Báo cáo khoa học: Investigations into the ability of an oblique a-helical template to provide the basis for design of an antimicrobial anionic amphiphilic peptide pot

Investigations into the ability of an oblique a-helical
template to provide the basis for design of an
antimicrobial anionic amphiphilic peptide
Sarah R. Dennison
1
, Leslie H. G. Morton
2
, Klaus Brandenburg
3
, Frederick Harris
4
and
David A. Phoenix
1
1 Faculty of Science, University of Central Lancashire, Preston, UK
2 School of Natural Resources, University of Central Lancashire, Preston, UK
3 Forschungszentrum Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany
4 Department of Forensic and Investigative Science, University of Central Lancashire, Preston, UK
Globally and particularly in developing countries [1],
antimicrobial drug resistance has become a major prob-
lem, resulting in a decline in the effectiveness of existing
antimicrobial agents [2]. As a consequence, infections
have been rendered more expensive and harder to treat,
and epidemics have been made more difficult to con-
trol. Moreover, many previously treatable infectious
diseases such as tuberculosis now have greatly
increased rates of morbidity and mortality [3]. In
response, the pharmaceutical industry has investigated
a number of compounds with the potential to act as
new and effective antimicrobial agents [4], ranging from
photosensitizing dyes [5] to nucleosides [6]. A recent

focus of these investigations has been a-helical anti-
microbial peptides (a-AMPs) which are components of
mammalian innate immune systems [7–10].
Generally, a-AMPs are cationic [11,12], which facili-
tates their interaction with the anionic membranes
of microbial cells, and they exert their antimicrobial
action by the use of nonreceptor-based mechanisms of
Keywords
anionic; antimicrobial; a-helical; membrane;
peptide
Correspondence
D. A. Phoenix, Deans Office, Faculty of
Science, University of Central Lancashire,
Preston PR1 2HE, UK
Fax: +44 1772 892903
Tel: +44 1772 893481
E-mail:
(Received 12 January 2006, revised 12 June
2006, accepted 20 June 2006)
doi:10.1111/j.1742-4658.2006.05387.x
AP1 (GEQGALAQFGEWL) was shown by theoretical analysis to be an
anionic oblique-orientated a-helix former. The peptide exhibited a mono-
layer surface area of 1.42 nm
2
, implying possession of a-helical structure
at an air ⁄ water interface, and Fourier transform infrared spectroscopy
(FTIR) showed the peptide to be a-helical (100%) in the presence of vesi-
cle mimics of Escherichia coli membranes. FTIR lipid-phase transition
analysis showed the peptide to induce large decreases in the fluidity of
these E. coli membrane mimics, and Langmuir–Blodgett trough analysis

found the peptide to induce large surface pressure changes in monolayer
mimics of E. coli membranes (4.6 mNÆm
)1
). Analysis of compression iso-
therms based on mixing enthalpy (DH) and the Gibbs free energy of mix-
ing (DG
Mix
) predicted that these monolayers were thermodynamically
stable (DH and DG
Mix
each negative) but were destabilized by the pres-
ence of the peptide (DH and DG
Mix
each positive). The peptide was found
to have a minimum lethal concentration of 3 mm against E. coli and was
seen to cause lysis of erythrocytes at 5 mm. In combination, these data
clearly show that AP1 functions as an anionic a-helical antimicrobial pep-
tide and suggest that both its tilted peptide characteristics and the com-
position of its target membrane are important determinants of its efficacy
of action.
Abbreviations
a-AMP, a-helical antimicrobial peptide; AP1, GEQGALAQFGEWL; FTIR, Fourier transform infrared spectroscopy; Ole
2
PtdEtn,
dioleoylphosphatidylethanolamine; Ole
2
PtdGro, dioleoylphosphatidylglycerol; SUV, small unilamellar vesicle.
3792 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS
membrane invasion [13,14]. The relatively nonspecific
nature of these mechanisms renders the development

of acquired microbial resistance to a-AMPs unlikely,
although several mechanisms of inherent resistance to
these peptides have been reported [11,15,16]. The most
common of these mechanisms is exhibited by both
Gram-positive and Gram-negative pathogens and
effectively involves the reduction of anionic lipid con-
centrations in the bacterial cell envelope, thereby inhib-
iting the membrane-binding ability of cationic a-AMPs
[17–19].
Most recently, theoretical studies have suggested
that many a-AMPs may destabilize bacterial mem-
branes by the use of oblique orientated a-helical struc-
ture [20], which has been experimentally demonstrated
for the amphibian a-AMPs: aurein 1.2, citropin 1.1
and caerin 1.1 [21]. These a-helices have been described
in a variety of proteins and peptides, most commonly
viral protein segments, and are differentiated from
other classes of membrane-interactive a-helices in that
they possess a hydrophobicity gradient along the
a-helical long axis. This structural feature causes an
a-helix to penetrate membranes at a shallow angle of
30–60 °, thereby disturbing membrane lipid organiza-
tion and compromising bilayer integrity [22,23].
Among the a-AMPs predicted to form oblique-
orientated a-helices [12] are a small number that are
negatively charged, such as the amphibian peptide
maximin H5 [24]. It has been suggested that anionic
a-AMPs may have evolved to counter microbe resist-
ance to cationic a-AMPs, which would seem to make
these former peptides well suited for development as

novel antimicrobial agents directed against such organ-
isms [24,25].
There appears to have been little research into the
mode of membrane interaction used by anionic
a-AMPs, although photodynamic antimicrobial studies
have shown nonpeptide anionic molecules to be effect-
ive against Gram-negative bacteria because of their
ability to penetrate the membranes of these organisms
[26,27]. Pathogenic Gram-negative bacteria are becom-
ing increasingly problematic in areas ranging from
health care to the food industry [28–31], therefore
in this study we analysed a novel synthetic peptide,
AP1 (GEQGALAQFGEWL), as a potential anionic
a-AMP against Escherichia coli. The sequence of AP1
was designed to form a membrane-interactive oblique
orientated a-helix, shown here by theoretical analysis,
and Fourier transform infrared spectroscopy (FTIR)
confirmed that the peptide was a-helical in the pres-
ence of lipid vesicles that mimicked membranes of
E. coli. A standard toxicity assay showed that
AP1 inactivated the organism, and the use of
Langmuir-Blodgett troughs showed that the peptide
inserted strongly into lipid monolayers that mimicked
E. coli membranes. Compression isotherm analysis
indicated that lipid monolayers mimicking E. coli
membranes were thermodynamically stable but were
destabilized by the presence of AP1. FTIR lipid-phase
transition analysis showed that the peptide induced
changes in the membrane fluidity of E. coli mem-
branes, which were consistent with penetration of the

hydrophobic core of these membranes. AP1 was found
to lyse erythrocyte membranes, and, on the basis of
these combined data, it is suggested that the peptide
functions as an anionic membrane-interactive a-AMP.
These data also suggest that the antimicrobial activity
of AP1 depends on both the structural characteristics
of its tilted peptide architecture and the lipid packing
of its target membrane.
Results
The influenza HA2 fusion peptide is known to form a
membrane-interactive oblique orientated a-helix [32], a
secondary-structural motif recently postulated to fea-
ture in the action of a range of a-AMPs [20]. A seg-
ment of the HA2 peptide (GLFGAIAGFIENG),
which is key to its structure and underlying hydro-
phobicity gradient, was used as a basis for the hydro-
phobicity gradient of the AP1 peptide, thereby giving
these peptides 62% sequence homology. The sequence
of AP1 is predicted to produce an a-helical peptide
with structural features that are characteristic of both
this oblique orientated a-helix and the established
anionic a-AMP, maximin H5. Figure 1A shows that,
when the sequences of these three peptides were ana-
lysed using extended hydrophobic moment plot meth-
odology, the resulting data points were proximal and,
along with those of % 50% of the a-AMPs studied,
are candidates to form oblique orientated a-helices.
Figure 1B shows that, in an a-helical conformation,
AP1 would possess a hydrophobic arc size of 90 °, and
Fig. 2 indicates that this value (Fig. 2A) and the mean

hydrophobic moment of the peptide (0.35; Fig. 2B) are
highly comparable to those of maximin H5. However,
Fig. 2A,B also show that, for both peptides, these val-
ues fall in the lower quartile of those observed for the
cationic a-AMPs tested.
Monolayer analysis showed that increasing con-
centrations of AP1 in the subphase of a Langmuir-
Blodgett trough led to progressively greater interfacial
surface pressures until, at 20 lm peptide, a maximal
value of 11.5 mNÆm
)1
was observed (Fig. 3). Above
this peptide concentration, surface pressures were
effectively constant, which indicates that 20 lm AP1
S. R. Dennison et al. Antimicrobial properties of an anionic a-helical peptide
FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3793
was the minimum bulk concentration required to sat-
urate the air ⁄ water interface with the peptide under
these experimental conditions. These data were used to
determine the corresponding interfacial surface area
per AP1 molecule (Table 1), and, for 20 lm peptide,
extrapolation provides an estimate of peptide surface
of 1.40 nm
2
, which is comparable to that found for
peptides that adopt a-helical structure [33].
When spread from chloroform on to the subphase
of a Langmuir-Blodgett trough, AP1 formed stable
monolayers. Under compression, these monolayers
showed collapse pressures in the region of 20 mNÆm

)1
(Fig. 4), indicating the presence of a well-ordered
monolayer [34]. Compressibility moduli, C
À1
s
, were
derived from these isotherms (Table 2) and generally
decreased with increasing surface pressure, indicating
that the monolayer is in the protein phase [35].
Figure 4 also shows that the area per AP1 molecule
1.5
1.25
1
0.75
0.5
0.25
0
-1 -0.5 0
0.5 1
1.5
<H>
<µH>
A
G10
Q3
B
A7
E11
G4
Q8

W12
G1
A5
F9
E2
L13
L6
Fig. 1. (A) Extended hydrophobic moment plot analysis of AP1 (m ),
the known anionic a-AMP, maximin H5 (d), and peptides of the
a-AMP dataset ( />all as described in the text. AP1, maximin H5, and % 50% of the
peptides in the dataset are represented by data points that lie in
the shaded region, delineating candidacy for oblique-orientated
a-helix formation. (B) Sequence of AP1 represented as a 2D axial
projection. This a-helix possesses a hydrophilic face, which is rich
in glycine residues and polar residues (circled), and a hydrophobic
face formed from bulky apolar residues with a centrally placed glu-
tamate residue.
AB
1.20
1.00
0.80
0.60
0.40
0.20
350
300
250
200
150
100

50
0
α-AMP
AP1
H5
α-AMP
AP1
H5
Hydrophobic arc size (º)
Hydrophobic Moment (µH)
Fig. 2. Box plot for the hydrophobic arc size (A) and mean hydro-
phobic moment (B) of AP1, maximin H5, which is a known anionic
a-AMP, and the a-AMP dataset, />bru/amp_data.htm, all determined as described in the text. AP1 and
maximin H5 show comparable amphiphilic properties, which gener-
ally lie in the lower quartile range of the dataset.
0
2
4
6
8
10
12
0 5 10 15 20 25 30 35
AP1 concentration (
M)
Surface pressure (mN·m
-1
)
Fig. 3. AP1 surface pressure as a function of peptide concentration.
Increasing concentrations of AP1 were injected into a Tris ⁄ HCl buf-

fer subphase (10 m
M, pH 7.5) of a Langmuir-Blodgett system. At
each AP1 concentration, the peptide was allowed to equilibrate,
and the surface pressure determined and plotted, all as described
in the text.
Table 1. Surface excess (G) and interfacial surface area per AP1
molecule (A) for various molar subphase concentrations (C) of the
peptide where p is the interfacial pressure increase. Values for
these parameters were derived using AP1 surface pressure data
from Fig. 3 with G computed using Eqn (1) and A computed using
Eqn (2), all as described in the text.
C
(l
M)
p
(mNÆm
)1
) G
A
(nm
2
)
00
–
–
0.5 0.9 4.8 · 10
)8
48.44
1 5.15 3.1 · 10
)7

5.44
2 6.5 4.3 · 10
)7
3.87
5 7.9 6.1 · 10
)7
2.72
10 9.32 8.3 · 10
)7
2.00
15 10.65 1.0 · 10
)6
1.59
20 11.26 1.2 · 10
)6
1.40
25 11.32 1.3 · 10
)6
1.27
30 11.33 1.3 · 10
)6
1.26
Antimicrobial properties of an anionic a-helical peptide S. R. Dennison et al.
3794 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS
corresponding to this collapse pressure was 0.33 nm
2
.
The extrapolated area at p ¼ 0mNÆm
)1
for the iso-

therm provides a measure of the mean monolayer
surface area per AP1 molecule [36]. This area was
1.42 nm
2
per AP1 molecule and is comparable to the
value of 1.40 nm
2
calculated above for the peptide
using Eqns (1) and (2) (Table 1). Although towards
the lower end of the expected range, this would
approximate to that predicted for AP1 if the peptide
was orientated perpendicular to the air ⁄ water interface
(1.77 nm
2
[37]), but may also indicate the presence of
some non-a-helical structure in AP1.
FTIR conformational analysis showed that AP1
adopted predominantly b-type structures in solution.
However, at lipid to peptide ratios of 50 : 1 and above,
the peptide adopted % 100% a-helical structure in the
presence of lipid assemblies that mimicked membranes
of E. coli (Fig. 5). This conformational behaviour is
similar to that shown by most a-AMPs, which are gen-
erally non-a-helical in solution but assume a-helical
structure at the microbial interface [38–40].
A standard toxicity assay established a minimum
lethal concentration of 3 mm for AP1 when directed
against E. coli. Analysing the number of colony form-
ing units over time showed that, at this concentration,
the peptide took 1 h to induce 100% cell death

(Fig. 6).
Microbial membrane invasion is the primary killing
mechanism used by most a-AMPs [41,42]. FTIR con-
formational analysis shows that, in the absence of
AP1, small unilamellar vesicles (SUVs) mimetic of
E. coli membranes underwent transition from the gel
phase to liquid crystalline phase over the temperature
range 20–70 °C with a concomitant increase in mem-
brane fluidity, as indicated by the rise in wavenumber
from % 2851.0 cm
)1
to 2852.3 cm
)1
. The presence of
AP1 caused no apparent shift in the temperature range
of these phase transitions but, over this temperature
range, induced a significant decrease in the membrane
fluidity of E. coli membranes (Fig. 7).
AP1 also interacted with lipid monolayers that were
mimetic of E. coli membranes (Fig. 8), inducing
Fig. 4. A pressure–area isotherm for an AP1 monolayer. The pep-
tide was spread from chloroform on to a Tris ⁄ HCl buffer subphase
(10 m
M, pH 7.5). The variation of surface pressure with area per
peptide molecule was monitored as monolayers were compressed
and plotted, all as described in the text.
Table 2. Compressibility moduli (C
À1
s
) of lipid monolayers at vary-

ing surface pressure (p) (all values mNÆm
)1
). Values of C
À1
s
were
computed using data from compression isotherms (Fig. 9) and Eqn
(3). Monolayers were formed from either Ole
2
PtdGro, Ole
2
PtdEtn,
cardiolipin, or lipid mixtures that corresponded to membranes of
E. coli, all as described in the text.
Pressure p
(mNÆm
)1
)
C
À1
s
(mNÆm
)1
)
Cardiolipin E. coli
Ole
2
PtdGro Ole
2
PtdEtn

5 17.3 29.3 11.1 26.6
10 14.5 28.8 13.8 25.3
15 12.1 24.5 16.2 23.7
Fig. 5. FTIR conformational analysis of AP1 in the presence of
SUVs with lipid compositions that correspond to those of E. coli
membranes, all as described in the text. The numbers annotating
spectra indicate peak band absorbancies. For each spectrum, the
relative percentages of a-helical structure (1650–1660 cm
)1
) and
b-sheet structures (1625–1640 cm
)1
) were computed, all as des-
cribed in the text. In aqueous solution, AP1 was predominantly
formed from b-type structures (A), but in the presence of E. coli
membrane mimics (B), the peptide was 100% a-helical.
S. R. Dennison et al. Antimicrobial properties of an anionic a-helical peptide
FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3795
maximal changes in surface pressure of 4.6 mNÆm
)1
after 6500 s. This was further investigated by thermo-
dynamic analysis of compression isotherms derived
from monolayer mimics of E. coli membranes in either
the absence (Fig. 9A) or presence of AP1 (Fig. 9B).
C
À1
s
were derived from these isotherms (Table 2), and
C
À1

s
is seen to be generally low, indicating that the
lipid monolayers analysed were in a liquid expanded
phase [35] and, thus, are more fluid and possess high
Fig. 6. Time course for the viability of E. coli represented as per-
centage death rate in the presence of AP1 (3 m
M). At these con-
centrations, the peptide is bactericidal, achieving a 100% death
rate after 1 h. The percentage death rate was determined by com-
parison with identical noninoculated control cultures, all as des-
cribed above, and error bars represent the standard error on three
replicates.
2854
2853
2852
2851
2850
01020304050607080
Wavenumber (cm
–1
)
Temperature (°C)
Fig. 7. FTIR lipid-phase transition analyses of SUVs with lipid com-
positions that correspond to those of E. coli membranes, all as des-
cribed in the text. In the absence of AP1 (,) model membranes of
E. coli underwent a transition from the gel phase to the liquid crys-
talline phase liquid over the temperature range 30–70 °C with a
concomitant increase in membrane fluidity s indicated by the rise in
wavenumber from % 2851.0 cm
)1

to 2852.3 cm
)1
. The presence of
AP1 caused no apparent shift in this temperature range but induced
a significant decrease in the membrane fluidity of E. coli mem-
branes, which is consistent with the peptide penetrating the hydro-
phobic core of these membranes.
Fig. 8. Time course of interactions between AP1 and monolayers
with lipid compositions that correspond to those of E. coli mem-
branes, all as described in the text. Monolayers were at an initial
surface pressure of 30 mNÆm
)1
, mimetic of naturally occurring
membranes, and the peptide was introduced into the subphase to
give a final concentration 20 l
M, all as described in the text.
Fig. 9. Compression isotherms of monolayers formed from: lipid
compositions that correspond to those of E. coli membranes (a),
Ole
2
PtdEtn (b), Ole
2
PtdGro (c) and cardiolipin (d). The variation of
surface pressure with area per lipid molecule was monitored as
monolayers were compressed on a Tris ⁄ HCl buffer subphase
(10 m
M, pH 7.5) either in the absence of AP1 (A) or containing AP1
with a final concentration of 20 l
M (B), all as described above.
Antimicrobial properties of an anionic a-helical peptide S. R. Dennison et al.

3796 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS
compressibility. Table 2 also shows that AP1 induced
a general decrease in C
À1
s
with rising monolayer sur-
face pressure, indicating expansion of these bacterial
membrane mimics because of peptide interactions.
Values for the Gibbs free energy of mixing (DG
Mix
)
(Table 3) were derived from the compression isotherms
shown in Fig. 9. It can be seen from Table 3 that
DG
Mix
for E. coli model membranes varies with surface
pressure and according to the presence or absence of
AP1. Table 3 shows that values of DG
Mix
for these
E. coli model membranes are much lower than RT
(2444.316 JÆmol
)1
), indicating that deviations from
ideal mixing behaviour are small. In the absence of
AP1, negative values of DG
Mix
were observed for
E. coli model membranes (Table 3), indicating a stable
monolayer. However, in the presence of AP1 (Table 3),

positive values of DG
Mix
are observed for E. coli model
membranes, indicating that, although the lipids form-
ing these monolayers are miscible, repulsive interac-
tions are established in the presence of the peptide,
thereby decreasing membrane stability. These values of
DG
Mix
become progressively more positive as surface
pressure increases, showing that, at higher surface
pressures, mutual interactions between the component
molecules of these membranes are weaker than those
occurring in monolayers formed by their pure compo-
nents [43], becoming increasingly less stable with
compression. This instability may contribute to the
susceptibility of E. coli model membranes to the action
of AP1.
An important determinant of the susceptibility of
membranes to a-AMPs is the packing characteristics
of the individual membrane lipids [44]. To evaluate the
nature of interactions between the component lipid
molecules in E. coli model membranes, the interaction
parameter, a, and the mixing enthalpy, DH, were com-
puted (Table 3). It can be seen from Table 3 that, in
the absence of AP1, values for a and DH are negative
for these model membranes, but, in the presence of the
peptide, they are positive. These results confirm that
E. coli membranes are thermodynamically less stable
in the presence of AP1, further supporting the sugges-

tion that this instability may contribute to the
susceptibility of E. coli to the antimicrobial action of
the peptide.
Discussion
The biological action of many pore-forming and lytic
peptides involves membrane destabilization by the use
of lipid-interactive oblique-orientated a-helical struc-
ture [22], and such structure also appears to be used
by many a-AMPs [20]. It has been suggested that
anionic a-AMPs and their analogues may serve as
complements to their cationic counterparts in some
therapeutic contexts [24,25]. Here, a synthetic peptide,
AP1, was prepared to observe whether anionic pep-
tides with tilted peptide characteristics could be
designed to act as potential anionic a-AMPs.
Theoretical analysis confirmed that the peptide pos-
sessed the potential to form an a-helix with a balance
between amphiphilicity and hydrophobicity, and struc-
tural characteristics that are associated with oblique-
orientated a-helices (Figs 1 and 2). It can be seen from
Fig. 1B that the AP1 a-helix possesses a glycine-rich
polar face, and it has previously been shown that simi-
larly located glycine residues are critical for maintaining
the hydrophobicity gradients associated with mem-
brane-interactive oblique-orientated a-helices [45]. It
can also be seen from Fig. 1B that the AP1 a-helix pos-
sesses a wide hydrophobic face rich in bulky amino-acid
residues, and, in combination with a glycine-rich polar
face, these structural characteristics give a-helices an
effective inverted wedge shape. It has been recently

shown that a number of a-AMPs, experimentally dem-
onstrated to penetrate membranes in an oblique orien-
tation, appear to possess this inverted wedge shape
[12,21]. In addition, it can be seen from Fig. 1B that a
glutamate residue is centrally located in the apolar face
of the AP1 a-helix, and previous studies have shown
that similarly located glutamate residues are important
for the antimicrobial action of other a-AMPs also
predicted to form an inverted wedge shape [46].
FTIR spectroscopy showed that AP1 was completely
a-helical in the presence of model membranes mimetic
of those of E. coli (Fig. 5), although molecular area
Table 3. Gibbs free energy of mixing (DG
Mix
), interaction parameter (a) and enthalpy of mixing (DH) at varying surface pressure (p) for lipid
mixtures that correspond to membranes of E. coli. Values for these parameters were computed either in the presence or absence of AP1
using data from compression isotherms (Fig. 9) in conjunction with Eqns (4), (5) and (6), respectively, all as described above.
Pressure
p (mNÆm
)1
)
DG
Mix
(JÆmol
)1
) aDH (JÆmol
)1
)
–AP1 +AP1 –AP1 +AP1 –AP1 +AP1
5 )106.11 0.60 )7.35 0.04 )8986.72 51.00

10 )258.02 7.91 )17.88 0.55 )21852.82 669.85
15 )387.10 12.51 )26.83 0.87 )32785.45 1059.37
S. R. Dennison et al. Antimicrobial properties of an anionic a-helical peptide
FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3797
determinations showed that the peptide may possess
some non-a-helical structure at an air ⁄ water interface.
It would seem that AP1 specifically requires the
amphiphilicity associated with the environment of a
membrane or lipid interface to form such structure.
Monolayer studies confirmed that AP1 was able to par-
tition into model membranes that were mimetic of
those of E. coli (Fig. 8), and toxicity assay showed that
AP1 was bactericidal at 3 mm (Fig. 6). In combination,
these data clearly show that the peptide is able to func-
tion as an anionic a-AMP. Moreover, these results sug-
gest that interaction with bacterial membranes features
in the antibacterial action of AP1, and it is well estab-
lished that perturbation of the microbial membrane is a
primary killing mechanism used by a-AMPs [41,42].
This suggestion is strongly reinforced by the observa-
tion that AP1 showed haemolytic ability, thereby
clearly confirming that the peptide is able to induce cell
bilayer disruption. AP1 was found to be haemolytic at
5mm, thereby showing a common characteristic of
a-AMPs in that higher concentrations of these peptides
are generally required for haemolytic action than for
bactericidal action [38]. The minimum lethal concentra-
tion of AP1 is far in excess of those normally required
by cationic a-AMPs to inhibit target micro-organisms
(< 20 lm), but is closer to those required by some ani-

onic a-AMPs (80 lm) [12], which are known to gener-
ally exhibit lower levels of antimicrobial efficacy than
their cationic counterparts. Lipid-phase transition ana-
lysis clearly suggested that the interactions of the pep-
tide with membranes of E. coli induced a significant
decrease in the membrane fluidity of E. coli membranes
(Fig. 7), which is consistent with the peptide penetrat-
ing deeply into the membranes hydrophobic core.
To investigate further the mechanism of bacterial
membrane interaction used by AP1, thermodynamic
analysis of compression isotherms for lipid monolayer
mimetics of E. coli membranes were undertaken
(Table 3, Fig. 9). These analyses gave negative values
for DG
Mix
, a and DH in the absence of AP1, indicating
membrane stability, but, in the presence of AP1, posit-
ive values of for DG
Mix
, a and DH were obtained, sug-
gesting that the monolayer had become less stable.
This shows that the association of AP1 with these
model membranes had a destabilizing effect, and, when
taken with the FTIR data above, suggests that the
peptide may promote toxicity to E. coli by a lytic-type
mechanism involving disturbance of lipid acyl chains
within the membrane core [47].
It is well established that the packing characteristics
of component lipids is an important factor in
determining the stability of membrane bilayers [44]. It

is interesting to note that E. coli membranes possess
high levels of phosphatidylethanolamine (% 85%),
which is effectively shaped like an inverted wedge and
is known to have a strong preference for the nonlamel-
lar H
11
phase [14]. Thus, according to the wedge hypo-
thesis of Tytler et al. [48], it may be that insertion of
the inverted wedge shape formed by the AP1 a-helix
into membranes of E. coli leads to the formation of
nonbilayer structures and thereby membrane destabil-
ization. Such a mechanism of membrane perturbation
would be consistent with the use of a lytic-type mech-
anism for antimicrobial action, as predicted by the
thermodynamic analyses above and the involvement of
oblique-orientated a-helical structure in AP1. The
higher concentrations of peptide required for haemo-
lysis would indicate that the membrane composition
plays an important role in activity.
In summary, AP1 was found to function as an ani-
onic a-AMP, indicating that it is possible to design
a-AMPs by the use of an oblique-orientated a-helical
template. It appears from the biophysical data that the
peptide uses this structure for the destabilization of
membranes of Gram-negative bacteria, thereby promo-
ting the inactivation of these organisms. The relatively
high concentration required for the minimum lethal
concentration indicates though that further lessons
with respect to the amino-acid composition are still to
be learnt. However, as a general lesson, the data pre-

sented in this study emphasize that, in development of
antimicrobial compounds, both the structural charac-
teristics and composition of their target membrane are
important determinants of their efficacy of action.
Experimental procedures
Reagents
AP1 (GEQGALAQFGEWL) was supplied by Pepsyn
(Liverpool, UK), produced by solid-state synthesis, and
purified by HPLC to greater than 95%, which was con-
firmed by MALDI MS. Buffers and solutions for monolay-
er experiments were prepared from Milli-Q water. Nutrient
broth was purchased from Amersham Bioscience (GE
Healthcare, Chalfont St Giles, UK). Dioleoylphosphatidyl-
glycerol (Ole
2
PtdGro) and dioleoylphosphatidylethanolam-
ine (Ole
2
PtdEtn) were purchased from Alexis Corporation
(Axxora, Bingham, UK). Cardiolipin, Hepes, Tris and all
other reagents were purchased from Sigma (Sigma-Aldrich,
Gillingham, UK).
Primary structure analyses
The sequences of 161 known a-AMPs were obtained from
Dennison et al. [41] ( />Antimicrobial properties of an anionic a-helical peptide S. R. Dennison et al.
3798 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS
amp_data.htm) and along with those of maximin H5
(ILGPVLGLVSDTLDDVLGIL [24]) and AP1 were ana-
lysed according to conventional hydrophobic moment
methodology [49]. Essentially, this methodology treats the

hydrophobicity of successive amino acids in a sequence, as
vectors and then sums these vectors in two dimensions,
assuming an amino-acid side chain periodicity of 100 °. The
resultant of this summation, the hydrophobic moment, pro-
vides a measure of a-helix amphiphilicity. The analysis of
the present study used a moving window of 11 residues,
and for each sequence under investigation, the window with
the highest hydrophobic moment was identified [49]. For
these windows, the mean hydrophobic moment, <lH>,
and the corresponding mean hydrophobicity, <H>, were
computed using the online program moment helix predic-
tion ( and
the normalized consensus hydrophobicity scale of Eisenberg
et al. [49]. These mean values were plotted on the hydro-
phobic moment plot diagram of Eisenberg et al. [50], as
modified by Harris et al. [23], to identify candidate oblique-
orientated a-helix-forming segments. Assuming an idealized
a-helix with a residue side chain angular periodicity of
100 °, a 2D axial projection of the peptide was generated
[51]. The angle subtended by the hydrophobic residue distri-
bution was taken as a measure of hydrophobic arc size.
Preparation of lipid unilamellar vesicles
SUVs with lipid compositions designed to mimic E. coli
membranes were prepared as described by Keller et al. [52].
Essentially, chloroform solutions of Ole
2
PtdGro, Ole
2
Ptd-
Etn and cardiolipin in molar proportions of 1 : 13.67 : 2

[53] were dried with nitrogen gas and hydrated with Hepes
buffer (10 mm, pH 7.5) to give final total lipid concentra-
tions of 150 mm. The resulting cloudy suspensions were
sonicated at 4 ° C with a Soniprep 150 sonicator (amplitude
10 lm) until clear (30 cycles of 30 s), centrifuged (15 min,
3000 g,4°C), and the supernatant decanted for immediate
use.
FTIR conformational analysis of AP1
To give final peptide concentration ranging from 3 mm
to1 mm, AP1 was solubilized in either Hepes buffer
(10 mm, pH 7.5) or suspensions of SUVs formed from
Ole
2
PtdGro, Ole
2
PtdEtn and cardiolipin as described
above. These samples were spread individually on a CaF
2
crystal, and the free excess water was evaporated at room
temperature. The single band components of the VAP1
amide I vibrational band (predominantly C¼O stretch) was
monitored using an FTIR ‘5-DX’ spectrometer (Nicolet
Instruments, Madison, WI, USA), and, for each sample,
absorbance spectra were produced. For these spectra, water
bands were subtracted, and the evaluation of peptide band
parameters (peak position, band width and intensity)
performed. Curve fitting was applied to overlapping bands
using a modified version of the curfit procedure written
by Dr Moffat, National Research Council, Ottawa,
Ontario, Canada. The band shapes of the single compo-

nents are superpositions of Gaussian and Lorentzian band
shapes. Best fits were obtained by assuming a Gauss frac-
tion of 0.55–0.6. The curfit procedure measures the peak
areas of single band components, and, after statistical
evaluation, determines the relative percentages of primary
structure involved in secondary-structure formation, all as
described by Dennison et al. [54].
FTIR analysis of phospholipid phase-transition
properties
To give a final peptide concentration of 3 mm, AP1 was solu-
bilized in suspensions of SUVs, which were formed from
Ole
2
PtdGro ⁄ Ole
2
PtdEtn ⁄ cardiolipin as described above. As
controls, corresponding lipid SUVs were prepared with no
peptide present. All samples were then subjected to automa-
tic temperature scans with a heating rate of 3 °C per 5 min
and within the temperature range 0–60 °C. For every 3 °C
interval, 50 interferograms were accumulated, apodized,
Fourier transformed, and converted into absorbance spectra
[55]. These spectra monitored changes in the b fi a acyl
chain melting behaviour of phospholipids, with these changes
determined as shifts in the peak position of the symmetric
stretching vibration of the methylene groups, m
s
(CH
2
), which

is known to be a sensitive marker of lipid order. The peak
position of m
s
(CH
2
) lies at 2850 cm
)1
in the gel phase
and shifts at a lipid specific temperature T
c
to 2852.0–
2852.5 cm
)1
in the liquid crystalline state [55].
Antimicrobial assay
Cultures of the E. coli strain W3110, which had been
freeze-dried in 20% (v ⁄ v) glycerol and stored at )80 °C,
were inoculated into 10 mL nutrient broth. After overnight
incubation in an orbital shaker (100 r.p.m., 37 °C), 100-lL
aliquots of these cultures were used to inoculate 100 mL
nutrient broth in 250 mL flasks, which were then incubated
with shaking (100 r.p.m., 37 °C) until growth in the mid-
exponential phase was reached (A ¼ 0.6; k ¼ 600 nm).
Aliquots (1 mL) of bacterial samples were centrifuged,
using a bench top centrifuge (15 000 g, 3 min, 22 °C), and
the centrifuged cells washed three times in 1-mL aliquots of
Tris ⁄ HCl buffer (10 mm, pH 7.5). These cells were then
suspended in 1 mL of this buffer containing AP1 at a final
concentration of 3 mm, which corresponds to its minimum
inhibitory concentration. These culture ⁄ peptide mixtures

were incubated at 37 °C, and samples taken at the begin-
ning of the experiment (time zero), and at 15 minute inter-
vals for 1 h and then hourly intervals for 7 h. At each time
interval, samples were surface-spread on to nutrient agar
plates, which were incubated at 37 °C for 12 h. As a
S. R. Dennison et al. Antimicrobial properties of an anionic a-helical peptide
FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS 3799
control, bacterial cultures were similarly treated but in the
absence of peptide. Colony counts were expressed as colony
forming units (CFU) ml
)1
. The percentage reduction in col-
ony counts for each time interval was then calculated, and
the results were presented graphically against time.
Monolayer technique
All experiments were conducted at 21.0 ± 1 °C using a
Langmuir trough measuring 5 · 16 cm, which was fitted
with two moveable barriers and was supplied by NIMA
Technology (Coventry, UK). Unless otherwise stated,
monolayer studies were performed using a Tris ⁄ HCl buffer
subphase (10 mm, pH 7.5), which was continuously stirred
by a magnetic bar (5 r.p.m.). Surface tension was monit-
ored by the Wilhelmy method using a Whatman’s (Ch1)
paper plate in conjunction with a microbalance, as des-
cribed by Brandenburg et al. [56]. Changes in monolayer
surface pressure ⁄ area were recorded as graphic output on a
PC using nima software version 5.16, which interfaces with
the Langmuir-Blodgett microbalance.
Peptide surface activity
The barriers of the Langmuir-Blodgett trough were adjus-

ted to their maximum separation (surface area 80 cm
2
), and
this position maintained. AP1 was then injected into the
buffer subphase to give final concentrations of 1–30 lm,
and, at each peptide concentration, changes in surface pres-
sure at the air ⁄ water interface were monitored for 1 h. The
maximal values of these surface pressure changes were then
plotted as a function of the peptide’s final subphase concen-
tration (Fig. 3). From these results, the surface excess, G,
was calculated by means of the Gibbs’ adsorption isotherm,
which is given by Eqn (1) [57]:
C ¼À
1
RT
Â
Dp
D ln C
ð1Þ
where R is 8.314 JÆmol
)1
ÆK
)1
, T ¼ 294 K, p is the interfa-
cial pressure increase (mNÆm
)1
), and c is the molar concen-
tration of peptide in the subphase. These values of G were
then used to determine values of the interfacial surface area
per AP1 molecule (A) according to Eqn (2):

A ¼
1
NC
ð2Þ
where N is Avogadro’s number (Table 1).
The ability of AP1 to spread on an aqueous surface and
to form a stable monolayer was investigated. The barriers
of the Langmuir-Blodgett trough were adjusted to their
maximum separation (surface area 80 cm
2
) and this posi-
tion maintained. A 10-lL aliquot of AP1 in chloroform
(1 mm) was spread on to a buffer subphase and allowed to
equilibrate for 1 h. The resulting peptide monolayer was
compressed using the moveable barriers of the trough to
produce a pressure ⁄ area isotherm, which was converted by
nima software in to an output plot of surface pressure vs.
monolayer surface area per AP1 molecule (Fig. 4).
Peptide interactions with lipid monolayers
The ability of AP1 to penetrate lipid monolayers at con-
stant area was studied. Monolayers were formed by spread-
ing on to a buffer subphase, chloroform solutions of
Ole
2
PtdGro, Ole
2
PtdEtn and cardiolipin in molar propor-
tions of 1 : 13.67 : 2 [53]. The solvent was allowed to evap-
orate off over 30 min and then the monolayer compressed
at a velocity of 5 cm

2
Æmin
)1
to give a surface pressure of
30 mNÆm
)1
. The barriers were maintained in this position,
and peptide was then injected into the subphase to achieve
the desired optimum peptide concentration of 20 lm which
was determined by analysis of surface activity data des-
cribed in Fig. 3. This subphase concentration of AP1 gave
rise to a lipid to peptide ratio of approximately 100 : 1,
which was used in all other monolayer studies. Interactions
of the peptide with lipid monolayers were monitored as
changes in monolayer surface pressure vs. time.
The ability of the peptide to interact with lipid monolay-
ers was also investigated using compression isotherms.
Monolayers were formed by spreading on to a buffer sub-
phase chloroform solutions of either Ole
2
PtdEtn, Ole
2
Ptd-
Gro, cardiolipin, or these lipids in molar proportions of
1 : 13.67 : 2 [53]. The solvent was allowed to evaporate off
over 30 min, and monolayers then compressed using a bar-
rier speed of 5 cmÆmin
)1
either with AP1 absent from the
subphase or included in the subphase at a final peptide con-

centration of 20 l m. Changes in monolayer surface pressure
vs. changes in area per lipid molecule of the monolayer
were monitored and recorded.
Thermodynamic analysis of compression
isotherm data
Thermodynamic analysis of compression isotherms was
used to investigate the molecular interactions and dynamic
behaviour of monolayers. The compressibility modulus,
C
À1
s
, provides a measure of the compressional elasticity of
a monolayer and can be used to characterize the phase state
of the isotherm, thereby providing information about the
compactness and packing of the model membrane [35]. Val-
ues of C
À1
s
(Table 1) were computed according to Eqn (3):
C
À1
s
¼ÀA
dp
dA

ð3Þ
where p is the surface pressure of the monolayer, and A
represents the area per molecule in the monolayer.
The Gibbs free energy of mixing (DG

Mix
) quantifies the sta-
bility of monolayer mixtures, thereby providing information
on interactions between the components of the monolayers.
Values of DG
Mix
were computed according to Eqn (4):
Antimicrobial properties of an anionic a-helical peptide S. R. Dennison et al.
3800 FEBS Journal 273 (2006) 3792–3803 ª 2006 The Authors Journal compilation ª 2006 FEBS
DG
Mix
¼
Z
p
0
½A
1;2 n
Àðx
1
A
1
þ x
2
A
2
þ x
n
A
n
Þdp ð4Þ

where A
1,2, . n
is the molecular area occupied by the mixed
monolayer, A
1
,A
2
A
n
are the area per molecule in the
pure monolayers of component 1, 2, n, x
1
, x
2
x
n
are the
molar fractions of the components and p is the surface
pressure. These data were then recorded as the variation of
DG
Mix
with monolayer surface pressure (Table 3). Numer-
ical data were calculated from compression isotherms using
the methodology of Simpson [58].
The interaction parameter (a) relates the interaction of
each molar fraction of components within a monolayer
with the free energy of mixing. Values of a were computed
(Table 3) according to Eqn (5):
a ¼
DG

Mix
RTðX
n
1
X
2
X
n
þ X
1
X
n
2
X
n
þ X
1
X
2
X
n
n
ð5Þ
where X are the molar fractions of the components, R is
8.314 JÆmol
)1
ÆK
)1
, and T is 294 K. These data were then
used to compute values of monolayer mixing enthalpy (DH)

(Table 3) according to Eqn (6):
DH ¼
RTa
Z
ð6Þ
where Z is the packing fraction parameter and calculated
using the Quikenden and Tam model [59].
Haemolytic assay of AP1
Haemolytic assay was conducted as described by Harris &
Phoenix [60]. Essentially, packed red blood cells were
washed three times in Tris-buffered sucrose (0.25 m sucrose,
10 mm Tris ⁄ HCl, pH 7.5) and resuspended in the same
medium to give an initial blood cell concentration of
% 0.05% (w ⁄ v). For haemolytic assay, this concentration
was adjusted such that incubation with 0.1% (v ⁄ v) Triton
X-100 for 30 min produced a supernatant with A
416
of 1.0,
which was taken as 100% haemolysis. Aliquots (1 mL) of
blood cells at assay concentration were then used to solubi-
lize various amounts of stock AP1 solution, which had been
added to a test-tube and dried under nitrogen gas. The
resulting mixtures were incubated at room temperature with
gentle shaking. After 30 min, the suspensions were centri-
fuged at low speed (1500 g, 15 min, 25 °C), and the A
416
of
the supernatants determined. In all cases, levels of haemoly-
sis were determined as the percentage haemolysis relative to
that of Triton X-100 and the results recorded. Background

haemolysis was less than 1% in all cases.
Acknowledgements
We thank Jo
¨
rg Howe, Division of Biophysics, Fors-
chunginstitute, Borstel, Germany for his assistance
with FTIR analysis. We would also like to thank Dr
Frank Grunfeld (NIMA Technology, UK) for his tech-
nical advice with the monolayer studies.
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