Direct visualization of the alamethicin pore formed
in a planar phospholipid matrix
Piotr Pietaa,b, Jeff Mirzaa, and Jacek Lipkowskia,1
a
Department of Chemistry, University of Guelph, Guelph, ON, Canada N1G 2W1; and bDepartment of Physical Chemistry of Supramolecular Complexes,
Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland
Edited by Royce W. Murray, University of North Carolina, Chapel Hill, NC, and approved November 7, 2012 (received for review September 9, 2012)
We present direct visualization of pores formed by alamethicin
(Alm) in a matrix of phospholipids using electrochemical scanning
tunneling microscopy (EC-STM). High-resolution EC-STM images
show individual peptide molecules forming channels. The channels
are not dispersed randomly in the monolayer but agglomerate
forming 2D nanocrystals with a hexagonal lattice in which the
average channel–channel distance is 1.90 ± 0.1 nm. The STM
images suggest that each Alm is shared between the two adjacent
channels. Every channel consists of six Alm molecules. Three or
four of these molecules have the hydrophilic group oriented to-
ward the center of the channel allowing for water column forma-
tion inside the channel. The dimensions of the central pore in the
images are consistent with the dimension of the water column in
a model of hexameric pore proposed in the literature. The images
obtained in this work validate the barrel-stave model of the
pore formed in phospholipid membranes by amphiphatic pep-
tides. They also provide direct evidence for cluster formation by
such pores.
| interface | Langmuir-Blodgett
antimicrobial
A ntimicrobial peptides (AMPs), including alamethicin (Alm),
are small (6–100 amino acids) molecules produced by many
living organisms (1, 2). AMPs are biocidally active against Gram-
negative and Gram-positive bacteria, fungi, enveloped viruses,
eukaryotic parasites, and even tumor cells (3). Importantly,
AMPs are effective against strains of antibiotic-resistant bacteria
(2). Their membranolytic activity involves nonspecific pore for-
mation mechanisms. Several models of the pores formed by
AMPS such as barrel-stave, torroidal or worm-like pore and
carpet models have been proposed (4). However, the mechanism
of membrane disruption by AMPs is still not quite understood.
Alm is a 20-residue peptide isolated from the Trichoderma
viride fungus (4). It has been thoroughly studied as a model of
channel formation in biological membranes (5), due to its activity
against Gram-positive bacteria and fungi (4, 6). Alm has a linear
sequence of residues with the C-terminal being phenylalaninol
(Pheol) and the N terminus being acetylated (6). Structural
analysis of Alm revealed by X-ray crystallography (7) indicates
that the molecule is predominantly α-helical. In the helical
conformation, the length of the molecule is ∼3.2 nm. The pres-
ence of α-helical conformation of Alm in organic solutions and in
membrane environments was observed by NMR (8), circular
dichroism (CD) (9), Raman (9), and FTIR (10) measurements.
In bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)
Alm is helical from residues 1–12 in the liquid crystalline state,
whereas in the gel state, the helix extends from residues 1–16 (9).
Alm helix is amphiphatic with one face more hydrophilic than
the other (11). The amphiphatic properties influence the in-
teraction of the helix in phospholipid bilayers. Depending on
experimental conditions, Alm can be oriented with its helical axis
parallel to the plane of the bilayer (surface or S state) or can be
inserted into the lipid bilayer with the helical axis pointing in the
vertical direction (inserted or I state) (12). The S state is observed
at low peptide to lipid ratios (P/L), whereas insertion of Alm into
the membrane takes place at higher peptide concentration, above
www.pnas.org/cgi/doi/10.1073/pnas.1201559110
a certain critical value (P/L*) (13), which depends on tem-
perature and on the lipid nature (11, 13). For most lipids, at
a concentration of P/L = 1:15 or higher, all peptides are in the
I state (14).
The dipole moment of Alm was determined to be 60–79 D,
corresponding to a net +1/2 charge at the N- and a −1/2 charge
(elementary charge units) at the C terminus of the helix (15). Due
to the large dipole moment, the insertion of Alm into the mem-
brane can be controlled by the transmembrane potential (16, 17).
Upon insertion, Alm molecules aggregate to form voltage-gated
channels described by the barrel-stave model (18). In that model,
Alm forms a cylindrical array of parallel helices with the hy-
drophilic sides of the helices oriented to the interior, creating a
CHEMISTRY
water-filled and ion-conducting central lumen, whereas the hy-
drophobic sides of helices are oriented toward the hydrophobic
portion of the membrane (6, 7).
The presence of multiple conductance levels observed for Alm
in bilayer lipid membranes (BLMs) suggests formation of pores
with various aggregation numbers of Alm molecules (11). The
COMPUTATIONAL BIOLOGY
number of Alm monomers per channel was estimated to vary
BIOPHYSICS AND
between 6 and 12 (7, 19). The pore size varies with hydration and
with lipid composition (12). For example, in bilayer of 1,2-
dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) the pores
are made of n = 8–9 monomers, with the inner and outer pore
radii of ∼0.9 and 2.0 nm, whereas in bilayer of diphytanoyl phos-
phatidylcholine (DPhPC) the pores are made of n = 11 monomers,
with the inner and outer pore radii of ∼1.3 and 2.5 nm, respectively
(12). For 1,2-dioleoyl-sn-glycero-phosphatidylcholine (DOPC) and
1,2-dierucoyl-sn-glycero-phosphatidylcholine (diC22:1PC) bilayers
was reported to be equal to 5 and 9 with the outer pores radii
1.36 and 1.96 nm, respectively (20). The aggregation of Alm is
affected by the phase transition of the lipid (21). In plane neutron
and X-ray scattering experiments performed on multiple bilayers
indicate that at temperatures below the phase transition and at
low water content, lateral distribution of Alm pores is correlated
and the pores are arranged in a 2D crystalline superstructure
(21–23). The immiscibility of Alm with phospholipids was observed
in monolayers at the air/water interface, indicating a tendency
of the peptide toward 2D crystallization (3). The segregation of a
mixture of hydrophobic helical peptides and phospholipid mol-
ecules into separate peptide and lipid phases was also predicted
by theoretical calculations (24). The elucidation of the lateral
distribution of Alm in the plane of the phospholipid membrane is
still under debate and it is essential to understand the mechanisms
of its action (22).
There is a wealth of information about the behavior of Alm
in bilayers composed of lipids with no net charge (e.g., typical of
mammalian cells). However, the bactericidal action of Alm can
be better understood by studying bilayers composed of negatively
Author contributions: J.L. designed research; P.P. and J.M. performed research; P.P. and
J.L. analyzed data; and P.P. and J.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: jlipkows@uoguelph.ca.
PNAS | December 26, 2012 | vol. 109 | no. 52 | 21223–21227
charged lipids such as those found in bacteria. A model mem-
brane composed of neutral DMPC and negatively charged
phosphatidylglycerol (PG) lipids was used to replicate the lipid
membrane of Gram-positive bacteria (25). PG lipids are a major
membrane component (up to 58%) in Gram-positive bacteria
(26). Negatively charged heads of PG give rise to an electrostatic
surface potential that promotes the insertion of Alm into the
membrane with the N terminus embedded into the bilayer and
the C terminus directed out of the membrane (21).
The objective of this work is to provide molecular resolution
images of the Alm pore in a phospholipid matrix using the
electrochemical scanning tunneling microscopy (STM). Re-
cently, we obtained molecular resolution images of a gramicidin,
a hydrophobic channel forming peptide, dispersed in a mono-
layer of DMPC (27). The goal of the present study was to use
similar methodology to provide complementary information
about the structure of the pore formed by the amphiphatic
peptide such as alamethicin. The Alm molecules were in-
corporated into the 1:1 mixture of DMPC and egg-PG (egPG).
The choice of egg-PG was serendipitous. However, it gave good
results. The negative charge of egg-PG molecules helped to in-
sert Alm with N terminus facing the gold surface and C terminus
oriented toward the solution. We succeeded in obtaining first
molecular resolution images of Alm aggregates in the DMPC/
egg-PG matrix. The images revealed that aggregates of Alm form
2D nanocrystals of variable size. The size of one pore in the
nanocrystal is in reasonable agreement with models of hexameric
pore proposed in the literature. The presence of a variable
number of pores in a nanocrystal correlates well with the ob-
servation of multiple conduction states for Alm incorporated
into a BLM. At present, high resolution STM images of a film of
phospholipid in solution can be acquired for a monolayer only.
However, molecular resolution STM images of a phospholipid
bilayer supported at a conductive surface in air have already
been reported (28–30). With further methodological improve-
ments, imaging of the supported bilayer in a solution should also
be possible in the future. This study illustrates how significant
information for the understanding of peptide aggregation in lipid
matrixes the STM experiments may provide.
Results
Before the transfer of the monolayer onto the gold electrode
surface its properties were characterized at the air–solution in-
terface. Fig. 1A shows compression isotherms recorded at the
air–solution interface of the Langmuir trough. Curve 1 plots the
isotherm of the mixed monolayer containing Alm and DMPC/
egPG at 1:15 mol ratio. For comparison, curves 2 and 3 plot the
Fig. 1. (A) Surface pressure/area isotherm of Alm/(DMPC/ePG) (1:15) (curve 1), DMPC/ePG (1:1) (curve 2), and Alm (curve 3), spread on aqueous subphase at
15 °C. Dotted line indicates the value of surface pressure at which monolayers were transferred onto the Au bead. (B) Molecular area vs. surface pressure
curve obtained by subtracting the isotherm of the DMPC/egPG mixture (curve 2 in A) from the isotherm of the Alm/DMPG/egPG mixture (curve 1 in A).
21224 | www.pnas.org/cgi/doi/10.1073/pnas.1201559110
compression isotherms for the DMPC/egPG (1:1) mixture and
the pure Alm, respectively. Curve 1 is shifted toward higher
values of molecular areas in comparison with curve 2, for the
DMPC/egPG (1:1) mixture without Alm. It displays a step
at ∼31 mN·m−1 whose position coincides with the collapse
pressure observed in the isotherm of the pure Alm. Such be-
havior suggests that Alm is not miscible with the mixed lipids.
For immiscible monolayer of noninteracting molecules, the
mean molecular area of the three-component mixture should
be equal to:
A1 = ð1 − xÞ A2 + x A3 ; [1]
where A1 and A2 are the mean molecular areas of the compres-
sion isotherms 1 and 2, A3 is the mean molecular area of the pure
Alm monolayer, and x is the mole fraction of Alm in the three-
component mixture. Fig. 1B compares the area per Alm mole-
cule in the three-component monolayer, calculated as:
½A1 − ð1 − xÞ A2 
A3 = ; [2]
x
to the area per molecule in the pure Alm monolayer. The
differences between the two curves are small but measurable.
The areas per Alm molecule in the mixture are about 15%
smaller than in the pure Alm monolayer, suggesting that
molecules in the three-component mixture are slightly com-
pressed. However, in the range of surface pressures between ∼5
and ∼30 mN·m−1 there is no significant change in the area per
molecule whose value is ∼3 nm2/molecule. This value is in good
agreement with the area for Alm molecule oriented with its
helical axis parallel to the interface (S state). These results are
consistent with the literature which reported that Alm is immis-
cible with phospholipids in monolayers spread at the air/water
interface and the Alm molecules are oriented with axis of the
helix parallel to the interface (3, 31, 32).
The mixed monolayer of Alm/(DMPC/egPG) (P/L = 1:15) was
transferred onto the Au(111) electrode surface at surface pres-
sure 27 mN·m−1 (below the Alm collapse pressure), using the
Langmuir–Blodgett technique. The data reported in Fig. 1 in-
dicate that this monolayer consisted of phase segregated phos-
pholipids and Alm molecules that were oriented with helical axis
in plane of the monolayer. This monolayer was dried at ∼17 °C
overnight. Next the gold electrode covered by the monolayer was
assembled into the electrolyte-filled cell of the electrochemical
STM and the monolayer-covered surface of gold was imaged in
solution. Fig. 2 A and B shows STM images of this monolayer.
Pieta et al.

Fig. 2. Electrochemical STM (EC-STM) images of a monolayer of Alm and
DMPC/egPG (1:15 molar ratio) deposited onto a Au(111) surface. Images
were acquired using tunneling currents of (A) 1.16 and (B) 0.78 nA.
The ability to image an insulating molecule by STM may be
explained in terms of a weak coupling between electronic states
in the adsorbate and in the substrate near the Fermi level that
gives the adsorbate a property of an antenna capable of receiving
tunneling electrons (33, 34). The electrolyte screens the negative
charge on PG lipids and assists in ordering of the monolayer.
Water molecules may play a role in tunneling through bio-
molecules. However, the role of solvent in STM imaging in so-
lution is still poorly understood (33). Two significantly different
structures can clearly be distinguished in these images. The first
consists of elliptical spots arranged into a flower-like pattern and
the second structure consists of long parallel stripes. In Fig. 2A
the flower-like aggregates are imbedded into the ordered film
consisting of long stripes. Fig. 2B shows that the aggregates can
also be observed in disordered film, indicating that formation of
these aggregates is not induced by the presence of the ordered
long stripe domains. The presence of two ordered structures
within the same monolayer indicates that the phase separation
occurs in the film.
To identify the nature of the two phases, higher resolution
images of each phase were acquired. Fig. 3A shows a domain
where only parallel stripes are seen. The average distance be-
tween the two adjacent spots within the stripe is 0.45 ± 0.1 nm
(Fig. 3B) and the mean area of the spot is 0.23 ± 0.1 nm2. These
dimensions are consistent with the average distance between two
lipid chains and cross-sectional area of a lipid chain in closely
Fig. 3. (A) EC-STM images of DMPC/egPG monolayers (1:1 molar ratio) deposited onto a Au(111) surface. The image was acquired using a constant tunneling
current of 1.16 nA. (B) Cross-sectional profile of the lipid stripes. The profile was taken along the black line shown on the STM image.
Pieta et al.
packed monolayers of phospholipids determined by STM and X-
ray diffraction (27, 35, 36). They suggest that the contrast in Fig. 3A
is formed by lipid molecules oriented with polar groups facing the
gold surface and the acyl chains directed to the electrolyte (35, 36).
Therefore, the stripes seen in Figs. 2A and 3A show rows of upright
oriented lipid molecules and each spot in the stripes corresponds
to the top view of a single acyl chain of a lipid molecule.
Fig. 4A shows a high resolution image of the domain with the
flower-like structure. The contrast is formed by pairs of spots
that are arranged into a hexagonal lattice whose unit cell has two
vectors equal to 1.90 ± 0.1 nm and the angle between these
vectors equal to 60°. The average area of the cell is equal to
3.13 ± 0.3 nm2. Each cell of this lattice contains three pairs of
spots with the pair–pair distance 0.8 ± 0.2 nm. The mean area of
the pair of spots is 0.6 ± 0.2 nm2. This is in agreement with the
effective cross-section area of the Alm molecule in I state (7, 22).
Fig. 4B is an image of the structure in which the shaded area
illustrates the hexagonal nature of the 2D lattice. A group of
flower-like six split spots, enclosed in the shaded area shown
in Fig. 4B, resembles a barrel-stave model of Alm channel pro-
posed in the literature (7). The average radius of the circle
shown in Fig. 4B is equal to 1.0 ± 0.2 nm. This number corre-
sponds to the sum of the radius of the central pore plus half of
the dimension of the split spot (each split spot is shared by two
CHEMISTRY
neighboring circles). In the literature, the channels are modeled
as a column of water surrounded by cylinders of Alm molecules.
For the model of six Alm molecules the radius of the water
column is equal to 0.55 nm, whereas the effective radius of the
individual cylinders of Alm molecules is equal to 0.5 nm (22).
This is in good agreement with the dimension of the radius of the
COMPUTATIONAL BIOLOGY
circle shown in Fig. 4B. We can therefore safely assume that the
BIOPHYSICS AND
flower-like domains are formed by Alm molecules oriented
perpendicular to the surface of the monolayer. This is a different
orientation than in the monolayer at the air–solution interface.
The film that was transferred from the air–solution interface
onto the gold electrode surface changed its structure upon drying
overnight and subsequent rehydration after immersion into the
solution. The STM image suggests that each Alm molecule is
shared between the two adjacent channels. Such an arrangement
is unusual in the Alm literature. All models describing Alm
channels in lipid bilayers suggest formation of individual bundles
of Alm arranged into the barrel-stave channel (22). In the pore
models, Alm molecules are arranged such that the interior of the
channel is hydrophilic, whereas the exterior is hydrophobic, a
PNAS | December 26, 2012 | vol. 109 | no. 52 | 21225
Fig. 4. (A) EC-STM image of the flower-like structures on a Au(111) surface. The image was acquired using a constant tunneling current of 1.16 nA. The
hexagonal lattice of the channels defined by two base vectors of length a = 1.90 ± 0.1 nm with an angle γ = 60 ± 5° between these vectors is superimposed on
the image. (B) Schematic arrangement of channels formed by Alm molecules.
result of the amphiphatic character of the peptide. In the wheel
model of the Alm helix (20), the polar groups occupy ∼40% of
the circumference of the Alm molecules. These polar groups
(the Gln7 and Glu18 side chains and the carbonyl oxygen atoms
of Aib10 and Gly11) are located along a strip parallel to the Alm
axis, whereas Glu19 is rotated by about 98° with respect to the
positions of Gln7 (11, 20). The Gly11 and Glu18 are rotated
about 33° and 44°, respectively, with respect to the position of
Gln7. The models assume that hydration and formation of hy-
drogen bonded network between the Gln7 residues play a pivotal
role in the channel formation (7, 11). This information was used
to build the model that explains the contrast of Alm domains in
STM images shown in Fig. 5A. In Fig. 5B the same structure is
shown without the background of the STM image. In this model,
hydrophilic groups of Alm molecules are directed toward the
center of the channels and Gln7 residues form a hydrogen-
bonded annulus. In this arrangement, the interior of the channels
is hydrophilic, whereas the Alm molecules from neighboring cells
of the 2D lattice are oriented to each other with the hydrophobic
groups. The hydrogen-bonded annulus formed by Gln7 residues
determines the narrower fragment of the channel and controls
the conductive behavior of the Alm channels. In a hexameric
bundle, the number of water molecules hydrogen bonded to six
Gln7 side chains is between 20 and 25, giving the minimum ra-
dius of water column inside the channel 0.20–0.25 nm (37, 38). In
our model each channel consists of six Alm molecules. However,
only three or four Gln7 residues are oriented toward the channel
Fig. 5. Proposed structural model for the Alm molecules assembly (A)
superimposed on the STM image and (B) without the STM image; image
dimensions 5.5 × 5.5 nm.
21226 | www.pnas.org/cgi/doi/10.1073/pnas.1201559110
center. Consequently, two different effective radii of the chan-
nels are observed ∼0.25 and ∼0.18 nm for the four and the three
Gln7 residues, respectively (Fig. 5B). The radius of the channel
formed by four Gln7 residues is in good agreement with the min-
imum radius of the water column predicted for the hexameric
bundle by molecular dynamics (MD) calculations (37).
Conclusions
We have provided unique direct images of Alm aggregates in
a Alm/DMPC/egg-PG matrix. The Alm molecules formed well-
ordered 2D nanocrystals surrounded by the lipids. The phos-
pholipids assist insertion of Alm molecules; however, they do not
template the nanocrystals’ formation. The nanocrystal size dis-
tribution is quite broad. The Alm molecules are arranged into
a flower-like pattern with hexagonal lattice. Each flower-like unit
has a central pore surrounded by six Alm molecules. This unit
resembles the barrel-stave model of a hexameric pore formed by
Alm in phospholipid bilayers. The radius of the central pore is
comparable to the minimum radius of a column of water in the
barrel-stave model of Alm aggregates predicted by MD calcu-
lations. The present data provide a unique direct visualization of
the barrel-stave aggregation of an amphiphatic peptide. We have
also provided direct evidence that individual channels may form
large clusters (nanocrystals). Cluster formation has been sug-
gested in the literature. However, it has not been proven. The
variable number of pores in the nanocrystals may explain mul-
tiple conduction states for Alm incorporated into a BLM. These
results constitute significant contribution to the understanding of
the pore formation by amphiphatic peptides.
Experimental Methods
The working electrode was a small Au bead formed by melting a gold wire
(Alfa Aesar, 99.999% purity). The bead was welded to a gold plate. The
atomically flat (111) facets at the bead surface were used for image acqui-
sition. Before each experiment, the gold electrode was cleaned in hot (80 °C)
piranha solution (concentrated H2SO4/30% H2O2 3:1 vol/vol) for 30 min and
rinsed thoroughly with Milli-Q utrapure water. (CAUTION: piranha solution
reacts violently with organic materials and should be handled with extreme
care). The gold electrode was then flame annealed and quenched in Milli-Q
water before the experiment. The Kel-F parts of the STM electrochemical cell
were cleaned in cool (20 °C) piranha solution. The STM images were acquired
using a Nanoscope II EC-STM connected to a Nanoscope IIIa controller (Digital
Instruments) with an A scanner. The constant current mode was used for
imaging. The tungsten STM tips were electrochemically etched in 2 M NaOH
and then coated with polyethylene to minimize the faradaic currents. The
STM experiments were carried out at 18 ± 1 °C. For all experiments 0.1 M NaF
(MV Laboratories) was used as the supporting electrolyte. Milli-Q ultrapure
Pieta et al.
water (final resistivity ≥18.2 MΩ cm) was used to prepare all solutions. During
the image acquisition, the electrode was kept at an open circuit potential
(OCP) of +0.2 V vs. Ag/AgCl with a bias voltage of −0.35 V.
DMPC, egPG, both from Avanti, and alamethicin, from Sigma-Aldrich,
were used without further purification to make 10 and 4 mg·mL−1 stock
solutions, respectively, in chloroform:trifluoroethanol (Sigma-Aldrich) (1:1,
vol/vol) mixed solvent. DMPC, egPG, and Alm solutions were added to a test
tube to obtain a 6.25% molar ratio of Alm with respect to the lipids. The
tube was then heated to about 40 °C for 1 h. During this time the solution
was mixed in a vortex (Fisher; Vortex Genie 2) and the solvent was evapo-
rated under an argon stream. A thin film of the lipid–peptide mixture
remained on the wall of the test tube. Further drying was achieved by
storing the test tube under vacuum for at least 12 h at room temperature.
The dry film in the test tube was dissolved in chloroform (Sigma-Aldrich) to
give the final concentration Alm 0.153 mM, DMPC 1.145 mM, and eggPG 1.145
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Pieta et al.
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at a surface pressure of 27 mN/m by the Langmuir–Blodgett technique. During
the compression the temperature of the aqueous subphase was kept at 15 °C.
After deposition, each sample was dried overnight at temperature ∼17 °C
and then transferred into the STM electrochemical cell.
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