Interactionsoftheantimicrobialb-peptideb-17with phospholipid
vesicles differfrommembraneinteractionsof magainins
Raquel F. Epand
1
, Naoki Umezawa
2
, Emilie A. Porter
2
, Samuel H. Gellman
2
and Richard M. Epand
1
1
Department of Biochemistry, McMaster University Health Sciences Centre, Hamilton, Canada;
2
Department of Chemistry,
University of Wisconsin, Wisconsin USA
We have studied the interaction of b-17, a potent synthetic
antimicrobial b-peptide, with phospholipids. We find that
unlike other antimicrobial peptides such as magainin II, b-17
facilitates the formation of nonbilayer phases, indicating
that the peptide promotes negative curvature. Studies of
liposomal leakage also indicate a different mode of mem-
brane interaction relative to magainin II, but both leakage
and membrane binding show that b-17, like magainin II, has
strong affinity for membranes containing anionic lipids. This
is likely to be an important factor contributing to the anti-
microbial specificity ofthe b-peptide.
Keywords: b-peptide; antimicrobial; peptide–lipid inter-
actions; membrane leakage; membrane intrinsic curvature.
There is currently extensive interest in developing new
antimicrobial agents to counter the growing clinical pro-
blem of bacterial resistance to traditional antibiotics [1]. A
large variety of natural peptides and synthetic derivatives
display antimicrobial activity [2], and these peptides have
been viewed as potential sources of new therapeutic agents.
Nearly all ofthe synthetic derivatives have been constructed
from a-amino acids, as are the natural host-defense
peptides. Recently, antimicrobial activity has been reported
for a number of b-amino acid oligomers (b-peptides) [3–8].
b-Peptides differfrom conventional peptides (a-amino acid
residues) in two important ways. First, the unnatural
backbone of b-peptides confers resistance to degradation
by proteolytic enzymes [8–10]. Second, b-peptides construc-
ted from appropriately rigidified residues display higher
conformational stability, on a per-residue basis, than do
conventional peptides [6]. It is therefore of interest to
compare the mechanism(s) ofb-peptide antibacterial action
with the antibacterial mechanisms of analogous conven-
tional peptides.
Here we characterize theinteractionsof one antimicrobial
b-peptide, b-17 [7,8], with lipid vesicles, which are simplified
models of cell membranes. b-17 was designed to mimic
natural host-defense peptides like the magainins, which are
thought to exert their antimicrobial effects by disrupting
bacterial membranes. The structure ofb-17 is shown in
Fig. 1. Magainins are cationic peptides that contain 23
a-amino acid residues and adopt an amphiphilic a-helical
conformation in the presence of membranes [11]. b-17
contains 17 b-amino acid residues and adopts a 12-helical
conformation, which is defined by a network of 12-mem-
bered ring C¼O(i) fi NH(i + 3) hydrogen bonds [12–15].
(The a-helix contains 13-membered ring C¼O(i) fi
NH(i + 4) hydrogen bonds.) Theb-peptide 12-helix has
approximately 2.5 residues per turn and a rise of 5.5 A
˚
per
turn; therefore, a 17-residue 12-helix should be similar in
length to a 23-residue a-helix formed by a conventional
peptide. b-17 contains only two types of b-amino acid
residue, hydrophobic trans-2-aminocyclopentanecarboxylic
acid (ACPC) and cationic trans-3-aminopyrrolidine-4-
carboxylic acid (APC). The repeating APC-ACPC-APC-
ACPC-ACPC pentad gives rise to a 12-helix with distinct
cationic and hydrophobic sides, which mimics the amphi-
philic a-helical conformation adopted by magainin peptides
[11].
We have characterized interactions between b-17 and
vesicles by differential scanning calorimetry, to gain insight
on lipid phase transitions, and by circular dichroism (CD),
to gain insight on b-peptide conformation in the presence
and absence of membranes. We have also examined the
ability ofb-17 to lyse model membranes as a function of
lipid composition, in an effort to explain the specificity of
action and characterize the energetics of binding to the
different liposomal systems used. Like magainins and other
host-defense peptides, b-17 is more effective at killing
bacterial cells than at inducing lysis of human red blood cells
(which are taken to represent eukaryotic cells in general)
[7,8]. The basis for this cell specificity among natural
peptides remains a subject of debate [1,16–19], although
electrostatic factors are thought to be important, as bacterial
cell surfaces generally have a greater negative charge density
than do eukaryotic cell surfaces [11]. Vesicle model studies
have been conducted with many conventional antimicrobial
peptides [18]; therefore, our results withb-17 have an ample
Correspondence to R. M. Epand, Department of Biochemistry,
1200 Main Street West, McMaster University Health
Sciences Centre, Hamilton, ON, L8N 3Z5 Canada.
Fax: 905 521 1397, Tel.: 905 525 9140,
E-mail: epand@mcmaster.ca
Abbreviations: DPam
2
PtdEtn, dipalmitoleoyl-phosphatidylethanol-
amine; Ole
2
PtdEtn, dioleoylphosphatidylethanolamine; Ole
2
PtdGro,
dioleoylphosphatidylglycerol; Ole
2
PtdSer, dioleoylphosphatidyl-
serine; eggPtdCho, phosphatidylcholine extracted from egg yolk;
LUV, large unilamellar vesicle; PamOlePtdSer, 1-palmitoyl-2-
oleoylphosphatidylserine; SUVs, small or sonicated unilamellar
vesicles; T
H
, bilayer to hexagonal transition temperature.
(Received 11 November 2002, revised 11 January 2003,
accepted 27 January 2003)
Eur. J. Biochem. 270, 1240–1248 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03484.x
basis for comparison. The lipids used in this study as
components of lipid mixtures have the structures shown in
Fig. 2.
Materials and methods
Materials
Phospholipids were purchased from Avanti Polar Lipids
(Alabaster, AL).
The labeled lipids were made by chemical modification
of phosphatidylethanolamine that was prepared from
egg phosphatidylcholine by transphosphatidylation.
[Ala8,13,18]-magainin II amide was purchased from the
Sigma Chemical Co (St Louis, MO, USA).
Synthesis of NBD-b-17
Fmoc-ACPC-OH and Fmoc-APC(Boc)-OH were synthe-
sized as previously reported [20,21]. b-17 was prepared with
a conventional automated solid-phase peptide synthesizer
(Synergy 432 A; Applied Biosystems, Foster City, CA)
using extended coupling and deprotection steps. After
removal ofthe N-terminal Fmoc protecting group, the
resin-bound peptide was treated with three equivalents of
4-fluoro-7-nitrobenzofurazan(4-fluoro-7-nitrobenz-2-oxa-1,3-
diazole) (Molecular Probes, Eugene, OR) in dry dimethyl-
formamide containing 5% v/v diisopropylethylamine. The
reaction vessel was covered with foil and shaken at room
temperature. After 48 h, the resin was washed with
dimethylformamide and then CH
2
Cl
2
.NBD-labeledb-17
was cleaved fromthe resin and purified by RP-HPLC using
a Vydac C4214TP510 column. Trifluoroacetic acid coun-
terions were replaced with chloride ions through three
dissolutions oftheb-peptide in 50 m
M
HCl followed by
lyophilization. The replacement of trifluoroacetic acid was
confirmed by
19
F-NMR. A small amount of decomposition
was seen during this step; the final purity of NBD-b-17 was
95%, and the purity ofb-17 itself was >98%.
Differential scanning calorimetry
Lipid films were made from dipalmitoleoylphosphati-
dylethanolamine (DPam
2
PtdEtn) dissolved in chloroform/
methanol (2 : 1, v/v). Increasing mole fractions of peptides
dissolved in methanol were added and the solvent removed
by evaporation with nitrogen. Final traces of organic
solvent were removed in a vacuum chamber attached to a
liquid nitrogen trap for 2–3 h. The lipid films were hydrated
at room temperature by vortexing with 20 m
M
Pipes buffer
containing 0.14
M
NaCl, 1 m
M
EDTA and 0.002% sodium
azide pH 7.4. The final lipid concentration was 5 mgÆmL
)1
.
Lipid suspensions were degassed under vacuum before
being loaded into a NanoCal high sensitivity calorimeter
(CSC, American Forks, UT). A heating scan rate of
Fig. 1. The structure of b-17.
Fig. 2. Structures of phospholipids used in
these studies. (1) Dioleoylphosphatidyletha-
nolamine (Ole
2
PtdEtn); (2) dielaidoylphos-
phatidylethanolamine; (3) dipalmitoleoylphos-
phatidylethanolamine (DPam
2
PtdEtn), and
(4) dioleoylphosphatidylcholine (Ole
2
PtdCho)
have zwitterionic headgroups. (5) Dioleoyl-
phosphatidylglycerol (Ole
2
PtdGro); (6) diol-
eoylphosphatidylserine (Ole
2
PtdSer) and (7)
1-palmitoyl-2-oleoylphosphatidylserine
(PamOlePtdSer) have anionic headgroups.
Lipids are sometimes referred to by their
headgroups, e.g. phosphatidylethanolamine,
phosphatidylcholine, phosphatidylglycerol,
phosphatidylserine.
Ó FEBS 2003 Antimicrobialb-peptide and phospholipidvesicles (Eur. J. Biochem. 270) 1241
0.75 KÆmin
)1
was employed. The observed phase transition
was fitted with parameters describing an equilibrium with a
single van’t Hoff enthalpy and the transition temperature
reported as that for the fitted curve. Data were analyzed
with the program Origin 5.0. For some curves the
endothermic peak was not symmetrical and it appeared
that including more than one component would provide a
better fit to the data. However, there was no progressive
trend showing a systematic increase in one component
relative to another as a function of b-peptide/lipid ratio.
Fitting the transition to one component provides a measure
of the average effect oftheb-peptide on the curvature
properties ofthe lipid.
Circular dichroism (CD)
The CD spectra were recorded using an AVIV model 61 DS
CD instrument (AVIV Associates, Lakewood, NJ). The
sample was contained in a 1-mm pathlength quartz cell that
was maintained at 25 °C in a thermostated cell holder. The
CD data are expressed as the mean residue ellipticity. All
CD runs were made withb-peptide dissolved in 10 m
M
sodium phosphate buffer containing 0.14
M
NaF and 1 m
M
EDTA at pH 7.4. For samples containing lipid, the lipid
was first made into a dry film from a solution of
chloroform/methanol, and then hydrated by vortexing with
buffer. The lipid suspension was then sonicated to clarity to
make small or sonicated unilamellar vesicles (SUVs).
Large unilamellar vesicles
Lipid films were made by dissolving appropriate amounts of
lipid in a mixture of chloroform/methanol (2 : 1, v/v) and
dried in a test tube under nitrogen. Final traces of solvent
were removed in a vacuum chamber attached to a liquid
nitrogen trap for 2–3 h. Dried films were kept under argon
gas at )30 °C if not used immediately. Films were hydrated
with buffer, vortexed extensively at room temperature and
then subjected to five cycles of freezing and thawing. The
homogeneous lipid suspensions were then further processed
by 10 passes through two stacked 0.1 lm polycarbonate
filters (Nucleopore Filtration Products, Pleasanton, CA) in a
barrel extruder (Lipex Biomembranes, Vancouver, BC), at
room temperature. Large unilamellar vesicles (LUVs) were
kept on ice and used within a few hours of preparation. Lipid
phosphorus was determined by the method of Ames [22].
Leakage studies
Aqueous content leakage from liposomes was determined
using the ANTS-DPX assay [23]. Lipid films were hydrated
with 12.5 m
M
8-aminonaphthalene-1,3,6-trisulfonic acid
(ANTS), 45 m
M
p-xylene-bis-pyridinium bromide (DPX),
68 m
M
NaCl, 10 m
M
Hepes at pH 7.4. The osmolarity of
this solution was adjusted to be equal to that ofthe buffer
as measured with a cryoosmometer (Advanced Model
3MOplus Micro-Osmometer, Advanced Instruments Inc.,
Norwood, MA). LUVs of 0.1 lm diameter were prepared
by extrusion as described above. After passage through a
2.5 · 20 cm column of Sephadex G-75, the void volume
fractions were collected and thephospholipid concentration
was determined by phosphate analysis. The fluorescence
measurements were performed in 2 mL of buffer composed
of 10 m
M
Hepes, 0.14
M
NaCl, 1 m
M
EDTA, pH 7.4, in a
quartz cuvette equilibrated at 37 °C with stirring. Aliquots
of LUVs were added to the cuvette to a final lipid
concentration of 25 l
M
. The fluorescence was recorded as
a function of time using an excitation wavelength of 360 nm
and an emission wavelength of 530 nm with 8 nm band-
width slits. A 490-nm cutoff filter was placed in the emission
path. Leakage was started by addition ofthe peptide in
buffer solution. The value for 100% leakage was obtained
adding 20 lL of a 10% Triton X-100 solution to the
cuvette. Runs were performed in duplicate.
Membrane binding
NBD-labeled compounds have a low quantum yield in
aqueous solution, but the quantum yield becomes higher
when the probe enters a hydrophobic environment such as
in a membrane [24]. To assess the degree ofb-17 association
with themembrane systems tested in leakage, a series of
LUVs were made as previously described and aliquots of a
suspension of LUVs were added sequentially to NBD-
labeled a-peptide in the cuvette and the fluorescence
intensity changes followed until a plateau was reached.
Fluorescence intensity was measured with an Aminco-
Bowman Series II spectrofluorimeter, with magnetic stir-
ring, at 25 °C; excitation was set at 467 nm and emission at
539 nm, using 4 nm bandpass slits in excitation and
emission and a 490-nm cut off filter in the emission path.
Polarizers were set at 90° for the excitation and 0° for the
emission to minimize scattering effects. Siliconized glass
cuvettes were used, containing 2 mL of 10 m
M
Hepes buffer
with 0.14
M
NaCl and 1 m
M
EDTA at pH 7.4. b-Peptide
solutions were made in the same buffer and added at a
concentration of 0.4 l
M
to the cuvette, before addition of
LUVs. After every addition of vesicles, the system was
allowed to incubate for a few minutes. Controls with
addition of LUVs to buffer only were also made and
subtracted. The intensity changes were used to calculate
binding affinity [25–27].
Isothermal titration calorimetry (ITC)
The experiments to measure the heat of reaction at 30 °C
were carried out on a VP-ITC isothermal titration calori-
meter manufactured by MicroCal, Northampton, MA. The
solutions were degassed under vacuum prior to being loaded
in the calorimeter. Theb-peptide solution (25–50 l
M
)was
placed in the reaction cell and was titrated with 3–10 lLof
5–10 m
M
lipid mixtures in the form of LUVs delivered from
a motor-driven syringe. The reference cell contained the
same buffer as the solution placed in the cell. A buffer
composed of 10 m
M
Hepes, 0.14
M
NaCl, 1 m
M
EDTA at
pH 7.4 was used in all titrations. The binding curves
obtained were fitted with software provided by Microcal.
Results
Differential scanning calorimetry
We measured how increasing mole fractions ofb-17 changed
the bilayer to hexagonal phase transition temperature (T
H
)
1242 R. F. Epand et al.(Eur. J. Biochem. 270) Ó FEBS 2003
of a model unsaturated phospholipid, DPam
2
PtdEtn, to
obtain an indication ofthe relationship between insertion
of the peptide and the stability ofthe bilayer phase.
DPam
2
PtdEtn is a zwitterionic lipid with a bilayer to
hexagonal phase transition at 43 °C. Small mole fractions of
additives that interact with this lipid have been shown to
either lower this transition temperature, thereby destabi-
lizing the lamellar phase and facilitating the conversion to a
more highly curved nonlamellar phase, or vice versa.The
change in transition temperature in the presence of small
amounts of additives has been therefore used as a measure
of propensity to induce curvature in themembrane [28].
Changes in membrane curvature are required for several of
the proposed mechanisms by which antimicrobial peptides
induce membrane leakage. Thus, the formation of a large
pore or a carpet-like disruption on the surface ofthe bilayer
requires increased positive membrane curvature. However,
not all lytic peptides promote positive membrane curvature.
For example, the peptide mastoparan, isolated from wasp
venom, has the opposite effect on membrane curvature. It
promotes negative curvature. How this effect of promoting
negative curvature is related to the lytic action of masto-
paran is not well understood, except for the fact that it
would tend to destabilize the bilayer phase toward the
formation of inverted phase morphology. This is not meant
to imply that a peptide that promotes negative curvature
will cause the formation of nonlamellar structures, but
rather that it will facilitate the formation of transient
structures with increased negative curvature. b-17 also
promotes negative curvature and lowers the transition
temperature of DPam
2
PtdEtn by )708 ± 104 °CÆmol
)1
fraction of peptide added. This is opposite to the direction of
the shift of T
H
of +1800 ± 300 °CÆmol
)1
found with the
addition of magainin II [29] and does not correspond to the
type of curvature required for a large toroidal pore. We have
also compared our previous differential scanning calori-
metry results using magainin II with those ofthe more
potent magainin analog, [Ala8,13,18]-magainin II amide
[30]. We find that the peptide-induced changes in the
transition of DPam
2
PtdEtn are similar for both magainin II
and for [Ala8,13,18]-magainin II amide (data not shown).
In a toroidal pore there are two types of curvatures to be
considered, i.e. one in the plane ofthemembrane and the
other along the bilayer normal; for large pores, the positive
curvature required along the bilayer normal predominates.
If b-17 promoted the formation of a similar pore arrange-
ment, but the diameter ofthe pore were smaller than that of
magainin, then the negative curvature in the dimension
around the rim ofthe pore would predominate. However,
the pore formed by b-17 does not appear to be very small as
it allows the release of b-galactosidase from bacterial cells at
a similar rate to magainin [8]. It is thus possible that b-17
lyses membranes by a pore-like mechanism, but the
properties ofthe pore would have to be different from that
formed with magainin in order to be consistent with the
different curvature tendencies of these peptides.
CD
Solutions ofb-17 in water were diluted into phosphate
buffer, and the far UV CD spectra were measured in the
presence and absence of SUVs (Fig. 3). When visually
transparent SUVs of lipid mixtures are added to a solution
of the b-peptide, there is a red shift in the CD spectra with
an increase in signal intensity. Similar changes in 12-helical
b-peptides are observed in going from water to methanol or
trifluoroethanol [31,32]. Trifluoroethanol and methanol are
known to stabilize helical conformations of both b-peptides
[33] and b-peptides [3,32]. Thus the CD spectra in Fig. 3
suggest that the 12-helical conformation ofb-17 is modestly
stabilized in the presence ofvesicles relative to aqueous
solution. DeGrado et al. [3] have observed strong helix
stabilization by vesicles for a different class of b-peptides
that are more conformationally flexible than is b-17.
Leakage
Leakage experiments were designed to obtain information
regarding the role of anionic headgroup, fatty acyl
chain unsaturation and vesicle curvature in the action of
b-17 on model membranes. For this purpose, different
anionic lipids dioleoylphosphatidylglycerol (Ole
2
PtdGro)
Fig. 3. Circular dichroism spectra ofthe b-17
peptide at a concentration of 100 l
M
. b-17
alone in buffer (solid line); b-17with SUVs of
Ole
2
PtdEtn : Ole
2
PtdSer (1 : 1) at a lipid to
peptide molar ratio (L : P) ¼ 10 (dotted line);
b-17 with SUVs of Ole
2
PtdEtn : Ole
2
PtdGro
(1:1)ataL:P¼ 10 (dashed line).
Ó FEBS 2003 Antimicrobialb-peptide and phospholipidvesicles (Eur. J. Biochem. 270) 1243
or dioleoylphosphatidylserine (Ole
2
PtdSer) were mixed with
dioleoylphosphatidylethanolamine (Ole
2
PtdEtn), dielaidoyl-
phosphatidylethanolamine or phosphatidylcholine extrac-
ted from egg yolk (eggPtdCho), as the zwitterionic lipid.
Phosphatidylethanolamine has a smaller headgroup and
therefore a larger tendency to promote negative curvature
compared to phosphatidylcholine. We included Ole
2
PtdEtn
and dielaidoylphosphatidylethanolamine, two species of
phosphatidylethanolamine, as components of these mix-
tures, because the trans double bond at position 9–10 in
dielaidoylphosphatidylethanolamine results in less deviation
of the acyl chain fromthe bilayer normal than does the cis
double bond of Ole
2
PtdEtn. Thus dielaidoylphosphatidyl-
ethanolamine has less intrinsic negative curvature than
Ole
2
PtdEtn, which is illustrated by the fact that pure
dielaidoylphosphatidylethanolamine has a T
H
of 65 °C
compared with 10 °C for pure Ole
2
PtdEtn, despite their
similarity in chemical structure. The presence ofthe anionic
lipids in themembrane allows for the formation of stable
liposomes at room temperature in the presence of
Ole
2
PtdEtn; pure PtdEtn does not easily form liposomes,
presumably due to limited hydration ofthe headgroup and
the higher energy cost of bending the membrane. In
addition, the presence ofthe anionic lipid PtdGro in these
mixtures is important as this is a major component of
prokaryotic membranes, while the use of phosphatidylserine
mimics the anionic lipid that becomes exposed to the
extracellular side ofthe bilayer in cancer cells or in apoptotic
mammalian cells. The zwitterionic lipid was mixed with the
negatively charged lipid Ole
2
PtdSer or Ole
2
PtdGro at a 1 : 2
molar ratio.
The rate and final extents of leakage is shown in
Fig. 4A,D. The leakage of aqueous contents from lipo-
somes of different lipid compositions is summarized by
showing the dependence ofthe percentage leakage at 300 s
on theb-peptide to lipid ratio (Fig. 5). In Fig. 6, leakage
Fig. 4. Leakage from 25 l
M
LUVs of Ole
2
PtdEtn/Ole
2
PtdGro (1 : 2) (A); dielaidoylphosphatidylethanolamine/Ole
2
PtdGro (1 : 2) (B); eggPtdCho/
Ole
2
PtdGro (1 : 2) (C) or eggPtdCho (D) induced by b-17. Numbers identifying each curve correspond to the micromolar concentration of b-17.
1244 R. F. Epand et al.(Eur. J. Biochem. 270) Ó FEBS 2003
from Ole
2
PtdEtn LUVs containing the anionic lipids
Ole
2
PtdGro, Ole
2
PtdSer, 1-palmitoyl-2-oleoylphosphatidyl-
serine (PamOlePtdSer) or phosphatidylserine extracted
from bovine brain is shown. Substituting either bovine
brain phosphatidylserine; dielaidoylphosphatidylethanol-
amine, or PamOlePtdSer for Ole
2
PtdSer had little effect,
highlighting again the predominance of headgroup charge
over chain length or degree of unsaturation ofthe fatty acid
in controlling a-peptide induced leakage. EggPtdCho alone
had a lower rate of leakage than did the lipid mixtures,
implying the requirement for an anionic lipid. This result is
expected for a peptide withantimicrobial activity, as
microbial membranes have a net negative surface charge.
Membrane binding
All vesicle systems tested that contained Ole
2
PtdGro were
found to bind strongly to NBD-b-17 peptide, as would be
expected of a cationic molecule (Fig. 7). Binding to LUVs of
Ole
2
PtdEtn/Ole
2
PtdGro and dielaidoylphosphatidyletha-
nolamine/Ole
2
PtdGro was virtually indistinguishable, while
the lowest binding affinity was found for Ole
2
PtdGro/
Ole
2
PtdSer.
Isothermal titration calorimetry (ITC)
Isothermal titration calorimetry was performed withb-17 in
the cell and incremental addition of vesicles. The results of
titration of LUVs of dielaidoylphosphatidylethanolamine/
Ole
2
PtdGro (1 : 2) into a solution ofb-17 is given in
Fig. 8A. Similar titrations were performed for mixtures of
Ole
2
PtdEtn/Ole
2
PtdGro(2:1)aswellasOle
2
PtdEtn/
Ole
2
PtdSer (2 : 1) (not shown). The titration for pure
eggPtdCho LUVs did not evolve significant heat with either
b-17 or NBD-b-17 (Fig. 8B). However, addition of Ole
2
Ptd-
Gro to eggPtdCho produced a titration curve similar to that
of the other LUV containing anionic lipids. The two modes
of binding observed could be explained by aggregation at
the high concentrations of peptide required for the ITC
experiment and in the presence of high local concentrations
of LUVs after each addition.
Fig. 5. Extent of leakage from 25 l
M
LUVs induced by b-17 after 300 s
as a function of P/L. The LUVs used were Ole
2
PtdEtn/Ole
2
PtdGro
(1 : 2) (j), dielaidoylphosphatidylethanolamine/Ole
2
PtdGro (1 : 2)
(m), eggPtdCho/Ole
2
PtdGro (1 : 2) (h), eggPtdCho (e).
Fig. 6. Extent of leakage from 25 l
M
LUVs induced by b-17 after
300 s as a function of P/L. TheLUVsusedwereOle
2
PtdEtn/
Ole
2
PtdGro (1 : 2) (j), Ole
2
PtdEtn/bovine brain phosphatidyl-
serine (1 : 2) (h), Ole
2
PtdEtn/Ole
2
PtdSer (1 : 2) (,), Ole
2
PtdEtn/
PamOlePtdSer (1 : 2) (.).
Fig. 7. Binding isotherms for NBD-b-17
with LUVs of Ole
2
PtdEtn/Ole
2
PtdGro (1 : 2)
(m), dielaidoylphosphatidylethanolamine/
Ole
2
PtdGro (1 : 2) (j), Ole
2
PtdEtn/Ole
2
Ptd-
Ser (1 : 2) (d), eggPtdCho/Ole
2
PtdGro (1 : 2)
(h).
Ó FEBS 2003 Antimicrobialb-peptide and phospholipidvesicles (Eur. J. Biochem. 270) 1245
For this reason, thermodynamic data was not extracted
to evaluate binding. The titrations however, allowed
discrimination between the behaviour ofthe peptide in
lipids containing Ole
2
PtdGro and with eggPtdCho alone.
Discussion
In this study we have characterized theinteractionsof b-17
with model membranes, in order to rationalize the obser-
vations that theantimicrobial activities and hemolytic
activities ofb-17 and those of [Ala8,13,18]-magainin II
amide [30], are similar [7,8]. This finding indicates that these
peptides must both cross the bacterial cell wall at similar
rates. It has been observed that although a cyclic derivative
of magainin has similar activity against liposomes when
bound to the membrane, its activity against biological
membranes can be altered by its ability to access the
membrane surface [34]. Both b-17 and [Ala8,13,18]-maga-
inin II amide have in common their affinity for anionic
lipids, but we found significant differences between b-17 and
magainin II or [Ala8,13,18]-magainin II amide in the way
these antimicrobial peptides interact with vesicle systems.
While both magainin II [29] and [Ala8,13,18]-magainin II
amide raise the T
H
of DPam
2
PtdEtn, b-17 lowers it. The fact
that magainin raises the T
H
is in accord withthe suggested
pore mechanism [35] in which the peptide has to promote a
large positive curvature ofthe lipid monolayer lining the
pore. It is less clear how the promotion of positive curvature
relates to the action oftheb-17 peptide. The fact that
homologous antimicrobial peptides can have different
effects on liposomal membranes is not unique to the present
paper. It has recently been shown that a group of model
Leu-containing diastereomeric peptides micellized vesicles
while the Ile-containing diastereomeric homologs fused
model membranes [36].
The lipid dependence of leakage is also very different for
magainin II and for b-17. In part, this reflects their different
curvature tendencies. For example, replacing Ole
2
PtdEtn
with dielaidoylphosphatidylethanolamine (Fig. 4A,B),
which has less negative intrinsic curvature, reduces the lysis
caused by b-17 as can be seen by the lower rate of contents
release caused by 5 l
M
b-17 with dielaidoylphosphatidyl-
ethanolamine:Ole
2
PtdGro (1 : 2) compared to 5 l
M
b-17
with Ole
2
PtdEtn:Ole
2
PtdGro (1 : 2). Similarly 10 l
M
b-17
is required for complete leakage to be achieved with
dielaidoylphosphatidylethanolamine/Ole
2
PtdGro (1 : 2) as
opposed to 6.5 l
M
b-17 with Ole
2
PtdEtn/Ole
2
PtdGro
(1 : 2). In contrast, magainin II promotes greater lysis of
vesicles when Ole
2
PtdEtn is substituted with dielaidoyl-
phosphatidylethanolamine [29]. The effects of changes in
lipid composition (to modulate membrane curvature) on
liposomal leakage rates have not been studied for
[Ala8,13,18]-magainin II amide. Magainin II also exhibits
much more lysis with liposomes containing PtdGro than
with liposomes containing PtdSer [29]. We found that b-17
(Fig. 6) did not discriminate, in terms of inducing leakage,
between vesicles containing Ole
2
PtdGro or Ole
2
PtdSer, even
though the binding affinity ofb-17 to PtdSer-containing
vesicles is lower than for PtdGro-containing vesicles.
Magainin II has been shown in several studies to have
antitumor activity [37–45]. Withthe greater interaction of
b-17 with PtdSer-containing liposomes, as compared with
magainin, one would anticipate that b-17 would be a more
potent anticancer agent.
Both magainin II and b-17 lyse anionic liposomes with
greater potency than they lyse zwitterionic lipsosomes
(Fig. 4C,D), and they both bind more strongly to anionic
membranes than to the zwitterionic membrane, as seen by
ITC. Therefore, their microbial specificity is likely a
consequence of improved electrostatic interactions with
microbial vs. eukaryotic membranes. For several anti-
microbial peptides it is believed that the basis ofthe selective
toxicity towards bacteria is that the peptide is cationic and
can therefore partition to a greater extent into microbial
membranes, which have exposed anionic lipids, than into
eukaryotic cell membranes. Alternatively, the antimicrobial
activity could result from interaction ofthe peptide with a
specific component of microbial membranes. Recently it has
been shown that theantimicrobial peptide nicin has greater
specificity for microbial membranes because of a specific
interaction with lipid II, a component of these membranes
[46–49].
Like other cationic antimicrobial peptides, b-17 displays
specificity for anionic lipids. However, b-17 differs from
Fig. 8. ITC titrations at 30 °C. (A) Successive injections of 10 lLof
3.7 m
M
LUVs of dielaidoylphosphatidylethanolamine/Ole
2
PtdGro
(1 : 2) into 12 l
M
b-17 placed in the calorimeter cell. Similar titrations
were obtained with other lipid mixtures containing Ole
2
PtdGro.
(B) successive injections of 10 lLof5m
M
eggPtdCho LUVs into
25 l
M
of b-17placedinthecell.
1246 R. F. Epand et al.(Eur. J. Biochem. 270) Ó FEBS 2003
several other such antimicrobial peptides, such as magainin
II and its analog [Ala8,13,18]-magainin II amide, in that
b-17 promotes negative rather than positive membrane
curvature. Therefore it is likely that there are differences in
the manner in which b-17 affects membrane properties,
compared with magainin. One manifestation of these
differences is the lack of specificity of interaction of the
peptide with PtdSer vs. PtdGro. Despite these mechanistic
differences, however, b-17 behaves as a potent antimicrobial
peptide having a minimal inhibitory concentration against
several bacteria at least as good as or better than that of
[Ala8,13,18]-magainin II amide [8]. [Ala8,13,18]-magainin II
amide is an analog ofthe natural peptide magainin II that
has a greater antimicrobial activity [30]. This analog is more
hydrophobic than magainin II, having three Ala substitu-
ting for a Ser and two Gly. Theantimicrobial activity of this
analog for several bacteria is of a similar potency to its
hemolytic activity [8] in contrast to magainin II where there
is approximately 100-fold greater antimicrobial activity
compared with hemolytic activity [50]. Being more hydro-
phobic, [Ala8,13,18]-magainin II amide will likely penetrate
more deeply into themembrane than the parent peptide,
magainin II. Deeper membrane penetration also facilitates
the promotion of negative curvature, as the regions of the
bilayer below the pivotal plane are expanded more than
regions close to the interface. Thus, although [Ala8,13,18]-
magainin II amide still induces positive curvature effects on
membranes, as measured by shifts in T
H
, its effects on
membranes would tend to be somewhat different from that
of magainin II. When the [Ala8,13,18]-magainin II amide
analog is compared withb-17 by HPLC analysis, both
peptides have similar overall hydrophobicities, with similar
retention times. However, the effects of these peptides on the
T
H
of DPam
2
PtdEtn suggest that the b-17hasagreater
depth of penetration. Nevertheless the two peptides exhibit
similar microbial specificity.
Acknowledgements
This work was supported by the Canadian Institutes of Health
Research, Grant 7654 and by the US National Institutes of Health
(GM56414). N.U. was supported in part by a Research Fellowship for
Young Scientists fromthe Japan Society for the Promotion of Science,
and E.A.P. was supported in part by a Biotechnology Training Grant
from NIGMS.
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. Interactions of the antimicrobial b-peptide b-17 with phospholipid vesicles differ from membrane interactions of magainins Raquel F. Epand 1 , Naoki Umezawa 2 ,. that there are differences in the manner in which b-17 affects membrane properties, compared with magainin. One manifestation of these differences is the lack of specificity of interaction of the peptide. predominates. If b-17 promoted the formation of a similar pore arrange- ment, but the diameter of the pore were smaller than that of magainin, then the negative curvature in the dimension around the rim of the