Article

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Amphipathic Membrane-Active Peptides Recognize and Stabilize

Ruptured Membrane Pores: Exploring Cause and Effect with Coarse-
Grained Simulations
Delin Sun,† Jan Forsman,‡ and Clifford E. Woodward*,†
†
School of Physical, Environmental and Mathematical Sciences, University of New South Wales, Canberra ACT 2600, Australia
‡
Theoretical Chemistry, Chemical Centre, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
*
S Supporting Information
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ABSTRACT: Induction of membrane pores has been suggested as

the common molecular action by which a variety of amphipathic
membrane-active peptides cause damage to cells. In this study, we
have performed coarse-grained molecular dynamics simulations to
establish two clear molecular processes that seem critical for the
activity of amphipathic peptides. They are (i) the recognition and (ii)
the stabilization of ruptured membrane pores. By considering 12
structurally different peptide types, we reveal that peptide secondary
structure content, hydrophobicity, and length are important
physicochemical factors that allow amphipathic peptides to aggregate
in and stabilize ruptured membrane pores. The simulated inner
diameters of peptide-stabilized membrane pores are in good
agreement with available experimental data. However, the orienta-
tions of α-helical peptides in the membrane pore were found to be quite dispersed. This supports recent challenges to the
traditional depictions to peptide orientations in the classical toroidal and barrel-stave pore models.

1. INTRODUCTION lipid vesicles loaded with fluorescent dye in order to establish

The damage incurred by cell membranes through induction of
nanoscale pores has been suggested as a common molecular
action for a wide variety of amphipathic membrane-active
peptides, including cell-penetrating peptides,1,2 antimicrobial
peptides,3,4 amyloidal peptides,5,6 and Baxα5 mitochondria-
apoptotic peptide.7,8 However, the mechanisms by which
amphipathic peptides induce membrane pores are still being
debated. Experimental studies using model lipid membranes
have provided valuable evidence that membrane-active peptides
generate membrane pores via a cooperative kinetic proc-
ess.1,2,4−7,9−13 Huang and co-workers, using X-ray diffraction
and oriented circular dichroism (OCD) techniques, established
that a minimum concentration of amphipathic peptides was
required for membrane pore formation, suggesting a cooper-
ative mechanism for these peptides.4,12 According to the “two-
state” model proposed by Huang et al., amphipathic peptides
first accumulate on the outer leaflet of the membrane with an
orientation parallel to the membrane surface (the S-state). The
asymmetric distribution of peptides on the lipid membrane
causes surface area expansion coupled with an internal
membrane tension. Above a threshold concentration of
peptides, the lipid membrane ruptures to form a pore.
Concomitantly, a fraction of membrane-bound peptides alter
their orientations to become perpendicular (the I-state) as they
presumably insert themselves into the membrane pore. Tamba
et al.9 and Fuertes et al.11 performed kinetic experiments with
the dynamic character of membrane pores induced by
magainin-2 (an antimicrobial peptide) and Baxα5 (a
mitochondria-apoptotic peptide). It was found that the
ruptured membrane pores were unstable, with initial diameters
of some tens of nanometers. However, within minutes the pore
diameters decreased to a smaller equilibrium value. This
phenomenon is consistent with the findings of Brochard-Wyart
and co-workers, who found that application of mechanical
tension could rupture a giant lipid vesicle to form a large
transient pore of micrometer size, which closes within seconds,
once the stress is removed.14 These experimental studies
suggest that amphipathic peptides induce and then stabilize
membrane pores, thus allowing persistent leakage of internal-
ized contents, such as fluorescent dyes.15 Several amphipathic
peptides, like magainin-2 and Baxα5, were found to exhibit so-
called “all-or-none” release kinetics,10,11 whereby vesicles either
remain intact or else completely release their contents. Other
pore-forming peptides which show this type of release kinetics
are the GALA peptide,16 cecropin A,13 human cathelicidin (LL-
37),17 human defensins,18 pardaxin,19 α-hemolysin,20 human
islet amyloid polypeptide (IAPP),6 α-synuclein,21 and cyto-
chrome c.22 The ability to significantly stabilize and maintain
Received: September 26, 2014
Revised: November 27, 2014
Published: December 9, 2014

© 2014 American Chemical Society 752 DOI: 10.1021/la5038266

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membrane pores would appear to be a general requirement for
the activity of these pore-forming peptides. Notwithstanding
the significant progress achieved by experimental studies, the
process by which these types of amphipathic peptides carry out
this action remains unclear.
Molecular simulations have increasingly been used to study
interactions of membrane-active peptides with lipid bilayers.
Indeed, some of that work has reported that high
concentrations of antimicrobial peptides are able to generate
membrane pores in lipid bilayers within a few microseconds or
only a few hundred nanoseconds.23−26 However, these
simulated pores have generally displayed very small internal
diameters (∼1−2 nm) which do not appear to correspond to
the large equilibrium membrane pores (diameter ∼ 3−5 nm)
reported in experiments.9,12 Furthermore, kinetic experiments
with fluorescent dye-loaded lipid vesicles have consistently
reported that amphipathic peptides trigger leakage of
fluorescent dyes on the time scale of seconds or even
minutes,9,10 which is several orders of magnitude slower than
the rate of pore formation observed in the aforementioned
simulation studies.23−26 This then raises doubt as to whether
the simulated mechanisms accurately represent the actual
behavior of these peptides. On the other hand, rapidly formed
small pores may play the role of nucleation sites for the sudden
rupture of the lipid membrane over a longer time scale.27
Simulations by Leontiadou et al. showed that the presence of a
small pore in a lipid bilayer could dramatically reduce the
threshold surface tension required to rupture the membrane.28
Furthermore, experiments by Lee et al. support the role of
precursor membrane defects for inducing stable membrane
pores.4 Those authors also reported that peptide translocation
could occur (presumably via transient pores) before a large and
stable membrane pore was created.12
The mechanisms by which amphipathic membrane-active
peptides induce membrane rupture, and the potential role
played by small transient pores, remain to be established. In the
simulation work reported here we do not attempt to answer
this question. Instead, we will focus on the later stages of
membrane rupture leading up to the formation of stable pores,
as observed in many experiments, e.g., Tamba et al. and Fuertes
et al.9,11 That is, we assume a mechanism whereby amphipathic
peptides rupture the membrane, presumably through surface
tension (though we do not discount other mechanisms) to
form a large unstable pore, which is then rapidly stabilized. We
focus on the role that amphipathic peptides play in the
transition of the large ruptured membrane pore to the smaller,
stable equilibrium pore. Our aim is to elucidate the key
molecular determinants that significantly affect the peptide’s
efficacy in this pore transition process.
2. SIMULATION METHODS
2.1. Model and Pore Creation. We performed molecular
dynamics (MD) simulations using the nonpolarizable coarse-
grained MARTINI force field developed by Marrink et al.29,30
Our purpose was to investigate amphipathic peptides in the
presence of a ruptured membrane pore. While there is evidence
that the MARTINI force field is inadequate for the simulation
of membrane defect (small pore) formation per se,31 we use it
here to model the response of amphipathic peptides to a
membrane pore which has already been formed. Thus, our
study relies on the MARTINI model’s ability to adequately
describe peptide interactions with the lipid bilayer. In this
regard, it is worth noting that the nonpolarizable MARTINI
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model predicts potentials of mean force for amino acid
translocation through zwitterionic bilayers,32 which are in
reasonable agreement with full atomistic simulations results33 as
well as the Wimley−White interfacial scale.34
In experiments, large membrane pores are purported to open
due to the surface tension caused by the asymmetric adsorption
of peptides. Such pores are not likely to be observed in the
relatively short time span of our simulations (microseconds).
To circumvent this problem, we created an initial pore in the
membrane using artificial means. In lipid vesicle experiments,
membrane rupturing is usually obtained through application of
a mechanical stress.9 Simulations by Tieleman et al. showed
that an electric field and mechanical stress are both able to
rupture a lipid bilayer.35 In our study, a pore was produced by
an electric field of magnitude of 0.7 V/nm, applied across a
lipid membrane (electroporation). For the latter we used a
model of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), containing 1152 lipids and solvated by 46 000 water
beads. The electric field induced a large pore, approximately 15
nm in diameter in the bilayer. To equilibrate the porated
bilayer, 200 ns of MD simulations was run in an isotropic
pressure (1 bar) ensemble. This ensemble constrains volume
fluctuations to be uniform in all dimensions and is not generally
used for bilayer simulations. Instead, it is usual to employ a
semi-isotropic pressure ensemble, which allows independent
volume fluctuations with a uniform pressure tensor (normal
and parallel to the membrane). However, we found that if the
semi-isotropic ensemble was used during the pore equilibration
simulations, rapid closure of the pore would ensue within 30 ns.
There is ample evidence that the MARTINI model tends to
accelerate pore closure dynamics. For example, simulations by
Bennett et al., using the united-atom Berger lipid model, found
that a small pore induced by lipid flip-flop in a lipid bilayer
could survive for hundreds of nanoseconds.36 Nevertheless, the
relatively rapid dynamics of the MARTINI model does allow us
to investigate phenomena, which may not be easily accessible to
more detailed atomistic simulations. During the 200 ns
equilibration simulations, the lipids rapidly adopted a
configuration consistent with a toroidal pore. We subsequently
added membrane-active peptides to the equilibrated system in
order to investigate their response to the presence of the pore.
2.2. Adsorption of Peptide to a Stabilized Pore. We
studied 12 structurally different peptides. They were the
antimicrobial peptides magainin-2 (PDB: 2MAG), melittin
(PDB: 2MLT), LL-37 (PDB: 2K6O), mastoparan (PDB:
1D7N), human-β-defensin-1 (PDB: 1IJV), protegrin-1 (PDB:
1PG1), lactoferricin (PDB 1LFC), indolicidin (PDB: 1G89),
tritrpticin (PDB: 1D6X); the amyloid human IAPP (PDB:
2L86), the apoptotic Baxα5 peptide (sequence of 108−126 of
the apoptosis-regulated BAX protein, PDB: 1F16), and the cell-
penetrating peptide transportan (PDB: 1SMZ). The terms in
parentheses represent the Protein Data Bank (PDB)
nomenclature. The atomistic structures of these peptides
were downloaded from the Protein Data Bank. The secondary
structures were determined using the DSSP program (http://
binf.gmu.edu/software/DSSPCMBI.html), and mapping to the
coarse-grained topology was finally achieved with the martinize
code (http://md.chem.rug.nl/cgmartini/). Here, we should
note that in the MARTINI model the predetermined peptide
secondary structures are artificially maintained by geometric
constraints throughout the simulations. Hence, the MARTINI
model precludes the study of phenomena involving secondary
structure changes of peptides upon adsorption onto lipid
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bilayers. This apparent “defect” of the MARTINI model
somewhat limits our study, as the adsorption of membrane-
active peptides to lipid bilayers is usually accompanied by
significant structural changes in the peptides. For example,
many α-helical antimicrobial peptides adopt a coiled structure
in solution and only fold into an α-helical form upon
adsorption onto cell membranes.37 The mechanism by which
the lipid membrane induces these structural changes is beyond
the scope of this simulation study. Instead, we focus on the role
played by adsorbed peptides that have already adopted their
lipid-associated structures. However, in this work, we do probe
the importance of peptide secondary structure on the peptide’s
ability to stabilize membrane pore.
The peptides were gradually introduced with random
configurations into the simulation box after the pore was
generated and equilibrated. In order to obtain a peptide to lipid
ratio (P:L) of 1:144, we initially placed eight peptides (and
neutralizing counterions) at random locations in the water
phase above one leaflet of the porated POPC bilayer. We then
equilibrated the system for 500 ns. During this equilibration
stage, we again used the isotropic pressure coupling method,
which stabilized the membrane pore, while the peptide
distribution reached equilibrium. If a higher P:L ratio was
required, eight more peptides and counterions were inserted as
before and the system equilibrated for a further 500 ns. This
process was repeated until the desired peptide concentration
was reached. The peptide concentrations we investigated are
given by P:L = 1:144, 1:72, 1:48, and 1:36. With the number of
peptides investigated, this amounted to 48 different systems in
total.
The process above describes equilibration of peptide
configurations in the presence of a pore, which was artificially
stabilized by the chosen ensemble. Hence, we denote it as the
constrained pore adsorption simulation stage (CPAS). The
modeling employed allows us to investigate the response of the
peptides to the generated pore over a time scale much longer
than it takes for the isolated pore to collapse. In experimental
scenarios, if peptides were already strongly adsorbed on the
bilayer surface when the membrane was ruptured, they would
already be proximal to the edge of the ruptured pore where
they could quickly act to reduce the line tension. They could
also further stabilize the ruptured pore by the generation of
lateral surface tension in the bilayer. It is indicative that the
lifetime of ruptured pores, in the presence of membrane-active
peptides, was reported to be of the order of seconds,9,11 which
is much longer than the 30 ns lifetimes seen in our
unconstrained coarse-grained simulations of isolated pores.
2.3. Relaxation of the Pore in the Presence of
Adsorbed Peptide. The final configurations from the CPAS
simulations, described above, initiated our next stage of
simulations, which aimed at determining the stability of the
membrane pore in the presence of the adsorbed peptides.
During these unconstrained pore stage (UCPS) simulations, we
performed a further 1.5 μs of MD simulations, while the
pressure was allowed to fluctuate according to the semi-
isotropic coupling method. Here, volume fluctuations parallel
and perpendicular to the bilayer plane were independent, with
the same value of 1 bar for the components of the average
pressure tensor. This maintains a zero surface tension, averaged
across the bilayer and (without peptide present) would lead to
rapid pore closure within 30 ns. For magainin-2, Baxα5, and
LL-37, simulations were performed using two different
scenarios: one where the peptides were constrained to α-
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helical secondary structures and the other where these
structural constraints were removed.
All simulations were run in the GROMACS 4.5.5 package.38
The temperature was maintained at 310 K using the Berendsen
thermostat.39 The system’s pressure was controlled with the
Berendsen barostat39 at 1 bar with a compressibility of 3.0 ×
10−5 bar−1. Nonbonded Lennard-Jones (LJ) and electrostatic
interactions were both truncated at a distance rcut = 1.2 nm. To
avoid generation of unwanted noise, the standard shift function
of GROMACS was used in which both the energy and force
vanished at the cutoff distance. The LJ potential was shifted
from 0.9 to 1.2 nm, and the electrostatic potential was shifted
from 0 to 1.2 nm. All systems were simulated with an
integration time step of 30 fs. We should note that for the
coarse-grained MARTINI model the simulation time is an
effective one, which corresponds to being about 4 times longer
than “real” time.
2.4. Calculating the Inner Diameter of Membrane
Pore. Two assumptions were made in order to calculate the
inner diameters of membrane pores: (i) the inner geometry of
the pore was viewed as a cylinder, as shown in Scheme 1A; (ii)
Scheme 1. (A) Inner Geometry of Membrane Pore Can Be
Viewed as a Cylinder; (B) Mass Density Distribution Profile
for D3B and C3A Beads of MARTINI POPC Lipid Bilayera
a
The distance between the two peaks is chosen to be the height of the
cylinder.
the density of water in the pore is the same as that of bulk water
(1 g/cm3). Similar assumptions were also used in the
simulation study of Leontiadou et al.28 The central region of
the pore was inscribed with a circular cylinder of height H. The
inner diameter, d (= 2r), of the pore was then calculated (in
nanometers) using the relation
NA 4N 72N
3
= × 1021, d = 2r = 2
18 cm πr 2H 602πH
where H is chosen as the distance between two peaks in the
density distribution profile for the D3B and C3A beads of the
POPC lipid bilayer (see Scheme 1B). NA is Avogadro’s
constant, and N is the average number of water beads in the
cylinder (note: the factor 4 appearing as one MARTINI water
bead is equivalent to four real waters).
2.5. Calculating α-Helical Peptide Tilt Angles. The tilt
angles for α-helical magainin-2 (9 ALA−16 PHE), Baxα5 (6
LEU−17 ALA), melittin (15 ALA−22 ARG), and LL-37 (10
LYS−21 VAL) were calculated in our simulations as follows.
The tilt angle is defined as that between the helix axis, as
determined by two backbone beads (listed in the parentheses
above) and the bilayer normal (see Scheme 2). In all cases, the
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average tilt angle distributions were obtained over the last 500
ns of the UCPS simulations.
Scheme 2. Tilt Angle of α-Helical Peptides in Membrane
Pore
3. RESULTS AND DISCUSSION
3.1. CPAS Simulations Show That Amphipathic
Peptides Recognize the Ruptured Membrane Pore. All
the membrane-active peptides studied here displayed the same
behavior during the course of the CPAS simulations: from the
bulk water above the porated membrane they selectively
adsorbed to the edges of the ruptured membrane. Figure 1
Figure 1. Top-viewed snapshots illustrating the selective adsorption of
amphipathic peptides to the artificially stabilized ruptured membrane
pore. Peptides are colored orange; lipids are colored blue.
shows snapshots of the final configurations of the CPAS
simulations for the different peptides at a concentration of P:L
= 1:36. In all cases, the peptides preferred to accumulate at the
edge and within the constrained membrane pores. At these
regions, the amphipathic peptides tend to insert their
hydrophobic residues into the lipid alkyl chain regions while
their hydrophilic residues are proximal to the lipid head groups.
Simulations by Mihajlovic and Lazaridis, using an implicit
membrane model, reported similar behavior. They found that
models for alamethicin, melittin, magainin-H2, and piscidin-1
antimicrobial peptides all adsorbed strongly to a membrane
pore.40 Experiments by Rakowska et al. also showed the
migration of antimicrobial peptide on a membrane surface to
the edge of pores leading to their expansion (E-state).41 Our
simulation results suggest that the ability to recognize
membrane pores is a common trait of membrane-active
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amphipathic peptides. While we have initiated membrane
rupture via electroporation, in experiments it appears to be the
excess peptide adsorption to the bilayer that induces pore
formation. However, once the pore has formed, our results
show that peptides would rapidly adsorb to it via diffusion. That
is, the pore provides a region of significantly lower chemical
potential for these peptides. We note that, unlike other
simulation studies,42−44 the peptides have not been purposely
inserted into the pores. Thus, we are able to demonstrate that
these peptides preferentially adsorb to the pore compared to
other environments, e.g., an intact membrane surface.
3.2. Nonelectrostatic Interactions Are Important for
Pore Recognition. We also performed modified CPAS
simulations in order to elucidate the mechanisms leading to
the peptide aggregation at membrane pores. The coarse-grained
POPC lipid model was divided into three structural
components: lipid head (two beads), glycerol region (two
beads), and alkyl chain (nine beads). Using melittin as a
representative peptide, we began by converting all arginine and
lysine residues to their neutral forms so that the peptides did
not interact electrostatically with the lipid heads. With this
model of “neutral” melittin, we repeated the CPAS simulations.
We found that, even with electrostatic interactions absent, the
peptides still adsorbed strongly to the membrane pore (Figure
2A). This is somewhat surprising, as it is often suggested that
Figure 2. Side views of the adsorption of melittin peptides to the
artificially stabilized ruptured membrane pore. (A) Electrostatic
interactions between peptides and lipids switched off. (B) Lennard-
Jones interactions between peptides and lipid tails are switched off.
Only lipid heads (blue) and peptides (orange) are shown for clarity.
electrostatic attractions between cationic amphipathic peptides
and lipids play a dominant role in peptide activity.45 For
example, simulation work by Sengupta et al. suggests that
electrostatic interactions are an essential component for the
formation of small transient pores in membranes26 due to the
creation of electrostatic potentials across membranes. While we
cannot rule out the importance of electrostatics for the
formation of pores in membranes, it appears not to play an
essential role in the subsequent adsorption of peptides to pores.
This conclusion may of course be different for the case of
negatively charged lipids. In this context, we note that melittin
(+5) has a charge at the higher end of the spectrum of peptides
we studied.
In a second set of simulations, the electrostatic interactions
were maintained while attractive hydrophobic interactions
(modeled by LJ potentials) between the melittin peptides
and the glycerol and alkyl chain regions were switched off.
Following CPAS simulations with this modified “hydrophilic”
melittin model, we found significantly less peptide bound to the
membrane pore (Figure 2B). Thus, hydrophobic interactions
between the peptide and the inner part of the lipids appear to
play a crucial role in peptide adsorption to pores. The
membrane pore is a region of high negative Gaussian curvature
and (due to bilayer distortion) is rich in packing defects, which
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expose hydrophobic lipid tails to the water. It is in such regions
of the lipid bilayer that opportunistic binding with the
hydrophobic regions of amphipathic peptides can occur.
Given that the structural features of melittin are common to
other amphipathic peptides, we expect this behavior to be
representative of all the peptides modeled in this study. Indeed,
virtually all amphipathic membrane-active peptides have a high
content of hydrophobic amino acids, allowing them to interact
favorably with the inner part of the bilayer leaflet. The ability to
sense membrane defects is a trait that should be shared by all
proteins, which contain amphipathic α-helices. Specific
examples such as α-synuclein,46 Golgi-associated Arf GTPase-
activating protein 1,47 and Bin/amphiphysin/Rvs (BAR)
domains48 have been shown in experiments to preferentially
bind to highly curved regions of lipid membranes.
We should note that similar findings to ours have been
reported in simulations by Vácha and Frenkel,49 who
investigated a rigid-rod model for peptides and a simplified
model for lipids. Those authors found that the rigid peptides
could form a “double-belt” structure, anchored to the pore
surface by hydrophobic interactions while associating with each
other along the helical axis. The MARTINI force field used
here provides an intermediate level of complexity that falls
between such very coarse-grained models and all-atom models.
3.3. UCPS Simulations Show That Amphipathic
Peptides Can Stabilize Membrane Pores. Experiments by
Peuch et al. have shown that adsorption of amphipathic
detergents can stabilize membrane pores by reducing their line
tension.50 It is plausible that amphipathic peptides may act in a
similar fashion. The subsequent stabilization of pores by
adsorbed peptides should be reflected in the pore lifetimes. We
tested this assertion with the 48 systems described above by
subsequently running unconstrained pore stage (UCPS)
simulations for a further 1.5 μs on the 48 systems described
above. We recall that in the UCPS simulations the pressure
coupling was switched to the semi-isotropic scheme, wherein
volume fluctuations in the directions parallel and perpendicular
to the bilayer are uncoupled. Hence, the isotropic volume
fluctuations that artificially stabilized the pores in the CPAS
simulations were removed. With UCPS simulations, a
preformed pore (in the absence of peptides) would rapidly
close within about 30 ns (for the MARTINI model). To
investigate the effect of adsorbed peptide, we initiated the
UCPS simulations with the final configurations of the CPAS
simulations described above. During the UCPS simulations, we
found that with adsorbed peptides the pore diameters did
decrease, though generally over a time scale much larger than
30 ns. The time dependence of the pore diameters is shown in
Figure S1 (Supporting Information). In the presence of some
peptide species, the pore remained open over the full length of
the UCPS simulations and appeared to reach an equilibrium
value (Figure S1). Figure 3 shows snapshots of the final
configurations of the UCPS simulations (with P:L = 1:36). The
peptides magainin-2, Baxα5, LL-37, defensin-1, protegrin-1,
human IAPP, and transportan accumulated strongly in the
pores, which remained open for the duration of the UCPS
simulations. For the peptides mastoparan, lactoferricin,
indolicidin, and tritrpticin the pore eventually closed, and the
peptides were dispersed on the bilayer. If an isolated pore is
unstable or at least weakly metastable, the presence of adsorbed
peptides appears to enhance the pore metastability as is
reflected in the increased pore lifetimes. The fact that the some
pores remained stable over the at least full length of the UCPS
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Figure 3. Top-viewed snapshots illustrating final configurations at the
end of the 1.5 μs unconstrained pore stage (UCPS) simulations.
Magainin-2, Baxα5, LL-37, defensin-1, protegrin-1, human IAPP, and
transportan are able to stabilize the membrane pores whereas
mastoparan, lactoferricin, indolicidin, and tritrpticin cannot stabilize
the pores.
simulations (an effective time of approximately 6 μs) suggests a
kinetic barrier to pore closure of several kBT. It is of interest to
investigate the molecular factors that determine the amount of
stability that adsorbed peptides can impart to a pore.
A number of experimental studies have reported that peptide
length, hydrophobicity, charge, and secondary structure are
important molecular factors governing the efficacy of
amphipathic peptides.51,52 In Table 1, we include key structural
parameters of the 12 peptides in our study. These are the
number of residues (peptide length), secondary structure
content, and net charge. From this table, it can be immediately
inferred that peptide charge seems to have only a small effect
on pore stabilization in the POPC bilayer. Magainin-2, Baxα5,
mastoparan, and indolicidin all carry a net charge of +3.
However, magainin-2 and Baxα5 gave open pores at the end of
the UCPS simulations, whereas for mastoparan and indolicidin
the pores closed. To further investigate if peptide charge is
necessary to maintain an open pore, we repeated a UCPS
simulation using the neutral form of magainin-2 peptide. As
shown in Figure 4A, the neutral magainin-2 peptide is still able
to maintain an open membrane pore over the length of the
UCPS simulation. We also found little correlation between a
larger peptide charge and the size of the stable pores. For
example, LL-37 and protegrin-1 both have a charge of +6, but
LL-37 forms much larger pores than protegrin-1. Furthermore,
lactoferricin with a charge of +8 gave rise to closed pores at the
end of the UCPS simulation.
The secondary structure content of peptides seems to have a
marked effect on the pore-stabilization property of amphipathic
peptides. Indolicidin, tritrpticin, and lactoferricin all have very
low or no secondary structure content, and we found that they
did not maintain open pores over the length of the UCPS
simulations, even at the highest concentration investigated.
Experimental evidence indirectly supports this result in that,
rather than large permanent pores, these three peptides have
been found to generate only transient ion-channel-like
membrane defects, which nevertheless are able to depolarize
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Table 1. Structural Parameters of 12 Amphipathic Peptides and the Calculated Inner Diameters of Peptides Stabilized
Membrane Pores at Varying P:L Ratios (1:144, 1:72, 1:48, and 1:36)
diameters of membrane pores (nm)
peptides number of residues secondary structure contentsa charges 1:144 1:72 1:48 1:36
magainin-2 23 0.87 +3 0 0.8 1.3 3.6
Baxα5 19 0.89 +3 0 0.7 1.6 3.9
melittin 26 0.92 +5 0 0.7 2.4 2.9
LL-37 37 0.82 +6 0 3.8 6.3 6.6
mastoparan 14 0.71 +3 0 0 0 0
defensin-1 36 0.72 +4 0 1.0 2.2 3.5
protegrin-1 18 0.78 +6 0 0 0 2.6
lactoferricin 25 0.40 +8 0 0 0 0
indolicidin 13 0 +3 0 0 0 0
tritrpticin 13 0 +4 0 0 0 0
human IAPP 37 0.77 +2 0 0.7 1.7 2.2
transportan 27 0.74 +4 0 0 0.8 1.8
a
Secondary structure contents were determined with the DSSP program.

Figure 4. (A) Stabilization of membrane pore by neutral α-helical
magainin-2 peptides. (B−D) Stabilization of membrane pores by
coiled forms of magainin-2, Baxα5, and LL-37 peptides.
bacterial cells.53−55 It is known that many amphipathic peptides
undergo a coil to α-helix transition upon adsorbing onto the
cell membrane.37 While the functional role of the helical
structure is elusive, our simulations imply that it helps to
stabilize the membrane pore. To further investigate this
conjecture, we repeated the UCPS simulations for α-helical
magainin-2, Baxα5, and LL-37. However, this time the model
constraints, which maintained the helical structures of the
peptides, were removed. The resulting snapshots of the
equilibrium membrane pores are shown in Figure 4B−D. We
found that the coiled forms of magainin-2 and LL-37 peptides
were still able to aggregate in the pore and keep it open for the
duration of the simulations, but the pore diameters were
significantly smaller than those with the structured peptides.
On the other hand, the coiled form of Baxα5 gave a closed
pore. Taken together, these results suggest that the
unstructured peptides impart less stabilization to the pore. It
is possible that a peptide must possess a molecular rigidity
(imparted by its secondary structure) in order to stabilize pores
effectively. These results are consistent with experimental
studies which have reported that peptide rigidity is an
important variable in modulating the ability of peptides to
disrupt membranes.56,57 The α-helical forms of the peptides are
also likely to aggregate with each other more readily, as there is
less configurational entropy cost to form aggregated structures
of rods, compared with random coils. Furthermore, α-helix and
β-sheet secondary structures give greater hydrophobicity to the
peptide. This has been shown for the MARTINI force field in
particular.29 The peptide length may also be a factor as to why
random coil magainin-2 and LL-37 are able to stabilize pores,
but not Baxα5. Both magainin-2 and LL-37 are relatively longer
than Baxα5. However, even in their folded forms, magainin-2,
Baxα5, and LL-37 display different equilibrium pore diameters.
757
From Figure 3, it can be seen that the size of the pore lined by
the longest peptide, LL-37, is noticeably larger than the other
two. The rodlike form of the α-helix promotes peptide−peptide
aggregation, with α-helices tending to associate with their long
axes parallel. The average size of such clusters will tend to
increase for longer peptide length, thus enhancing cooperative
adsorption to the pore. Consistent with this is the behavior of
mastoparan. Despite its high secondary structure content, it is
the shortest α-helical peptide we have studied and is unable to
maintain an open membrane pore over the UCPS simulation
time (see Table 1). This is supported by the experiments of
Arbuzova and Schwarz which showed that, while mastoparan
can generate pores in POPC lipid vesicles, they have a small
average diameter (∼1 nm) and relatively short lifetimes.58
3.4. Stable Pore Size and Release Kinetics Induced by
Peptides. Table 1 lists the inner diameters of the pores at the
end of the UCPS simulations, calculated with the method
described above. The time evolution of the pore diameters (see
Figure S1) indicates that when the pores persist, they seem to
fluctuate about an equilibrium value over the final one-third of
the simulation time. So we used the last 500 ns in order to
estimate the pore diameters. The results indicate that, for a
given peptide, the average size of the membrane pore (if it
persists at the end of the simulation) increases with the peptide
concentration. This is consistent with several experimental
findings.15,59 Furthermore, the calculated diameters are in good
agreement with experimental data, when available at similar
peptide concentrations. For example, at P:L = 1:48, the
simulated inner diameter of the LL-37-stabilized membrane
pore is 6.3 nm, which compares favorably with the
experimentally measured value of 6.6 nm.17 At P:L = 1:36,
the inner diameter of a magainin-2-stabilized pore was
determined to be 3.6 nm, which is close to the experimental
value of 3.7 nm (albeit for a mixed DMPC/DMPG bilayer).60
The simulated inner diameter of the melittin stabilized pore is
2.4 nm, at a peptide concentration of 1:48, and this result is
close to the experimentally reported range of 2.5−3.0 nm for
melittin with a POPC bilayer.61 Finally, at P:L = 1:36, the
simulated inner diameter of a protegrin-1-stabilized pore is 2.6
nm, while Mani et al. reported an experimental value of 2.1 nm
(for a POPE/POPG bilayer).62 Given the approximate form of
the MARTINI model, it gives surprisingly good predictions for
the ultimate diameters of these peptide-stabilized pores. This
perhaps reflects the fact that the peptide properties that
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determine pore size (and stability) are generic ones, which can
be effectively modeled at the coarse-grained level.
The size and lifetime of pores have been investigated in
several kinetic studies on the release of fluorescent dyes from
the interior of vesicles.63 Wheaten et al.64 have investigated
release kinetics using kinetic Monte Carlo simulations and
established that the character of the dye release kinetics can be
explained by the rates of pore opening and closing, relative to
the rate of dye efflux. In particular, faster rates of pore closure
can lead to so-called graded release, and slower rates of closure
give all-or-none release. In graded release, vesicles are
homogeneous, so at around the half-life of the dye efflux
process most vesicles contain half the original amount of dye.
In all-or-none release, at the same time point, most vesicles
either contain all the original amount of dye or are empty.
Four of the peptides studied here have been reported to
exhibit all-or-none kinetics; they are magainin-2, Baxα5, LL-37,
and defensin-1. These peptides also give rise to large stable
membrane pores in our simulations. On the other hand,
mastoparan and transportan, which are known to give graded
release kinetics, give either no stable pore (mastoparan) or a
pore with only a small radius (transportan) even at the highest
peptide concentration. It is tempting to use the final pore
diameter of our UCPS simulations as a predictor for the type of
release kinetics induced by a peptide. Those peptides that give
large, relatively stable pores with long lifetimes allow unfettered
transport out of vesicles via all-or-none kinetics. The peptides
that give small stable pores at high peptide concentrations can
induce graded dye release by transient pores that are large when
initially nucleated, then either rapidly shrink to a size, too small
to allow dye efflux, or else close altogether. In this context, it is
interesting to note that there is some uncertainty regarding the
release kinetics induced by melittin. Benachir and Lafleur65
reported that melittin induced all-or-none release of fluorescent
dyes whereas Rex and Schwarz66 asserted a graded mechanism.
Somewhat consistent with these findings is our observation that
melittin gives a membrane pore of intermediate size at the
highest peptide concentration.
3.5. Human IAPP: Prefibrils and Membrane Pores. Of
course, we cannot expect that our simulations will always
provide reliable predictions for release kinetics induced by
specific peptides. For example, human IAPP peptide has been
reported to exhibit all-or-none release kinetics,6 even though we
predict the final pore diameter, induced by IAPP, to be
generally smaller than that of mellitin. However, IAPP is
noteworthy for another reason. We found that this peptide
exhibits an interesting supramolecular structuring at the pore,
which is completely different to that seen for the other peptides
we investigated. As with the other the peptides, human IAPP
readily adsorbed within the pore and at its edge. However,
when the peptide aggregated at the pore, they also appeared to
nucleate a stalklike structure of stacked molecules with aligned
α-helices that projected away from the pore into the
surrounding solution (Figure 5). This stacking seems to be
caused by the combination of the rigidity and low electrostatic
repulsion between peptides. As noted earlier, rigid peptides
(with high α-helical content) pay less entropy cost with
aggregation. Additionally, IAPP has a charge of only +2, which
means that its stacking will be dominated by the attractive LJ
interactions. In this context, it is worth noting that the peptide
LL-37 is very similar to human IAPP, except for its charge. Both
peptides contain 37 residues (14 hydrophobic residues for LL-
37 and 12 hydrophobic residues for human IAPP) and high
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Article
Figure 5. Simulation derived prefibrillar IAPP structure. Individual
peptide is colored differently for clarification.
secondary structure contents (0.82 for LL-37 and 0.77 for
human IAPP). On the other hand, LL-37 contains 5 anionic
residues and 11 cationic residues (with a net +6 charge),
whereas human IAPP contains only 2 cationic residues. The
reduced electrostatic repulsions between peptides as well as the
weakened affinity of the peptide to the membrane head groups
would lead to human IAPP aggregation at the pore edges, while
LL-37 displays no such structuring. While IAPP peptide
aggregation is also possible in the bulk solution, it is the
higher concentration of peptide at the pore edges that
promotes their nucleation. It is well-known that human IAPP
is able to form fibrillar structures in its amyloidal form.67
However, it is the prefibrillar structures which have been
implicated in their cytotoxic activity, via a mechanism as yet
undetermined.68 The stalklike α-helical aggregate that we
observe in our simulations is a plausible first member of
prefibrillar structures, which are formed in a cascade of
reactions that ultimately lead to amyloidal fibrils via cooperative
crossed β-sheet formation within the peptide stack. The
nucleation of the stalk-like α-helical aggregate by the membrane
pore may thus explain the association of prefibrils with
cytotoxicity (brought about by long-lived membrane pores).
3.6. Orientations of α-Helical Peptides in the
Membrane Pore. While the literature contains much debate
on the subject, it is often asserted that the majority of pore-
forming peptides create toroidal pores, characterized by
outwardly arranged lipid heads throughout the pore surface.
It is also probably fair to say that prevailing opinion asserts that
peptides in pores are oriented (mainly) perpendicularly to the
bilayer plane.3 Contradicting this “classic” picture are
simulations by Marrink and co-workers, who reported a so-
called disordered toroidal pore structure.26 This type of pore is
characterized by α-helical peptides being randomly oriented
within the pore. Vácha and Frenkel (using a very coarse-grained
peptide rod model) also observed a novel “double-belt”
arrangement for some peptides.49 This structure seems
characteristic of longer peptides, such as the 189-residue
apolipoprotein Apo-A1 peptide, which appears to stabilize
pores by adsorbing onto the inner surface in a parallel
orientation. Indeed, two rows of peptides in this configuration
were observed (hence the nomenclature “double belt”). Figure
6 shows typical pore configurations that we obtained for α-
helical magainin-2, Baxα5, melittin, and LL-37 peptides, with a
peptide concentration of P:L = 1:36. As discussed earlier, our
simulated membrane pores had diameters which were very
close to experiments, so these configurations give a realistic
representation of the surface area available to the adsorbed
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Langmuir
Figure 6. Side views of α-helical peptide-stabilized membrane pores:
(A) magainin-2, (B) Baxα5, (C) melittin, and (D) LL-37. Waters are
colored cyan, lipid phosphate groups are colored tan, and peptides are
colored differently.
peptides.17,60,61 The average numbers of magainin-2, Baxα5,
melittin, and LL-37 peptides that we observed were 19, 27, 22,
and 25, respectively. The number for melittin is much larger
than the 1−2 peptides observed in earlier simulations of a much
smaller disordered toroidal pore.26 OCD measurements of Lee
et al. for melittin-induced pores in a POPC bilayer suggest that
only 4−7 peptides are found on average in a perpendicular
orientation (presumably in the membrane pore). In this case,
the experimentally determined pore diameter was 4.4 nm at P:L
= 1:15.12 This diameter was obtained at a much higher peptide
concentration than was used in our work (which may account
for its larger reported size). However, it does appear
inconsistent with the larger number of pore-bound peptides
we observed in our simulations. This discrepancy can be largely
traced to the orientational distribution of peptides in pores. In
the classic toroidal pore structure, peptides are usually assumed
to lie perpendicular to the plane of the membrane (I-state). In
reality, however, this may not be the case.
From the final 500 ns of the UCPS simulation, we calculated
the tilt angle distributions of peptides within the pore. In Figure
7, we have plotted the distributions for the α-helical peptides
magainin-2, Baxα5, melittin, and LL-37. It is clear that the
orientations of magainin-2, Baxα5, and melittin peptides in the
membrane pore are quite disperse. For example, the tilt angles
for magainin-2 span the range 22°−85°, with an average value
of 50.3°. This distribution is very similar to that of a disordered
toroidal pore observed by Marrink et al.26 OCD measurements
determine an effective fraction of pore-bound peptides in the
so-called I-state, i.e., having a perpendicular orientation. This
fraction corresponds to averaging cos2 θ over the tilt angle
distribution. Hence, the actual number of peptides in a
membrane pore (as predicted by OCD) will be usually
underestimated if the orientation of all peptides is assumed
to be perpendicular. For example, from our simulations, we
determine an average value of 7−8 magainin-2 peptides in the
I-state (for a pore with a diameter of 3.6 nm), much smaller
than the actual number of peptides present (approximately 19).
Interestingly, the tilt angle distribution profile for LL-37
peptides in the membrane pore suggests that LL-37 peptides
are orientated more parallel to the membrane surface,
Figure 7. Tilt angle distribution profiles for α-helical peptides in the membrane pore: (A) magainin-2, (B) Baxα5, (C) melittin, and (D) LL-37.
759
Article
somewhat similar to the longer peptide orientations observed
by Vácha and Frenkel.49 Such a distribution is caused by the
mismatch between the bilayer thickness and the relatively
longer peptide. The parallel orientation means that LL-37
creates an equilibrium pore with a larger diameter than shorter
peptides.
4. CONCLUSION
Using MARTINI coarse-grained simulations, we have shown
that amphipathic peptides are able to recognize and stabilize
membrane pores by aggregating with high affinity within and at
the edge of membrane pores. Our conclusions rely on the
ability of the MARTINI model to adequately describe peptide
interactions with zwitterionic lipid bilayers. We have already
noted that this force field is able to predict translocation of
amino acid residues through zwitterionic bilayers with
reasonable accuracy.32 Additionally, we have carried out further
simulations to ascertain if the polarizable MARTINI model
predicts similar results. This force field seems to give a better
description of bilayer thermodynamics, though it is computa-
tionally much more expensive to implement.69 We tested it
with the magainin-2 peptide, with the result that the
recognition and stabilization of the ruptured membrane pore
by the peptide were still captured (see Figure S2).
We found that the secondary structure content, peptide
length, and hydrophobic interactions significantly affect the
accumulation of peptide in the pore. On the other hand,
peptide charge seemed to have little influence, at least for the
zwitterionic lipids used in this study. The simulated membrane
pore sizes were in good agreement with available experimental
data, which lends some credence to our findings. Our results
suggest that α-helical peptides in the pore are generally
oriented in a largely disordered fashion, consistent with the
disordered pore model, at least for peptides of moderate length.
The longer LL-37 peptide adopted a parallel orientation, similar
to the “double-belt” configurations first described by Vácha and
Frenkel.49 Finally, we note that for the human IAPP peptide the
presence of the pore appears to nucleate prefibrillar structures
made up of stacked α-helices, which may explain why
cytotoxicity and prefibrillar structures seem to be associated
for these peptides.
■
ASSOCIATED CONTENT
*
S Supporting Information
Time evolution of the inner diameters of pores in the presence
of different peptides during the unconstrained pore stage
(UCPS) simulations; the recognition and stabilization of
membrane pore by magainin-2 peptide modeled with the
DOI: 10.1021/la5038266
Langmuir 2015, 31, 752−761
Langmuir
polarizable MARTINI force field. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: c.woodward@adfa.edu.au (C.E.W.).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
An allocation time from the Lunarc Computing Center at Lund
University is gratefully acknowledged. J.F. acknowledges
financial support from the Swedish Research Council.
■
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■
NOTE ADDED IN PROOF
While we cannot discount the possibility that the pores that are
observed during the UCPS simulations do not always achieve
equilibrium, many of the peptides we have investigated
significantly prolong the pore lifetimes, indicating their ability
to increase pore stability.
DOI: 10.1021/la5038266
Langmuir 2015, 31, 752−761