require that the
peptides have a defined amphipathic structure. The role of
the peptide secondary structure in these interactions has
been studied extensively. The peptides that have been
studied adopted predominantly a helix or b sheet structures
[1–8]. Incorporation of one or two D-amino acids into the
a helical regions of some of these peptides was found to
destabilize the a helical structure in solution but the
diastereomeric peptides retain most of their helical structure
upon membrane binding [8–12].
Interestingly, the incorporation of several D-amino acids
into the a helical cytolytic peptides pardaxin [13] and
melittin [14] clearly disrupted the a helical structure. However, the resulting diastereomers retained high antibacterial
activity but lost their cytotoxic effects on mammalian cells.
These results correlated with the ability of the peptides to
bind and induce leakage preferentially from negatively
charged lipid membranes. A melittin diastereomer was used
to analyze the role of the random coil to secondary structure
transition as a driving force for membrane binding and
insertion of diastereomeric peptides into lipid bilayers [7].

The energetic constraints of secondary structure formation
associated with D-amino acid incorporation appear to play


Ó FEBS 2002

3870 Z. Oren et al. (Eur. J. Biochem. 269)

a role in the preferential binding of the diastereomers to the
negatively charged outer surface of bacteria. The role of
secondary structure formation in cell selectivity was further
demonstrated by a comparison of leucine–lysine short
model peptides composed of either all L-amino acids or
the diastereomeric analog [15]. Here, we show that the
electrostatic interaction between the positively charged
diastereomer and negatively charged bacterial membranes
might allow the peptides to cross the energy barrier and
adopt a stable secondary structure that would enable them
to insert into the membrane. Furthermore, the results
demonstrate the feasibility of a novel approach, based upon
incorporation of D-amino acids into the peptide sequence,
for regulating cell-selective membrane lysis through modulating the transformation from a random coil into secondary structures. This approach differs from the prevalent
method of varying the net positive charge or the hydrophobicity of antimicrobial peptides [16–18].
The unique structural features induced by incorporating
D-amino acids that enable short diastereomeric antimicrobial peptides to retain membrane binding and lytic capabilities have not been determined. In this study we analyze in
detail the structural properties and interactions of a model
amphipathic a helical cytolytic peptide and its diastereomeric analog with zwitterionic and negatively charged
membranes. Previous studies of the biological functions of
these peptides revealed that both peptides have similarly
high antibacterial activity against most Gram-positive and

Gram-negative bacteria examined, however, only the wildtype peptide exhibits hemolytic activity towards human red
blood cells (hRBC) [19]. Here, calculations based on highresolution NMR data yield three-dimensional structures of
both peptides in membrane models. Further insight into the
membrane-binding properties of the peptides and their
depth of penetration into the lipid membrane has been
obtained through tryptophan quenching experiments and
the recently developed lipid/polydiacetylene (PDA) colorimetric assay [20,21]. The combined NMR, FTIR, fluorescence, and colorimetric studies point to the significance of
segregation between the charged and hydrophobic regions
of lytic peptides to membrane binding and its disruption,
compared with the formation of a helical or b sheet
structures.

EXPERIMENTAL PROCEDURES
Materials
4-Methyl benzhydrylamine resin (BHA) and butyloxycarbonyl (Boc) amino acids were purchased from Calibochem–
Novabiochem (La Jolla, CA, USA). Other reagents used for
peptide synthesis included trifluoroacetic acid (Sigma),
methylene chloride (peptide synthesis grade, Biolab, IL,
USA), dimethylformamide (peptide synthesis grade, Biolab,
IL, USA), piperidine (Merck, Darmstadt, Germany), and
benzotriazolyl-n-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP, Sigma). Egg phosphatidylcholine (PtdCho), egg phosphatidylglycerol (PtdGro),
phosphatidylethanolamine (PtdEtn) (type V, from Escherichia coli) and cholesterol were purchased from Sigma.
Dimyristoylphosphocholine (Myr2Gro-PCho), dimyristoylphosphatydilglycerol (Myr2Gro-PGro), and sphingomyelin
(SM) were purchased from Avanti Polar Lipids (Alabaster,

AL, USA). The diacetylene monomer 10,12-tricosadiynoic
acid was purchased from GFS Chemicals (Powell, OH,
USA). All other reagents were of analytical grade. Buffers
were prepared in double glass-distilled water. d38-DPCho
and d25-SDS were purchased from Cambridge Isotope

Laboratories (Cambridge, MA, USA).
Peptide synthesis and purification
The peptides KLLLKWLLKLLK-NH2 (K4L7W) and
KLllKWLlKlLK-NH2 (K4L3l4W, bold and lowercase letters indicate D-amino acids) were synthesized by a solid
phase method on a 4-methyl benzhydrylamine resin
(0.05 milliequivalents) [22]. The resin-bound peptides were
cleaved from the resins by hydrogen fluoride (HF), and after
HF evaporation and washing with dry ether, they were
extracted with 50% acetonitrile/water. HF cleavage of the
peptides bound to the 4-methyl benzhydrylamine resin
resulted in C-terminus amidated peptides. Each crude
peptide contained one major peak, as revealed by RPHPLC, which was 50–70% pure by weight. The peptides
were further purified by RP-HPLC on a C18 reverse phase
˚
Bio-Rad semipreparative column (250 · 10 mm, 300 A
pore size, 5 lm particle size). The column was eluted in
40 min, using a linear gradient of 10–60% acetonitrile in
water, both containing 0.05% trifluoroacetic acid (v/v), at a
flow rate of 1.8 mLỈmin)1. The purified peptides were
shown to be homogeneous (> 97%) by analytical HPLC.
The peptides were further subjected to amino acid analysis
and electrospray mass spectroscopy to confirm their composition and molecular mass.
Preparation of liposomes
Small unilamellar vesicles (SUV) were prepared by sonication of phospholipids dispersions as described in detail
previously [23]. Vesicles were visualized using a JEOL
JEM 100B electron microscope (Japan Electron Optics
Laboratory Co., Tokyo, Japan) as follows. A drop of
vesicles was deposited on a carbon-coated grid and negatively stained with uranyl acetate. Examination of the grids
revealed that the vesicles were unilamellar with an average
diameter of 20–50 nm [24].

NMR experiments
Peptides were dissolved in 90% H2O/10% 2H2O, to a total
concentration of 5 mM with d38-DPCho or d25-SDS at a
lipid/peptide molar ratio of 100 : 1. NaCl was added to a
total concentration of % 10 mM. Short centrifugation was
carried out to remove undissolved aggregates. The pH was
adjusted to 4.0 in all the samples. NMR experiments were
carried out at 303 K to achieve optimal spectral resolution.
Under these conditions, there was no doubling of peaks,
indicating all peptide is bound to the micelle homogeneously.
The NMR spectra were recorded on a Bruker DMX-500
spectrometer operating at an 11.7 Tesla magnetic field.
Two-dimensional (2D) NOESY [25] experiments were
carried out using WATERGATE water suppression [26],
with 8192 data points acquired for each free induction decay
(FID), and 256 points in the indirect dimension. The mixing
time used, 200 ms, was confirmed to have a negligible
contribution from spin-diffusion. Hydrogen bonding was


Ó FEBS 2002

Mode of interaction of lytic peptides (Eur. J. Biochem. 269) 3871

evaluated using H–D exchange experiments [27]. Hydrogen
bonds were assigned by recording 1D 1H spectra after
dissolution of lyophilized peptide/micelle assemblies in
2
H2O; hydrogen bonds were assigned only to amide protons
that still yielded visible 1H signals after more than 6 h. 2D

TOCSY experiments [28], using 8192 data points acquired
for each free induction decay (FID), and 256 points in the
indirect dimension, were carried out using a mixing time of
125 ms, applying the MLEV-17 pulse sequence [29]. All 2D
NMR data were obtained in the phase-sensitive mode using
the TPPI method [30]. TMS was used as an external
chemical shift reference. The NMR spectra were processed
using FELIX 98 software (MSI, Inc.). Zero-filling and a
quadratic sine-bell window function were applied in both
dimensions before Fourier transformation. Automatic baseline corrections with a fourth order polynomial function
were applied to all spectra.
Structure calculations
Assignment of the proton resonances to their respective sites
in the peptides was carried out using both TOCSY and
NOESY data. Cross peaks in the 2D spectra were classified
according to their volume, which was referenced to the
distance between the internal Trp-6 ring protons (H5-H6/
H4-H5) of the peptide. Three categories were defined
(strong, medium and weak), which resulted in restraints on
˚
the upper limits of proton distances of 2.7, 3.3 and 5.0 A,
respectively [27]. With hydrogen bonds, the distance
between the amide proton and receptor carbonyl oxygen
˚
was restrained to 1.6–2.3 A, and the distance between the
amide nitrogen and the carbonyl oxygen was determined as
˚
2.3–3.2 A.
Structure calculations were carried out using XPLOR
(version 3.851), applying a distance geometry-simulated

annealing protocol [31]. Initially, 40 structures were generated using a full-structure distance geometry protocol to
scan the conformational space. The distance geometry
calculation was followed by simulated annealing in which
the structures were annealed at 1000 K for 10 ps and cooled
to 300 K in 50 K steps over 10 ps. The Knoe was scaled
at 50 kcalỈmol)1 throughout the calculations. The final
refinement included energy minimization (4000 steps using
POWELL algorithm). The calculated structures were examined visually using the INSIGHTII (version 98.0) molecular
graphics program (MSI Inc.). Quality and accuracy of
calculated structures were evaluated using the program
PROCHECK [32].
ATR-FTIR measurements
Spectra were obtained with a Bruker equinox 55 FTIR
spectrometer equipped with a deuterated triglyceride sulfate
(DTGS) detector and coupled with an ATR device. For
each spectrum, 200 or 300 scans were collected, with a
resolution of 4 cm)1. During data acquisition, the spectrometer was continuously purged with dry N2 to eliminate the
spectral contribution of atmospheric water. Samples were
prepared as previously described [33]. Briefly, a mixture of
PtdCho/cholesterol [10 : 1 (w/w), 1 mg] alone or with
peptide (% 30 lg) was deposited on a ZnSe horizontal
ATR prism (80 · 7 mm), establishing a 1 : 60 peptide/lipid
molar ratio. The aperture angle of 45° yielded 25 internal

reflections. Before preparing the sample, we replaced the
trifluoroacetate (CF3COO-) counterions, which strongly
associate to the peptide, with chloride ions through several
lyophilizations of the peptides in 0.1 M HCl. This allowed
the elimination of the strong C¼O stretching absorption
band near 1673 cm)1 [34]. Lipid–peptide mixtures were

prepared by dissolving them together in a 1 : 2 MeOH/
CH2Cl2 mixture and drying under a stream of dry nitrogen
while moving a Teflon bar back and forth along the ZnSe
prism. Polarized spectra were recorded and the respective
pure lipid in each polarization was subtracted to yield the
difference spectra. The background for each spectrum was a
clean ZnSe prism. The sample was hydrated by introducing
excess deuterium oxide (2H2O) into a chamber placed on
top the ZnSe prism in the ATR casting, and incubating for
2 h before obtaining the spectra. H/D exchange was
considered complete due to the complete shift of the amide
II band. Any contribution of 2H2O vapor to the absorbance
spectra near the amide I peak region was eliminated by
subtracting the spectra of pure lipids equilibrated with 2H2O
under the same conditions.
ATR-FTIR data analysis
Prior to curve fitting, a straight base line passing through
the ordinates at 1700 and 1600 cm)1 was subtracted. To
resolve overlapping bands, we processed the spectra using
TM
PEAKFIT
(Jandel Scientific, San Rafael, CA, USA)
software. Second-derivative spectra accompanied by
13-data-point Savitsky–Golay smoothing were calculated
to identify the positions of the components bands in the
spectra. These wavenumbers were used as initial parameters for curve fitting with Gaussian component peaks.
Position, bandwidths, and amplitudes of the peaks were
varied until: (a) the resulting bands were shifted by no
more than 2 cm)1 from the initial parameters; (b) all the
peaks had reasonable half-widths ( 20–25 cm)1); and

<
(c) good agreement was achieved between the calculated
sum of all components and the experimental spectra were
achieved (r2 > 0.99). The relative contents of different
secondary structure elements were estimated by dividing
the areas of individual peaks, assigned to a particular
secondary structure, by the whole area of the resulting
amide I band. The results of four independent experiments
were averaged.
Lipid/polydiacetylene vesicle colorimetric assay
Preparation of vesicles composed of Myr2Gro-PCho/SM/
cholesterol/polydiacetylene (PDA) (16 : 16 : 5 : 60, w/w)
and Myr2Gro-PCho/Myr2Gro-PGro/PDA (20 : 20 : 60,
w/w) was carried out in a similar way as described
previously [20,21]. Briefly, the lipid constituents are dried
together in vacuum, followed by adding deionized water
and probe-sonication at around 70 °C. The vesicle solution
is then cooled and kept at 4 °C overnight, and polymerized
using irradiation at 254 nm. The resulting solution is intense
blue. Samples for UV/visible measurements were prepared
by adding peptides to 0.06 mL vesicle solutions at concentrations of 0.5 mM total lipid, 2 mM Tris. The pH of
the solutions was 7.8 in all experiments. After adding the
peptides, the solutions were diluted to 0.2 mL and the
spectra were obtained. All measurements were carried out at


3872 Z. Oren et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

27 °C on a Hewlett–Packard 8452 A diode-array spectrophotometer, using a 1 cm optical path cell.
A quantitative value for the extent of blue-red transition
is given by the colorimetric response (%CR), which is
dened [21]:
%CR ẳ ẵPB0 PBi =PB0 ị 100
and
PB ẳ Ablue =ẵAblue ỵ Ared ;
where A is the absorbance at either the ÔblueÕ component in
the UV/visible spectrum (640 nm) or the ÔredÕ component
(500 nm). ÔBlueÕ and ÔredÕ refer to the appearance of the
material, not its actual absorbance. PB0 is the red/blue ratio
of the control sample (without peptides), whereas PBI is the
value obtained for the vesicle-peptide solutions.

Fig. 1. Schiffer Edmundson wheel projection [36] of K4L7W and
K4L3l4W. The dotted background indicates hydrophilic amino acids
(Lys), the empty background indicates hydrophobic amino acids, and
the grey background indicates hydrophobic D-amino acids.

Tryptophan fluorescence and quenching experiments
To determine the environment and the depth of penetration
of the peptides, changes in the intrinsic Trp fluorescence
were measured in NaCl/Pi and upon membrane binding
[35,36]. Emission spectra were measured on a SLMAminco, Series 2 Spectrofluorimeter, with excitation set
at 280 nm, using a 4 nm slit, recorded in the range of
300–400 nm (4 nm slit). In these studies, SUV were used to
minimize differential light-scattering effects [37], and the
lipid/peptide molar ratio was kept high (1000 : 1) in order to
assure that spectral contributions of free peptides would be
negligible.

Tryptophan emission maxima
Peptide (1 lM) was added to NaCl/Pi, or NaCl/Pi containing 1 mM PtdCho/SM/cholesterol (5 : 5 : 1, w/w) SUV.
The wavelength at the maximum intensity of the tryptophan
emission was determined by fitting the emission spectra to a
log-normal distribution. Nonlinear least-squares (NLLSQ)
analyses and data simulations were performed with
ORIGIN 6.1 software package (Microcal, Inc., Northampton,
MA, USA).
Tryptophan Quenching Experiment
Peptides, containing one intrinsic tryptophan residue, were
added to Br-PtdCho/PtdCho/cholesterol (2.5 : 7.5 : 1, w/w)
or Br-PtdCho/PtdEtn/PtdGro (2.5 : 4.5 : 3, w/w) SUV at a
lipid/peptide ratio of 1000 : 1. After the emitted fluorescence was stabilized (10–60 min incubation at room
temperature), an emission spectrum of the tryptophan was
recorded. SUVs containing either 6,7-Br-PtdCho or 9,
10-Br-PtdCho, were used. Three separate experiments were
conducted for each peptide. In control experiments,
PtdCho/cholesterol (10 : 1, w/w) or PtdEtn/PtdGro (7 : 3,
w/w) SUV without Br-PtdCho were used.

RESULTS
Figure 1 depicts the Schiffer & Edmundson wheel projections [38] of the peptides studied, KLLLKWLLKLL–NH2
(K4L7W) and KLllKWLlKlLK-NH2 (K4L3l4W), where
bold and lowercase letters indicate D-amino acids. Both
peptides were amidated and display a net positive charge

of +5. The peptide containing only L-amino acids
(K4L7W) was designed to fold into an ideal amphipathic
a helix. In the diastereomer, D-amino acids were substituted
in positions likely to disrupt formation of a helical

structure [19].
Resonance assignment of the peptide and secondary
structure determination
Figure 2 summarizes sequential and medium range NOEs
as well as slow-exchanging amide protons for K4L7W and
its diastereomer in DPCho and SDS micelles. Micellar
systems have been widely used as model systems for
structural studies of membrane peptides and proteins [39].
DPCho, in particular, was selected in this study as it
resembles natural zwitterionic phospholipids, while SDS,
which has been extensively used in NMR as a model for
membrane environments [39,40], was employed here as a
mimic for negatively charged membranes. The assignment
of backbone resonances assignments was carried out using
conventional methods [25] based on TOCSY and NOESY
spectra, with trypthophan resonances, in particular, used as
the starting points in the sequential assignment analysis [25].
The NOE patterns shown in Fig. 2A,C feature several
strong and medium NOE cross peaks. The NOE patterns,
combined with the appearance of hydrogen bonds, indicate
relatively defined folded structures for the K4L7W peptide in
both micelle environments. The connectivity pattern of
the diastereomer on the other hand, (Fig. 2B,D), points
to less ordered structures, in particular in SDS. Significantly,
slow-exchange protons associated with hydrogen bond
formation were detected only in K4L7W, while no such
protons were detected in the diastereomer. This observation
confirms that the all L-residue peptide contains stable
helical domains. Further evidence of helical structures can
be inferred from the observation of medium-range NOEs,

such as dNN(i (r)i + 2), dbN(i(r)i + 1), daN(i (r)i + 3) and
daN(i (r)i + 4) [25].
Figure 3 superimposes the 15 lowest energy backbone
structures calculated from the NMR data using a
distance-geometry/simulated annealing protocol. The 15
selected structures incurred no NOE violations greater
˚
than 0.5 A. The statistical parameters pertaining to the
calculated structures, obtained using the software PROCHECK


Ó FEBS 2002

Mode of interaction of lytic peptides (Eur. J. Biochem. 269) 3873

Fig. 2. Schematic diagrams summarizing the NOE connectivities observed for the peptides in DPCho micelles: (A) K4L7W and (B) K4L3l4W; and in
SDS micelles: (C) K4L7W and (D) K4L3l4W. The slowly exchanging amide protons are marked with filled circles. The intensities of the NOE
connectivities are indicated by the widths of the stripes.

[32] are shown in Table 1. The calculated structures
shown in Fig. 3 exhibit a relatively good convergence,
consistent with the NOE patterns shown in Fig. 2. In
particular, the superimposed structures of the L-type peptide,
Fig. 3A,C, clearly show that the peptide exhibits a helical
structure, noticeably apparent in DPC micelles as a righthanded a helical conformation between residues 4–10
(Fig. 3A). Circular-dichroism (CD) spectroscopy indicated
high populations of similar helical structures of the L-peptide
in both neutral PtdCho vesicles and PtdEtn/PtdGro vesicles
(data not shown). In K4L3l4W, a nascent helical domain
within the central region of the peptide was detected when

the peptide was reconstituted in DPCho micelles, Fig. 3B.
However, all protons in the peptide were rapidly exchanged
in water, indicting that the putative helix is not stabilized.
More disordered structural features are apparent in the
diastereomer recosntituted in SDS micelles, Fig. 3D. The
data presented in Figs 2 and 3 confirm that the helical
content is reduced in K4L3l4W compared with the all
L-amino acids peptide.

Amphipathic peptide organization
Further insight into the structural properties of the
interactions between the peptide and membranes was
obtained upon examination of the NMR-calculated
average peptide conformations, displaying the relative
positions of the two central lysine residues (Lys-5 and
Lys-9) and adjacent leucines (Leu-4 and Leu-8), as
shown in Fig. 4. The structural features previously
discussed for the superimposed structures (Fig. 3) are
apparent in the average conformations. An a helix is
clearly observed for K4L7W in the DPCho micelles
(Fig. 4A), and other helical-type structures, albeit less
defined, are observed for K4L3l4W in the DPCho
micelles (Fig. 4B) and K4L7W in the SDS (Fig. 4C). A
conformation resembling a wide turn is obtained for
K4L3l4W in the SDS micelles (Fig. 4D). Importantly,
the positions of the leucine and lysine side-chains
emphasize the amphipathic organization of the peptides.
The relative orientations of the side-chain clearly

3874 Z. Oren et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 3. Superposition of backbone atoms of the
15 lowest energy structures of (A) K4L7W in
DPCho micelles; (B) K4L3l4W in DPCho
micelles; (C) K4L7W in SDS micelles; (D)
K4L3l4W in SDS micelles. The superposition
has been based upon the backbone
conformation of residues 4–10.

reveal segregation between charged interfaces (lysine sidechains) and hydrophobic domains (leucine side-chains)
in the membrane-associated peptides.
Secondary structures of the peptides in zwitterionic
lipid membranes determined by FTIR spectroscopy
Figure 5 depicts FTIR analysis of the two peptides in model
membranes. The FTIR analysis is aided by the NMR
results, which allow assigning specific secondary structures
to the amide I peaks of the peptides. In the FTIR
experiments, the peptides were incorporated into a zwitterionic lipid membrane composed of PtdCho/cholesterol
(10 : 1, w/w). Helical and disordered structures might
contribute to the amide I vibration at almost identical
wavenumbers, and it is difficult to determine from the IR
spectra alone the exact ratio between the helix and random
coil populations. However, exchanging hydrogen with

deuterium makes it possible, in some instances, to differentiate a helical components from random structures, as the
absorption of the random structure undergoes greater shifts
compared to the a helical components following deuteration. Thus, we examined the IR spectra of the peptides after

complete deuteration. The amide I region spectra of
K4L7W and K4L3l4W bound to PtdCho/cholesterol
(10 : 1, w/w) multibilayers are shown in Fig. 5A,C, respectively. The second-derivative, combined with a 13-datapoint Savitsky–Golay smoothing, was calculated in order to
identify the positions of the component bands in the spectra,
and are given in the corresponding panels in Fig. 5B,D.
These wavenumbers were used as initial parameters for
curve fitting with Gaussian component peaks. The assignments, wavenumbers (t), and relative areas of the component peaks are further summarized in Table 2.
Assignment of the different secondary structures to the
various amide I regions was based on the values taken from


0
10
0
0
0
0
0
30
50
60
70
60
1.12
1.12
1.41
1.77
0.045
0.045
0.045

0.051
±
±
±
±
0.520
0.610
0.419
0.550
0.030
0.045
0.027
0.037
±
±
±
±
0.457
0.657
0.757
0.687
0.0001
0.0003
0.0002
0.0006
±
±
±
±
0.004

0.006
0.005
0.008
8
0
8
0
D-SDS

L-SDS

96
80
90
67
D-DPC

L-DPC

Angles (deg)
˚
Bonds (A)
H-Bonds
Peptide

Number
of
restraints

1.58

1.61
1.94
2.54

2.70
2.98
3.16
3.86

50
30
30
10

DA
(%)
GA
(%)
AA
(%)
MF
(%)
Mean RMSD
to mean
˚
structure (A)
MP RMSD
(backbone)
˚
(A)

Impropers
(deg)

Convergence

MP RMSD
(heavy atoms)
˚
(A)

Structural Quality (Derived from
Ramachandran map)

Mode of interaction of lytic peptides (Eur. J. Biochem. 269) 3875

Deviations from Idealized geometry

Table 1. Structral Statistics for the L-peptides and their diastereomers in DPCho and SDS micelles. MP, mean pairwise. MF, most favored region. AA, additional allowed region. GA, generously allowed region.
˚
DA, disallowed region. None of the structures had NOE violation greater than 0.5 A and a dihedral angle violation greater than 5°.

Ó FEBS 2002

a study by Jackson & Mantsch [41], the results of the NMR
analysis, and the CD data obtained for K4L7W in PtdCho/
cholesterol (10 : 1, w/w) vesicles (data not shown). The
amide I region between 1610 and 1628 cm)1 is characteristic
of an aggregated b sheet structure and the region between
1640 and 1645 cm)1 corresponds to a random coil. The
amide I region of an a helical structure is located between

1650 and 1655 cm)1, and the amide I region from 1656 to
1670 cm)1 is characteristic of a 310 helix or distorted/
dynamic helix [42].
The band at approximately 1624 cm)1, observed in both
K4L7W and K4L3l4W, probably corresponds to small
populations of peptides forming aggregated b sheets. The
major amide I band of K4L7W in the PtdCho/cholesterol
multibilayers (% 1652 cm)1) is centered in the a helical
region, similarly to data reported previously in PtdEtn/
PtdGro membranes [19]. These results are in accordance
with the results of the NMR data in both membrane environments. Incorporation of four D-amino acids in K4L3l4W
disrupts the helical structure in PtdCho/cholesterol, as
confirmed by both the major amide I band shift to
% 1657 cm)1, as well as the peak width, which is similar
to previous reports for negatively charged membranes [19].
Based on the results of the three-dimensional structure of
K4L3l4W, this band might arise from a distorted/dynamic
helical structure [42].
Peptide interactions with membranes determined
by a lipid/polydiacetylene colorimetric assay
Application of the newly developed lipid/PDA colorimetric
assay [20,21] further illuminates the interactions of the
peptides with lipid membranes. The colorimetric assay was
shown to correlate blue-red transitions of lipid/PDA vesicles
with the degree of membrane penetration and lipid disruption by membrane-associated peptides [21]. In particular, it
has been shown that the colour changes, which are due to
structural rearrangements of the PDA matrix, depend upon
the depth of peptide insertion into the hydrophobic bilayer
lipids incorporated within the PDA vesicle framework [20].
Here, the lipid/PDA vesicle assay was used to evaluate the

interactions and association of the peptides with the lipid
domains.
Figure 6 features titration curves correlating the colorimetric response (%CR) with peptide concentrations,
recorded for K4L7W and K4L3l4W, respectively, interacting
with two vesicle compositions. An assembly consisting of
PDA, Myr2Gro-PCho, SM, and cholesterol (6 : 3 : 3 : 1
mol/mol/mol/mol) resembles a zwitterionic membrane surface, while Myr2Gro-PGro/Myr2Gro-PCho/PDA aggregates have been employed to mimic negatively-charged
membranes [34]. The %CR is a quantitative parameter that
measures the blue/red changes from the UV/vis spectra of
the vesicle solution [20].
Different chromatic responses are recorded following
peptide interactions with the two vesicle systems. In the case
of Myr2Gro-PCho/SM/cholesterol/PDA, for example, the
colorimetric titration curves show similar blue-red transitions induced by both peptides. These results indicate that
the peptides penetrate and disrupt the membrane to a
similar extent. However in the negatively-charged membrane model [Myr2Gro-PCho/Myr2Gro-PGro/PDA],
K4L3l4W seems to induce more pronounced blue-red


Ó FEBS 2002

3876 Z. Oren et al. (Eur. J. Biochem. 269)

tryptophan emission of K4L7W (338 ± 1 nm) and
K4L3l4W (342 ± 1 nm) were observed, reflecting their
relocation to more hydrophobic environments [45]. Blue
shifts in the tryptophan emission were similarly recorded for
both peptides within PtdEtn/PtdGro vesicles [19].
Depth of peptide penetration determined
by tryptophan-quenching

Fig. 4. Calculated average structures of the peptides (based on the 15
lowest energy structures presented in Fig. 3), showing the orientation of
residues Leu-4, Leu-8, Lys-5 and Lys-9. (A) K4L7W in DPCho micelles;
(B) K4L3l4W in DPCho micelles. (C) K4L7W in SDS micelles; (D)
K4L3l4W in SDS micelles.

transitions, i.e. higher %CR, compared to the L-peptide.
These data suggest that K4L7W penetrates deeper into the
lipid bilayer compared to the diastereomer, a result that is
consistent with the FTIR data discussed above.
Characterization of the tryptophan environment
using fluorescence spectroscopy
More information upon the membrane environment of the
peptides was obtained by recording the fluorescence emission spectra of tryptophan [43,44] in NaCl/Pi at pH 7.4, and
in the presence of vesicles composed of PtdCho/SM/
cholesterol (5 : 5 : 1, w/w/w). In these studies, SUVs were
used in order to minimize light-scattering effects [37], and a
high lipid/peptide molar ratio was maintained (1000 : 1) to
assure that spectral contributions of the free peptide would
be negligible. In buffer the tryptophan within both peptides
gave rise to a fluorescence peak at around 351 nm. When
PtdCho/SM/cholesterol vesicles were added to the aqueous
solutions containing the peptides, blue shifts in the

In this set of experiments the depth of penetration of the
peptides into lipid membranes was estimated through
quenching of the tryptophan fluorescence by bromine, a
quencher with an r6 dependence and an apparent R0 of
˚

9 A [35]. Tryptophan, which is sensitive to its environment, has been utilized previously in combination with
brominated lipids (PtdCho brominated at various positions within the alkyl chains, denoted Br-PtdCho) to
evaluate peptide localization within membranes [35,36].
Br-PtdCho quenchers of tryptophan fluorescence are
suitable for probing membrane insertion of peptides, as
they act over a short distance and do not drastically
perturb the lipid bilayers.
Significant quenching (Fig. 7) was observed for both
peptides with 6,7-Br-PtdCho/PtdCho and 6,7-Br-PtdCho/
PtdEtn/PtdGro (% 30–35% reduction of fluorescence signal), while less quenching was detected in the case of 9,10Br-PtdCho/PtdCho and 9,10-Br-PtdCho/PtdEtn/PtdGro
(20–25% reduction in zwitterionic membranes and 5–15%
in negatively charged environments). These results are
consistent with the blue shifts observed in the tryptophan
emission spectra decribed above, and suggest that the
peptides do not penetrate deeply into the membrane, but are
rather located closer to the membrane interface. Furthermore, the results indicate that the peptides penetrate less
into negatively – charged membranes compared to zwitterionic membrane environments. This might be related to the
electrostatic attraction between the positive interface of the
peptides and the negative headgroups of the phospholipids,
which is expected to position the peptides closer to the
membrane surface.

DISCUSSION
Membrane binding and lysis by cytolytic peptides are
influenced by their secondary structure. In previous studies
we have used FTIR spectroscopy to examine the membrane-bound structures of a group of newly designed short
diastereomeric antimicrobial peptides [10,15]. The data
showed increased flexibility of the secondary structure of
the diastereomers, compared with their all L-amino acids
analogs. However, FTIR spectroscopy can only provide an

average and approximate measure of the secondary structure content. Furthermore, assignment of secondary structures to the amide I peaks in the diastereomeric peptides has
been ambiguous due to the lack of correlation with other
structure determination methods. In the present study we
have carried out a detailed structural and functional analysis
for the native [all L-amino acid] model peptide K4L7W and
its diastereomeric analog. The data shed new light on the
structural features of the membrane-bound peptides and
their organization, and point to possible permeating mechanisms of diastereomeric antimicrobial peptides.


Ó FEBS 2002

Mode of interaction of lytic peptides (Eur. J. Biochem. 269) 3877

Fig. 5. FTIR spectra deconvolution of the fully
deuterated amide I band (1600–1700 cm)1) of
K4L7W (A) and K4L3l4W (C) in PtdCho/cholesterol (10 : 1, w/w) multibilayers. The second
derivatives, calculated to identify the positions
of the component bands in the spectra, are
shown in (B) for K4L7W and in (D) for
K4L3l4W. The component peaks are the result
of curve-fitting using a Gaussian line shape.
The sums of the fitted components superimpose on the experimental amide I region
spectra. In (A) and (C), the continuous lines
represent the experimental FTIR spectra after
Savitzky–Golay smoothing; the broken lines
represent the fitted components. A 60 : 1 lipid/
peptide molar ratio was used.

Table 2. Assignment, wavenumbers (m), and relative areas of the component peaks determined from the deconvolution of the amide I bands of the

peptides incorporated into PtdCho/cholesterol (10 : 1, w/w) multibilayers. A 1 : 60 peptide/lipid molar ratio was used. The results are the average of
four independent experiments. All values are given as mean ± SEM.
Aggregated b sheet
and a sheet
Peptide
designation
PtdCho/cholesterol
K4L7W
K4L3l4W
PtdEtn/PtdGroa
K4L7W
K4L3l4W
a

Random coil

m (cm– 1)

area (%)

m (cm– 1)

area (%)

1624 ± 2
1624 ± 2

15 ± 5
13 ± 2

1640 ± 1

27 ± 3

1621 ± 2
1634 ± 1

15 ± 5
25 ± 5

a Helix

Distorted/dynamic helix

m (cm– 1)

area (%)

1652 ± 1

85 ± 5

area (%)

1657 ± 1

60 ± 2

1658 ± 1

1651 ± 1

m (cm– 1)

75 ± 2

85 ± 5

The results were obtain from D. Avrahami [19].

Incorporation of D-amino acids into a short, amphipathic
a helical model peptide results in reorganization of the
backbone and side-chains.
The NMR-calculated structures of K4L7W indicate that
the peptide exhibits a right-handed a helical conformation
between residues 4–10 in zwitterionic environments
(Fig. 3A), and a less-defined helical structure in negativelycharged micelles (Fig. 3C). In the case of K4L3l4W,
extended conformation that might include a nascent helical
domain spanning the central region was detected
(Figs 3B,D). Previous NMR studies on the effect of
incorporating D-amino acids on a helical structures were
conducted on a 26-residue diastereomer analog of melittin,
and on an 18-residue model amphipathic a helical peptide.

The NMR structure of a melittin diastereomer (four
acids replaced by their D-enantiomers) revealed
an amphipathic a helix at its C-terminal region in trifluoroethanol/water, methanol, and DPCho/Myr2Gro-PGro
micelles, similar to native melittin [11]. However, double
D-amino acid replacement in the middle of a model
amphipathic a helical peptide resulted in the formation of

two separate helices [46]. Although in the above cases
significant sections of the diastereomers retain their a helical
structure, incorporation of four D-amino acids into K4L7W
has a substantial effect upon the secondary structure,
resulting in a distinct organization of the peptide backbone
and side-chains that, although not a helical, still maintains
its ability to disrupt membranes.
L-amino


3878 Z. Oren et al. (Eur. J. Biochem. 269)

Fig. 6. Colorimetric data for (A) Myr2Gro-PCho/SM/cholesterol/
PDA vesicles; and (B) Myr2Gro-PCho/Myr2Gro-PGro/PDA vesicles
following the addition of K4L7W (solid line) and K4L3l4W (broken line).
The graph depicts the change of colorimetric response (%CR, see
Experimental procedures) of the vesicle solution as a function of
peptide concentration.

Fig. 7. The quenching of tryptophan fluorescence by brominated
phospholipids. The experiment was conducted with two types of
liposomes PtdEtn/PtdGro (7 : 3, w/w) and PtdCho/cholesterol (10 : 1,
w/w) each contains 25% of either Br-PtdCho6,7 (light grey) or
Br-PtdCho9,10 (dark grey).

The results of the FTIR study in PtdCho/cholesterol and
PtdEtn/PtdGro membranes [19] have revealed that the
major amide I band of K4L3l4W is located at % 1657 cm)1,
as compared to % 1652 cm)1 within the a helical peptide
K4L7W (Fig. 5 and Table 2). Previous studies that examined structural changes in phospholipase A2 [42], bacteriorhodopsin, and other proteins [47,48] have correlated

increased amide I frequencies in this region with distorted/
dynamic a helical structures. Combined with the threedimensional structural analysis of K4L3l4W presented here,
we suggest assigning the band at % 1657 cm)1 to a distorted/
dynamic helical structure.
Contribution of amphipathic organization and interface
location to membrane disruption by the peptides
One of the most intriguing observations addressed by this
work was the similar high antimicrobial activities of the
peptide and its diastereomer [19]. The data obtained using
the lipid/PDA colorimetric assay further confirm those
results, and reveal that both peptides disrupt lipid mem-

Ó FEBS 2002

branes to a similar degree (Fig. 6). The major structural
differences between the peptides point to two properties that
may underlie their similar membrane-permeating activities,
namely amphipathic organization and interface location.
The structural analysis presented here reveals that
both peptides adopt amphipathic organization within the
membrane. In a previous study it was shown that the
antimicrobial peptide tritrpticin exhibits an amphipathic
organization that maximizes both electrostatic and hydrophobic interactions with the membrane, although the
peptides does not display either a helical or b sheet structures [49]. In the case of K4L7W the a helical structure
orients the hydrophobic and polar amino acids onto
separate surfaces, thus allowing simultaneous interactions
of the peptides with both the hydrophobic core of the
membrane and the polar headgroup region. Importantly,
despite the absence of a a helical structure, similar
segregation between the polar and hydrophobic surfaces

was observed for the diastereomer K4L3l4W (Figs 3 and
4). The conformation of membrane-bound K4L3l4W is
most likely affected by competing factors – ones that
favor disordered structures on the one hand, while
inducing more defined conformations, on the other hand.
Specifically, occurrence of disordered structures would
likely result from an electrostatic repulsion between
positively charged amino acids [50,51] and the destabilizing effect of D-amino acids incorporated into a helical
structures. In contrast, hydrophobic interactions between
nonpolar amino acids and the lipid hydrocarbon core
combined with the electrostatic interactions between the
charged amino acids and the polar headgroup region are
expected to induce formation of organized structures. Our
results indicate that the capability of K4L3l4W to adopt a
defined amphipathic secondary structure in the membrane, despite the incorporation of D-amino acids, is most
likely attributed to such hydrophobic and electrostatic
interactions.
Numerous studies have demonstrated the important
roles of the a helical structures in binding and incorporation of peptides within membranes [9,12,52,53]. However,
the results obtained here for K4L3l4W suggest that a
stable a helical conformation might not be the essential
requirement for membrane association and permeation
processes. The free energy of the hydrophobic stretches
within amino acids is probably a major driving force for
membrane binding, compensating for the reduced a helical
structure. Furthermore, both the blue shift in tryptophan
fluorescence observed following membrane binding, and
the quenching of Trp fluorescence by brominated lipids,
indicate that the all-L-residue peptide and its diastereomer
are located at the membrane interface. Taken together,

these results strongly suggest that the a helical structure is
not a prerequisite for maintaining an interface localization
of a peptide.
In summary, the structural and functional analyses of the
all-L-amino acids peptide and its diastereomeric analog
indicate that a defined a helical structure of K4L7W is not an
essential factor determining membrane binding and disruption. Furthermore, this work indicates two main properties
that contribute to membrane disruption capabilities of
cytolytic peptides, namely, amphipathic organization and
interface localization of the peptides. Our findings support
a detergent-like effect for both peptides upon cellular


Ó FEBS 2002

Mode of interaction of lytic peptides (Eur. J. Biochem. 269) 3879

membranes, described in the carpet model (reviewed in [54]),
rather than organized pores.

ACKNOWLEDGEMENTS
We are grateful to Dr Sofiya Kolusheva for assistance with the
colorimetric assay. R. J. is grateful for the financial support from the
US-Israel Binational Science Foundation. This research was supported
by the Israel Academy of Sciences and Humanities.

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