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Current Topics in Medicinal Chemistry, 2016, 16, 25-39 25

Antimicrobial Peptide Structure and Mechanism of Action: A Focus on the

Role of Membrane Structure

Tzong-Hsien Lee, Kristopher N. Hall and Marie-Isabel Aguilar*

Department of Biochemistry and Molecular Biology, Monash University, Wellington Rd, Clayton, Vic,
3800, Australia

Abstract: Antimicrobial peptides (AMPs) are showing increasing promise as potential candidate anti-
bacterial drugs in the face of the rapidly emerging bacterial resistance to conventional antibiotics in
recent years. The target of these peptides is the microbial membrane and there are numerous models to
explain their mechanism of action ranging from pore formation to general membrane disruption. The
interaction between the AMP and the target membrane is critical to the specificity and activity of these
peptides. However, a precise understanding of the relationship between antimicrobial peptide structure
and their cytolytic function in a range of organisms is still lacking. This is a result of the complex na-
ture of the interactions of AMPs with the cell membrane, the mechanism of which can vary considerably between differ-
ent classes of antimicrobia peptides. A wide range of biophysical techniques have been used to study the influence of a
number of peptide and membrane properties on the cytolytic activity of these peptides in model membrane systems. Cen-
tral to characterisation of this interaction is a quantitative analysis of the binding of peptide to the membrane and the co-
herent dynamic changes in membrane structure. Recently, dual polarization interferometry has been used to perform an in
depth analysis of antimicrobial peptide induced membrane perturbation and with new mass-structure co-fitting kinetic
analysis have allowed a real-time label free analysis of binding affinity and kinetics. We review these studies which
describe multi-step mechanisms which are adopted by various AMPs in nature and may advance our approach to the de-
velopment of a new generation of effective antimicrobial therapeutics.
Keywords: Antimicrobial peptides, dual polarisation interferometry, membrane bilayer anisotropy, membrane structure plastic-
ity, supported lipid bilayer.

1. INTRODUCTION only been characterised for a small portion (about 13%) of

Antibiotics were one of the greatest discoveries of the
20th century and have been used extensively with great suc-
cess to treat microbial infections since their introduction in
the 1950s. Unfortunately their effectiveness has diminished
over time due to the steady increase in resistant microbial
strains. Factors such as increased selective pressure from the
use of antibiotics, increased disease transmission and a de-
cline in development of novel antimicrobial therapeutics has
seen the rise of once treatable infections becoming untreat-
able. For example, the emergence of increased resistance to
antibiotics such as methicillin and drugs of last resort such as
vancomycin of strains such as enterococci, staphylococci (eg
Staphylococcus aureus) pose a serious global health problem
and urgent action is required [1-4].
Antimicrobial peptides (AMPs) are ancient weapons used
by organisms to combat microbial infections and have that
have remained effective, surviving the slow process of evo-
lution over time [5, 6]. With more than 5000 sequences char-
acterised to date [7], these peptides show a marked variation
in sequence, length and structure. The 3D structures have
*Address correspondence to this author at the Department of Biochemistry
and Molecular Biology, Monash University, Wellington Rd, Clayton, Vic.
3800, Australia; Tel: +61-3-9905-3723; Fax: +61-3-9902-9500;
E-mail: Mibel.Aguilar@monash.edu
AMPs primarily determined by solution nuclear magnetic
resonance (NMR) spectroscopy. Based on the Antimicrobial
Peptide Database, 13.8% are predicted to be helical, 4% β-
strand and 4% mixed. In contrast, the updated Collection of
Antimicrobial Peptide (CAMP) database consists of 682
high resolution AMP structures of which 18.7% are helical,
18.9% are β-strand, 60.1% are mixed or coil [7]. Thus, a
high resolution structure is only available for a low propor-
tion of AMPs with the majority of those being helical. The
diversity of AMP structures also suggests that the action of
these peptides on the target cells can be complex and beyond
the pore mechanisms based on the helical structure which
have been widely adopted in the design and development of
AMPs exhibiting antimicrobial activities. Furthermore, given
that only a small proportion of AMPs are helical in structure,
we know even less about the majority of AMPs and the reli-
ance on pore mechanisms as the sole or dominant mecha-
nism may not be valid. Ultimately, this may restrict the de-
sign and development of novel antimicrobial agents as it is
becoming increasingly evident that the mechanism of action
is far more complex than a defined pore or carpet mecha-
nism.
Structure activity relationship studies of antimicrobial
peptides have revealed that there are many factors that affect
the specificity and biological activity of these peptides. Fac-

1873-5294/16 $58.00+.00 © 2016 Bentham Science Publishers

26 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1
tors such as charge, secondary structure, hydrophobicity,
amphipathicity and hydrophobic moment are all critical to
function and are so interdependent that altering one property
will often result in significant changes to one or more of the
others. What many of these studies have in common is the
focus on the structure of the AMP during membrane binding.
However, the membrane also undergoes substantial changes
in structure but has, to date, remained relatively poorly un-
derstood. In this review, the details of mechanisms by which
AMPs exert their activity are explored with a specific focus
on the analysis of membrane structure changes during AMP
binding: from the initial interaction of AMPs with their
membrane target, conformational changes upon interaction
with the membrane, accumulation of critical concentration
and self-association, direct mechanisms of action such as
pore formation or general disruption to downstream effects
that lead to cell death. Target selectivity and possible resis-
tance is discussed and then finer details of factors that modu-
late the biological activity of these peptides such as: charge,
secondary structure, hydrophobicity, amphipathicity, hydro-
phobic moment and polar angle are discussed in detail. Fi-
nally, we focus on studies using multi-parameter-based opti-
cal biosensors that provide new information on the structure
of the membrane and the concept of membrane plasticity
during AMP binding.
2. PHYSICOCHEMICAL PARAMETERS THAT
MODULATE ANTIMICROBIAL PEPTIDE ACTIVITY
AND SPECIFICITY
It is well known that the biological activity and specific-
ity of AMPs are determined by numerous physicochemical
parameters of both peptides and membrane lipids [8]. The
exact relationships between these factors such as the se-
quence length, amino acid composition and structural char-
acteristics of AMPs and the bilayer composition and proper-
ties of the target cells for their mechanisms of action are not
well understood. A better understanding of the relationship
between these factors is critical to designing novel peptides
with increased potency and tailored species specificity.
2.1. Charge
The majority of antimicrobial peptides characterized to
date carries a net positive charge, ranging from +1 to as
much as +9 and can contain highly defined cationic domains.
The initial interaction with the membrane is primarily elec-
trostatic and there is a strong correlation between cationic
charge and biological activity with increasing charge corre-
lating with increased activity and also with increased peptide
concentration bound to the membrane interface [9-14]. There
is often an optimum charge for activity however; with
greater charge beyond this value resulting in decreased activ-
ity. For example, a charge dependence was demonstrated for
analogues of magainin 2, with activity increasing with
greater charge while hydrophobicity and structure remained
constant. However, any further addition of cationic residues
beyond a threshold point effectively decreased the peptide
selectivity for microbial targets with enhanced hemolysis
[15]. The effect of positive charge on activity has also been
shown for melittin analogues in which removal of the cati-
onic C-terminal tail of melittin resulted in a five-fold de-
Lee et al.
crease in hemolytic activity and a significant drop in mem-
brane binding [3, 16, 17].
Membrane charge density also plays critical role in me-
diating the binding and selectivity of cationic AMPs. Parti-
tion of cationic AMPs into zwitterionic lipids is generally
weak with a high dissociation constant. The presence of
negatively charged lipids such as phosphatidylglycerol (PG)
and cardiolipin in microbial membranes mediate an electro-
static interaction with the cationic peptides with a remarka-
bly reduced dissociation rate. However, while the contribu-
tion of electrostatic interactions forms the basis of selective
peptide binding to the bacterial membrane, this alone cannot
be used to explain the selectivity towards bacteria over host
cells. As described above, enhanced electrostatic interaction
is accompanied by a non-linear concentration-dependent cell
toxicity, once a charge threshold is exceeded. In addition,
there have been some rare cases where an indirect or inverse
relationship between charge and activity has been observed
[9, 18].
2.2. Secondary Structure
Various structures have been characterized for AMPs
which can be generally classified into four major structural
groups: helical peptides, β-strand/sheet peptides, mixed heli-
cal/sheet peptides and extended non-helical/sheet peptides.
Most AMPs undergo a conformational transition from flexi-
ble unstructured in solution to a specific structured or more
rigid conformation upon interacting with a membrane. In the
case of helical peptides, amino acid substitutions that signifi-
cantly disrupt the helical structures can lead to a dramatic
decrease in activity. For example; studies with proline sub-
stitutions with an active rabbit CAP18 fragment resulted in
complete loss of activity due to disruption of peptide struc-
ture [19]. Furthermore, many studies have shown that all-D
AMPs exhibit activity that is generally comparable to that of
the parent peptides [20, 21]. However, the introduction of
single or double D-amino acid substitutions which disrupt
the secondary structure can have a dramatic effect on peptide
activity. An example of this is in a study by Oren and Shai
[22], who showed that single-D substitutions within melittin
partly altered the secondary structure, essentially eliminating
hemolysis without having a great effect on the antimicrobial
action. Other investigations [23] into linear and cyclic ana-
logues of melittin and magainin showed that the cyclized
peptides were less efficient in initial binding compared to the
linear peptides. When similar amounts were eventually
bound however, the peptides exhibited similar ability to
permeabilize the membrane. Interestingly the cyclic melittin
analogue showed increased antibacterial activity with de-
creased hemolysis whereas the cyclic magainin analogue
showed increased hemolytic lysis and decreased antibacterial
activity. Chen et al [24] have also shown that substituting
residues of low helical propensity for those of high helical
propensity with magainin analogues can increase activity. A
series of peptides derived from Australian frogs have pro-
vided an important family of related antimicrobial peptides
which have been analysed in terms of effect of net charge,
length, and number of helical turns [25]. In particular, the
membrane binding properties of aurein 1.2, citropin 1.1,
maculatin 1.1 and caerin 1.1 have been studied using a range
of techniques [26, 27]. These studies have further demon-
Antimicrobial Peptide Structure and Mechanism of Action
strated that conformation has a strong effect on both the ini-
tial AMP binding with the membrane as well as the subse-
quent disruption process.
2.3. Hydrophobicity
Hydrophobicity is essential for interaction with mem-
branes as it controls the extent to which the peptide can par-
tition into the membrane layer. AMPs typically contain ap-
proximately 50% hydrophobic residues. Similarly to charge,
studies have shown that increasing hydrophobicity to opti-
mal percentage can increase activity against microbial cell
membranes [28]. Further increasing the hydrophobicity be-
yond this optimum point has been correlated with a loss of
antimicrobial activity and an increase in mammalian cell
toxicity [29]. For example, studies with magainin 2 ana-
logues showed that increasing mean hydrophobicity had lit-
tle effect on purely anionic PG liposomes, but even slight
increases in hydrophobicity showed a dramatic increase in
ability to lyse zwitterionic/anionic mixtures PC/PG 3:1 [30],
indicating the substantial influence that hydrophobicity has
on the membrane interaction.
2.4. Amphipathicity, Hydrophobic Moment and Polar
Angle
Charge, structure and hydrophobicity have been shown to
be important, but it is also the way that these three factors
come together and form an amphipathic structure that is of-
ten of greater importance [13, 31-33]. Almost all AMPs form
some kind of amphipathic structure upon interaction with
their target membrane. Amphipathicity refers to the relative
proportion and topographic location of hydrophilic and hy-
drophobic domains within the peptide. A quantitative meas-
ure of this is the hydrophobic moment (µH)which is the sum
of the amino acid vectorial hydrophobicities [34]. Hydro-
phobic moment also has a proportional relationship to activ-
ity with increasing hydrophobic moment increasing both
bacterial membrane disruption and hemolytic activity [6,
35]. An amphipathic α-helix is one of the most efficient am-
Fig. (1). a) An α-helical representation of an amphipathic structure with charged (red) areas aligning down one side of the helix and hydro-
phobic resides (blue) down the other side. b) Clustering of cationic and hydrophobic residues of several antimicrobial peptides of different
structural classes. Again red residues are positively charged and blue are hydrophobic amino acids. Other amino acids are not shown. Ma-
gainin 2 is depicted in its α-helical form. (b) Is modified from [6].
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1 27
phipathic structures with hydrophobic residues down one
side of the helix and cationic/hydrophilic residues down the
other side as shown in Fig. (1).
While the degree of helicity can influence peptide activ-
ity [24], it also has been shown that a high degree of helicity
and amphipathicity with a segregated hydrophobic domain,
correlates with increased hemolysis and toxicity to cells
composed of zwitterionic neutral lipids (36). The hydropho-
bic moment was also studied in the role of hydrophobicity of
magainin 2 analogues. Increasing hydrophobic moment also
had little effect on anionic PG liposomes, but had a dramatic
effect on lipid mixtures of PC/PG (3:1) [30]. The same study
also showed an increase in binding. The β -sheet AMPs are
also usually amphipathic, with a number of β -strands (fre-
quently anti-parallel) organized to create both hydrophilic
and hydrophobic surfaces. They are usually stabilized by a
series of disulphide bonds with as many as eight cysteines in
some peptides (eg plant defensins [36]) or by cyclization of
the backbone (eg protegrins [37], gramicidin [38] or the cy-
clotides [39]). The polar angle (θp) is closely related to am-
phipathicity and the hydrophobic moment and is defined as
the relative angular proportion of the polar to non-polar resi-
due sides of the amphipathic helix. Fig. (2) shows a hypo-
thetical example. If one side of the helix is hydrophobic and
the other side hydrophilic then the polar angle would be 180°
(Fig. 2a). If one third of the helix is hydrophilic and the two
thirds hydrophobic then polar angle would be 120° (Fig. 2b)
and so on.
Hp(2-20) is an AMP derived from the N-terminus of
Helicobacter pylori ribosomal protein L1 (RpL1), and pos-
sesses broad-spectrum activity against bacteria, fungi and
protozoa at low micromole concentration without causing
hemolysis [40]. The importance of various structural charac-
teristics of Hp(2-20), including sequence, length, conforma-
tion and amphipathicity/ hydrophobicity on the antimicrobial
activity has been assayed via antifungal, antibacterial and
haemolytic tests of a range of peptide analogues [41, 42].
One such analogue, HPA3, which has a double
28 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1
Fig. (2). A helical wheel diagram showing two hypothetical amphipathic peptides with different polar angles. Charged hydrophilic residues
are red and hydrophobic residues are blue. a) One side of the helix has hydrophilic residues and the other hydrophobic. This gives a polar
angle of 180˚. b) One third of the helix is hydrophilic and the two thirds hydrophobic, giving a polar angle of 120°.
substitution of Q17W and D19W, showed 4-16 fold in-
creased activity against bacteria and 2-4 fold higher potency
at inhibiting fungal growth [24, 26]. The enhanced antimi-
crobial activity was attributed to the higher overall hydro-
phobicity and amphipathicity and longer linear helical struc-
ture as determined by CD and NMR spectroscopy in mem-
brane-mimetic environments [25, 26].
3. MECHANISM OF ACTION OF ANTIMICROBIAL
PEPTIDES
The mechanism of AMP action has been extensively
studied on various membrane models with much fewer stud-
ies focussing on the action of AMPs on whole microbial
cells with the use of membrane potential sensitive dyes and
fluorescently labelled peptides. These studies have collec-
tively shown the primary interaction of AMPs with a mem-
brane and can be divided into two mechanistic classes:
membrane disruptive/permeabilizing and non-membrane
disruptive/permeabilizing [43]. The proposed models for the
mechanism of action extend from classical pore formation to
structural rearrangements such as molecular shape or interfa-
cial activity models [43]. Since more than one proposed
model may be relevant for a specific AMP, it is therefore
important to understand the mechanistic steps that occur se-
quentially on the membrane leading to disruption.
3.1. Initial Attraction and Interaction
AMPs are usually unstructured in solution and the initial
interaction of AMPs with the membrane is brought about via
electrostatic attraction between cationic residues within the
AMP and the negatively charged lipids in the bacterial
membrane target [9, 11, 15, 44]. Lysine and arginine have
been shown to interact strongly with phosphate groups in
lipid bilayers [45]. This observation, along with the fact that
most bacteria have a strong electrochemical gradient and that
electrostatic forces are relatively strong over long molecular
distances likely contributes to the membrane targeting initial
attraction of many AMPs [8]. Once in proximity to the target
membrane, coil-helix transitions have been characterised for
most AMPs [46, 47] as depicted in Fig. (3a). On the other
hand, β -sheet AMPs are typically much more structured in
solution. They are stabilized by disulphide bonds or cycliza-
tion of the peptide backbone. The cyclic β-sheet AMP tachy-
plesin is an example that has little change in structure as it
Lee et al.
goes from an aqueous environment to that of a membrane
mimic [48]. With such a stable structure in solution it is
likely that these AMPs will maintain the same stable con-
formation when they interact with the membrane surface.
These structures are usually amphipathic and this has been
found to be important for lytic activity [18]. Increasingly, it
is emerging that antimicrobial activity is not dependent on
specific peptide structure but more on the amino acid com-
position and physicochemical properties. Moreover, the ac-
tivity can depend more on the balance of electrostatic inter-
actions in the initial steps and hydrophobic interaction in the
later steps during binding-disruption process.
3.2. Concentration Dependant Threshold
Once the AMPs have targeted and bound to the mem-
brane a second stage of membrane interaction requires a cer-
tain ‘critical’ concentration in order to proceed [49, 50]. As
the concentration increases in the local area the peptides
have the potential to self-associate and penetrate deeper into
the membrane core and to exert the peptide’s effect via a
variety of mechanisms. Factors such as the composition of
the lipid head groups and the fluidity of the membrane as
well as the propensity of the peptide to self-assemble or mul-
timerize will influence the determination of such critical
concentration and the subsequent kinetic steps [51].
3.3. Self Association and Multimerization
Once the membrane-bound AMPs reached the critical
concentration, some AMPs self-associate or multimerize to
achieve the final membrane disruption / cell inactivation as
discussed in the barrel-stave/toroidal pore, aggregation and
amyloid models [52, 53]. The AMPs create complex struc-
tures through peptide-peptide and peptide membrane interac-
tions associated with specific AMP mechanisms of action.
The likelihood of the peptide forming these super structures
is dependent on amino acid composition and conformation of
the peptide in its monomeric form. Examples of AMPs that
have been shown to form oligomers that play a role in their
activity are cathelicidin LL-37 [53], alpha-defensin-6 [52]
and plant-derived kalata B2 [54]. The effectiveness of the
complex formed depends on the ability of peptides to align
hydrophobic and hydrophilic domains toward the membrane
or adjacent peptide in the complex. In this way they are able
to achieve more as a whole than the individual peptide can
Antimicrobial Peptide Structure and Mechanism of Action Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1 29

achieve by itself such as deeper penetration into the mem- ously or interchange sequentially, and that the mode of ac-
brane core. tion can be affected by multiple factors such as the physico-
chemical properties described above, the solution concentra-
tion of peptides, membrane-bound peptide/lipid ratio, com-
position and structure of both peptide and membrane and the
kinetics of binding and structural changes of all interacting
partners. Among all various models (Table 1), the barrel-
stave/toroidal pore models and carpet-like mechanisms have
been extensively applied to the mechanisms of AMPs action
on membrane and are discussed below and depicted sche-
matically in Fig. (3).

Table 1. Proposed models for the membrane interactions of

α -AMPs (adapted from [43]).

Representative
Antibacterial Mechanism
α-AMPs

Non-permeabilizing

1 Translocating via a transient pore Buforin II

2 Translocating via sinking raft δ-lysin

Permeabilizing

1 Barrel-stave pore Almethicin

2 Toroidal pore Magainin 2

3 Huge toroidal pore Lacticin Q

4 Disordered toroidal pore Melittin

5 Aggregation model Magainins

6 Interfacial activity model Magainin 2

7 Chaotic or non-stoichiometric model Magainin 2

Aurein 1.2,
8 Carpet mechanism
Cecropin
Fig. (3). A schematic of the main classical models depicting the
mechanisms of helical AMPs action. (a) peptides are unstructured 9 Detergent model
in solution; (b) initial binding to membrane interface mediated by
10 Membrane discrimination model V13KL
the electrostatic interaction with structural changes in peptides; (c)
once the membrane-bound peptides reached a critical concentration, 11 Shai-Huang-Matsazuki (SHM) model
they proceed through a stochastic cooperative transition in a multi-
step process destabilizing the membrane via either forming (d) 12 Membrane thinning/thickening model LL-37
barrel stave pore; (e) toroidal pore or (f) carpet-like complex with Magainin
extensive surface coverage up to a point resulting in (g) the mem- 13 Charged lipid clustering
analogues
brane disintegration. (e-f) The formation of toroidal pore can also
proceed through a carpet-like process once a critical concentration Synthetic α-
14 Sand in a gearbox model
of extensive surface coverage is reached (h) The overall outcome of AMPs
these membrane-destabilizing processes is leading to cell death. 15 Oxidized phospholipid targeting Temporin L

3.4. Models of Antimicrobial Action 16 Electroporation NK-lysin

Various molecular models have now been used to de- 17 Tilted peptide mechanism Aurein 1.2
scribe the action of AMPs on a membrane, and are listed in
18 Amyloid formation Temporin B
Table 1. These models range from structurally defined
mechanisms such as barrel-stave, toroidal and detergent-like 19 Leaky slit model
carpet models to structurally less defined mechanisms, such
as lipid segregation into domains, interfacial activity and
3.4.1. Pore Formation
formation of non-lamellar phases and the non-permeabilizing
sinking raft model. With such a wide variety of models it is There are two main peptide induced pore formation mod-
possible that multiple modes of action may occur simultane- els. One is the ”barrel stave” mechanism and the other, the
30 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1
”toroidal wormhole” mechanism. The barrel-stave model
was the first to be proposed to account for the single channel
conductance induced by alamethicin in black lipid mem-
branes [55]. In this model, the AMP binds to the membrane
surface through electrostatic attractions where it then under-
goes a conformational change adopting an amphipathic
structure. When a critical threshold of peptide concentration
is reached, the individual peptides self-assemble and create a
larger structure made up of a variable amount of peptides
that inserts more deeply into the membrane forming a ring
like a “barrel” pore (Fig. 3d). “Stave” refers to the individual
spokes within the barrel. In this pore structure, the hydro-
phobic domains face outward toward the hydrophobic re-
gions of the membrane core and the hydrophilic regions fac-
ing inward toward the aqueous pore. This allows for minimal
exposure of the hydrophilic residues to the hydrophobic tails
of the lipids and the hydrophobic regions of the complex to
avoid the hydrophilic aqueous pore [56, 57]. The “toroidal
pore” or “wormhole” mechanism is similar to the “barrel-
stave” mode of pore formation. This model has been pro-
posed primarily from studies of α-helical AMPs such as the
magainins and PGLa [58-60]. The peptides are initially at-
tracted to the membrane and undergo similar secondary
structural changes as the barrel stave model. The helices
formed are initially orientated parallel to the membrane with
the hydrophobic side of the helix binding and displacing the
phospholipid head groups. This causes a breach in the hy-
drophobic region and induces a positive curvature strain in
the membrane. This strain, along with membrane thinning
makes the surface more vulnerable to the AMPs by destabi-
lizing the integrity of the membrane [8]. Once a critical con-
centration of peptide is reached, they self-associate and ori-
ent perpendicular to the membrane surface with the hydro-
phobic residues no longer exposed to the lipid head groups
and form the toroidal pore complex as shown in Fig. (3e).
This differs from the barrel stave pore as the peptides still
interact with the lipid head groups and are not situated within
the membrane hydrophobic core. This arrangement is not as
stable as a barrel stave pore and is therefore more transient.
The stability of the pore has been shown to be affected by
peptide charge, with a greater number of positive side chains
causing repulsion and producing pores with shorter half-lives
[3]. Upon disintegration of the pore complex, some AMPs
have been shown to translocate to the luminal side of model
membranes [16, 61] and suggests that this mechanism may
also be a way in which peptides can access potential intracel-
lular targets.
3.4.2. Carpet-Like Mechanism
AMPs do not necessarily have to act via pore formation,
and in many cases they are believed to act through non-
specific membrane permeabilization. In a similar way to the
other two models described here, the peptides are initially
attracted to specific target membranes through electrostatic
forces. Upon binding there is a conformational change and
the hydrophobic regions of the peptide may or may not insert
into the membrane core. Upon a critical concentration, they
effectively cover the surface of the membrane in a “carpet-
like” manner [3, 31, 60, 62]. There is no further quaternary
structure formation and once the critical concentration has
been reached, this causes a change in the membrane energet-
ics and fluidity until eventually the membrane structure is
Lee et al.
destabilized and no longer can be maintained (Fig. 3f-g). The
collapse of membrane structure into micelles at the final
stage of the carpet mechanism where the peptides saturate
the membrane surface has also been described as a detergent-
like model while the carpet-like mechanism is mostly con-
sidered as the prerequisite step of the toroidal pore forma-
tion. Studies have shown that AMPs such as aurein 1.2 [63],
cecropin [36] and indolicidin [64] act in this manner.
3.5. Downstream Biological Effects
Irrespective of the mode of action; the downstream effect
of these mechanisms leads to cellular inactivation and death.
The integrity of the cytoplasmic membrane is of vital impor-
tance in regulating many functions of microbes. An AMP
that undergoes either a pore formation or membrane perme-
ablization mechanism above could lead to a loss of one or
more functions of the membrane - such as loss of metabolites
and ion gradients, membrane depolarization with phosphol-
ipid flip-flopping and a scrambling of the usual distribution
of lipids between the leaflets of the membrane, or loss of
respiration - all of which would lead to cellular death [44,
64-69]. This can often happen rapidly and may be only a
matter of minutes after exposure [70]. As mentioned previ-
ously, this is in stark contrast to traditional antibiotics that
can take days to be effective. Human defensins have been
shown to penetrate both the outer and inner membranes of
Gram-positive bacteria with cell death attributed to the
breaching of the inner membrane [71]. Defensins have also
been shown to destabilize the electrostatic gradient of S.
aureus [72]. Moreover, in E. coli, the inhibition of RNA,
DNA and protein synthesis coincided with the membrane
permeabilization. These assays indicate that permeabilization
leading to loss of other downstream cellular function is es-
sential for bacterial killing [71, 73]
There is also a growing body of evidence that there may
be additional or complementary mechanisms where mem-
brane permeabilization alone is not enough to cause micro-
bial death. AMPs have also been linked to the inhibition of
vital intracellular functions of microbes [71, 74, 75]. For
example, Nisin and Pep 5 were shown to liberate hyrolases
that degrade the cell wall [76]. Thrombin-induced platelet
microbicidal proteins are believed to cause inhibition of
DNA and/or RNA synthesis, with studies showing cell death
occurring at least half an hour after membrane permeabiliza-
tion of S. aureus [77]. Microcin has also been found to in-
hibit DNA replication by targeting DNA gyrase in E. coli.
[78]. Loss of cell viability of C.albicans has also been ob-
served after internalization by the antifungal peptide tenecin-
3 without any membrane permeabilization[79]. Pyrrhoco-
ricin has been shown to inhibit the ATPase actions of DnaK
and prevent chaperone assisted protein folding [80]. These
studies suggest that depending on individual factors, AMP-
induced cell death may occur via several independent or co-
operative "multi-hit" [81] mechanisms of action. Having
multiple and complementary ways of killing a wide variety
of microbes would be useful in suppressing the ability of the
individual pathogen to avoid cell death.
3.6. Antimicrobial Peptide Selectivity
If the high potency of AMPs is to be exploited for the
development of novel therapeutics, it must be selective i.e. it
Antimicrobial Peptide Structure and Mechanism of Action
must show high antimicrobial activity and low host toxicity
[82, 83]. AMPs use fundamental differences between pro-
karyotic and eukaryotic membranes as a way of selective
toxicity. Bacterial membranes are organized with their outer
most membrane layer heavily populated with net negatively
charged acid phospholipids such as PG. This is quite differ-
ent to the membranes of animals and plants that consist
mainly of neutral zwitter ionic phospholipids such as phos-
phatidylcholine (PC) which decreases the binding strength of
the cationic AMPs [67, 84]. The negatively charged lipids
that eukaryotic cells possess are usually found more on the
inner leaflet side of the membrane facing the cytoplasm, and
as such not exposed to the extracellular space. The presence
of cholesterol and other sterols that are not present in bacte-
rial membranes are also believed to inhibit AMP activity by
maintaining membrane integrity, and making permeabliza-
tion more difficult [84] and these factors may prove useful in
drug targeting.
Overall, the interaction of AMPs with a membrane in-
volves structural and topological changes in both the pep-
tides and the membrane. Complex factors determine the ac-
tivities of AMPs in destabilising membrane structure and
function and the ability of AMPs to discriminate between
pathogen and host cell membranes. Modifications in the
membrane surface charge, packing order and acylation have
been identified in the AMPs-resistant bacteria [85-87]. Cor-
relating the changes in membrane packing ordering as a
function of the membrane-bound peptide mass with those
modifications in the membrane properties will provide more
information for their impact on the interaction of AMPs and
dynamic changes in membrane structures and more impor-
tantly to assist the development of peptide-based therapeu-
tics against drug-resistant microbial infection.
3.7. Changes in Membrane Structure and Packing – The
Real Issue
Even with the present knowledge of the molecular basis
of antimicrobial and cytolytic peptide mechanism of action,
the design of new peptides with a narrow spectrum of activ-
ity against microbial infection with minimal toxicity to the
host cells is still a major challenge. This is partially due to
the fact that the target sites of action for both bacterial and
mammalian cells are the membrane lipids, so complete
specificity represents a demanding task to achieve. However,
the design of peptides having physicochemical properties
that result in optimal therapeutic results has been the goal of
a number of studies. At the heart of this problem is the need
to measure target membrane properties in terms of structure
and stability during AMP binding.
As membrane destabilization is one of the main actions
for AMPs, the molecular organization of membrane lipids, in
addition to the membrane surface charge and curvature, play
an important role in the selectivity of AMPs. Therefore, to
fully understand the mechanisms of membrane destabiliza-
tion by AMPs, it is important to elucidate how membrane
properties that control and maintain membrane shape, struc-
ture and dynamic stability are affected by the binding of
AMPs. Thus, characterizing the membrane changes associ-
ated with peptide binding becomes important for understand-
ing their mechanism of action.
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1 31
However, while the different mechanisms described
above involve changes in the membrane structure, the spe-
cific changes in the membrane structural properties and mo-
lecular organization of membrane lipids are not often explic-
itly described in these mechanisms. Moreover, the various
mechanistic actions of AMPs on membrane destabilization
have been mainly established for helical peptides with physi-
cal properties and structural characteristics discussed in Sec-
tion 2. These well-characterized structural properties may
not be adopted by non-helical AMPs.
Delineating the mechanisms of AMPs action thus re-
quires the dynamic changes in membrane structure to be
characterised quantitatively in response to the peptide bind-
ing and structural changes. There is a significant gap in our
understanding of membrane-mediated interactions particu-
larly in view of the role of lipid molecular organisation. This
is partly due to the limited understanding of the influence of
structure, molecular organisation and physicochemical prop-
erties of biomembranes on the mechanism of AMP action. In
particular, the quantitative analysis of these properties is
central to characterising the mechanistic steps of AMP bind-
ing to membrane and will be discussed below.
4. EXPLORING REAL TIME CHANGES IN LIPID
MOLECULAR ORGANISATION ASSOCIATED
WITH AMP MEMBRANE BINDING USING
WAVEGUIDE-BASED OPTICAL BIOSENSORS
In principle, the binding of AMPs to the membrane re-
sulting in membrane damage involves a degree of change in
membrane packing organization of lipid molecules. The
changes in membrane packing order have been studied by
the incorporation of probes in lipid bilayer or use a probe-
labeled (tagged) lipids with the fluorescence spectroscopy
and NMR [88-90]. However, these changes in the membrane
order must be correlated with the amount of peptide bound to
the membrane to differentiate between the molecular
changes associated with surface bound and inserted peptides.
Label-free optical biosensors have been widely used for real-
time qualitative and quantitative measurement of pep-
tide/protein-membrane interactions [91]. While the com-
monly used biosensor instruments such as SPR determine
mass-only measurements [91, 92], simultaneous membrane
structural information can be obtained by waveguide-based
instruments as described below.
4.1. Simultaneous Measurement of Membrane Anisot-
ropy (Birefringence) and Peptide Binding
The first step in characterising the structure of a mem-
brane is the accurate measurement of the bilayer properties
such as thickness, density and mass. The successful deposi-
tion of defect–free lipid bilayers of various compositions
onto the planar sensor chip can be characterised using optical
waveguide-based sensors, such as dual-polarisation interfer-
ometry (DPI) [93-96]. In particular, the structural properties
of the lipid bilayer in terms of changes in the optical thick-
ness and mass can be monitored in real time for lipid bilayer
formation. However, a single parameter such as mass or
thickness alone is not sufficient to represent the dynamic
changes in the structure of the lipid bilayers. What is now
possible is the measurement of the disordering of the bilayer,
32 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1 Lee et al.

that is, the changes in the molecular packing characteristics
of the lipid chain surrounding the membrane-adsorbed pep-
tides. Owing to the liquid crystal properties of lipid mole-
cules self-assembled into uni-axial aligned bilayers, one of
the unique structural properties of phospholipid bilayers is
that the ordered orientation of lipid molecules in a membrane
creates a unique optical anisotropic property, which shows
differences in refractive index (RI) for two orthogonal po-
larisations, ie, ne and no in a uniaxial optical axis, and shown
schematically in Fig. (4). The difference between these two
RIs for a lipid film is defined as the birefringence (∆nf),
where ∆n f = n e − n o . Furthermore, the degree of molecular
order, S, of the uniaxial lipid bilayer as defined by the ratio
of the principal polarizabilities of the bilayer to the molecu-
lar polarizabilities is proportional to the birefringence values
[97]. Thus, the birefringence values represent an averaged
measurement of lipid molecular orientation order and the
lipid acyl chain packing order. High ∆nf values are obtained
for a fully aligned lipid bilayer with fully saturated acyl
chains whereas low ∆nf values are determined for those bi-
layers with unsaturated acyl chains assembled in a random
and disordered system. A typical bilayer has a birefringence
of 0.015-0.025 refractive index units as determined by DPI
[93, 95, 96, 98, 99]. Thus, simultaneous measurement of
both mass and birefringence with high sensitivity can there-
fore characterise very subtle dynamic changes in orientation
and packing order of lipid molecules as a function of mem-
brane-bound peptide mass. The simultaneous measurement
of mass and structural parameters can also assist in delineat-
ing mechanisms of interaction that are difficult if not impos-
sible to observe by other means. The ability to measure bire-
fringence is thus the most significant feature of the DPI in-
strument in terms of investigating membrane-mediated
events.
Due to the direct action of AMPs on the cell membrane,
the main challenge is to understand the exact interplay be-
tween factors that control AMP structure and binding and
cell membrane organsiation that underpin phospholipid
selectivity and toxicity to cells, particularly in terms of the
changes in bilayer structure. The ability to characterise
both mass and birefringence simultaneously using DPI al-
lows the analysis of the relationship between the two prop-
erties to be measured in the interactions between AMPs and
lipids (Fig. 4). In the first instance, the real-time changes of
the mass response yields information on binding which is
analogous to other optical biosensors such as SPR. How-
ever, the simultaneous dependence of birefringence and
mass on time (as depicted in Fig. (4) provides new informa-
tion on the simultaneous changes in membrane structure
during the binding event. More significantly, the combina-
tion of these plots to give the birefringence vs. mass plots
(as illustrated in Figs. (5 and 6) reveals an enormous
amount of new information. In particular, a number of tran-
sitions can be described which can be used to evaluate pep-
tide behaviour and mechanism of action (discussed below
in Section 5).

Fig. (4). The dual polarisation interferometer (DPI) consists of a dual slab waveguide guiding two orthogonal polarisations light through
high-refractive index sensing and reference waveguide. (A) The two polarization modes, TM and TE, pass through the waveguide forming
two evanescent fields which can effectively result in phase shift for each TM and TE by the molecules binding onto the surface. Such
changes in phase shift are detected as changes in the interference fringe pattern at the far-field. Thus, changes in TM/TE phase shift can be
calculated as the layer thickness and RI for characterizing the lipid bilayer formation and the dynamic impact of peptide binding on the lipid
bilayer structure. Birefringence is a measure of molecular packing order. (A) A fully aligned/ordered bilayer has larger optical birefringence
(∆nf) than (B) a disordered unilamellar supported lipid bilayer which can effect from a peptide insertion. Thus, the changes in the packing,
alignment and degree of order of lipid molecules assembled on the surface can be determined from quantitative analysis of changes in bire-
fringence. (B) The simultaneous measurement of birefringence and mass provides novel method in delineating the mechanisms of the impact
of bound peptides on membrane structure.
Antimicrobial Peptide Structure and Mechanism of Action
4.2. Lipid Bilayer Perturbation and Disruption by AMPs
Both the initial binding of an AMP, and any subsequent
processes, may affect both the mass and structural ordering
of the membrane and analysis of mass-birefringence plots,
derived from the phase changes of DPI containing both mass
and structural axis reveals the changes in membrane ordering
that occur during peptide binding. This graphical analysis of
the correlation between binding and structural changes has
been used to evaluate the accumulated impact of a number of
peptides to effect progressive changes in membrane structure
and has greatly assisted in describing the membrane pertur-
bation process and disruption by AMPs.
The binding of AMPs to a membrane always involves
some increase in mass (at least initially) and may also be
accompanied by changes in birefringence and the depend-
ence of birefringence on the mass of bound peptide can be a
simple linear relationship or can lead to a series of more
complex binding-disorder profiles as shown in Figs. (5 and
6). The simpler plots can range from a horizontal to shallow
slope (corresponding to surface binding that has only a small
effect on birefringence), through to a near vertical decrease
in birefringence (corresponding to a substantial disruption of
the membrane structure), and several other cases that fall
between the two extremes [26, 89, 94-96, 100-103].
The simplest plot of AMP binding to membranes results
in an essentially linear decrease in the bilayer birefringence
with increasing peptide mass bound to membrane, as shown
for citropin 1.1 binding to DMPC (Fig. 5) and also seen for
HPA binding to DMPC and DMPC/DMPG (80:20) [94,
102]. This linear decrease in birefringence immediately after
peptide binding is a characteristic pattern for peptides incor-
porated into the membrane without a threshold of coverage
on the membrane surface [94]. This simple profile without a
Fig. (5). Schematic profiling the different effects of Australia tree frog AMPs, aurein1.2, citropin 1.1, maculatin1.1 and caerin1.2, on the
structure of DMPC and DMPC/DMPG bilayers which can be delineated by studying the correlation of bilayer disordering to the peptide
mass bound to the bilayer. The red-dotted arrows in birefringence (∆nf)–mass plots indicate the direction of changes in birefringence with
changes in mass during the association phase. (Adapted from [102])
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1 33
threshold point in the mass-birefringence plot has been ob-
served for aurein 1.2 binding to E coli lipid extract and
DMPE/DMPG [95], HPA3P binding to DMPC and
DMPC/DMPG [94], citropin 1.1, maculatin 1.1 and caerin
1.1 binding to DMPC (Fig. 5) [102], and a series of peptides
with antimicrobial activity including peptides derived from
various coagulation factors binding to DOPE/DOPG [104],
C-terminal peptides from S1 peptidases to DOPE/DOPG
[104], novicidin to DOPC/DOPG [105] and KYE28, KYE21
and NLF binding to DOPE/DOPG [106].
By comparison, biphasic binding-disorder plots can be
observed with changes in peptide sequence or changes in
lipid composition and gel-liquid phase. For example, the
binding of HPA3 and maculatin 1.1 to a gel-phase DMPC
bilayer can be compared where HPA3 displayed a simple
linear plot while maculatin 1.1 displayed a biphasic plot. In
particular, for maculatin 1.1, linear behaviour with bilayer
disordering [26, 89, 94, 96] was preceded by mass increases
but no bilayer disorder at the point of initial binding, which
was then followed by an abrupt non-linear disordering upon
further increases in mass. This biphasic change (correspond-
ing to the transition from a surface bound to an inserted state
above a threshold concentration) was caused by a proline-
induced helical kink in maculatin 1.1, as analogues in which
proline was replaced required much higher concentrations of
peptide to achieve this membrane disruption. Moreover, the
biphasic mass-birefringence plots were not apparent in the
fluid state (ie at higher temperatures) suggesting that the
kinked peptide structure was able to insert more easily into
the more fluid bilyaer. In contrast, the proline-substituted
HPA3 analogue, HPA3P, showed a simple linear decrease in
bilayer order with the fluid and the gel-DMPC/DMPG bi-
layer [96]. These studies clearly demonstrate that the role of
proline in various AMPs in bilayer perturbation can be de-
34 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1 Lee et al.

lineated by the changes in birefringence which is not possi-
ble by mass-only measurements. These distinctively differ-
ent features in binding to DMPC/DMPG compared to similar
binding behaviour with DMPC between HPA3 and macu-
latin 1.1 also demonstrate the importance in differentiating
the membrane structural changes from the peptide binding,
since in the absence of the birefringence data, the main con-
clusion would be that both peptides have similar binding
mechanisms.
The birefringence-mass profiles can also provide signifi-
cant new insight that redefines current models of AMP ac-
tion [95, 100, 102]. In particular, the sequential steps of
binding, insertion and bilayer disruption can be examined in
real time at continuous increments of peptide:lipid (P:L) ra-
tio. While many studies focus on defining the critical con-
centration for membrane destruction, characterising the steps
associated with different extents of bilayer perturbation at a
distinctive P:L threshold provides a much clearer under-
standing of the action of AMPs on the membrane. Aurein 1.2
and magainin 2 are two examples of naturally isolated AMPs
from the dorsal secretion of an Australian tree frog and an
African frog, respectively. Despite the difference in sequence
and in particular the length, with 13 and 23 amino acids for
aurein 1.2 and magainin 2, respectively, both peptides adopt
an amphipathic helix in an anionic membrane environment,
while no structure was found for magainin2 in a neutral
membrane environment. The carpet mechanism-linked de-
tergent model has been proposed for aurein 1.2 and a carpet-
induced pore formation for magainin peptides lyse mem-
branes based on similar critical peptide:lipid (P:L) ratios
measured by conventional means. However, information on
membrane structure changes immediately prior to, during
and after membrane lysis obtained from DPI showed appar-
ent differences for these two peptides which were previously
described as acting by the same carpet mechanism. The bire-
fringence-mass plots for magainin2 are shown in Fig. (6A-
D) and can be compared to the data for aurein 1.2 in Fig. (5).
Firstly, the P:L ratio determined at the point of disruption for
an anionic membrane was markedly lower for aurein 1.2
than for magainin 2 (Table 2). Moreover, an alanine-
substituted analogue, Ala8,13,18-mag2 with enhanced bacteri-
cidal activity also showed disruption of the anionic mem-
brane at a P:L ratio similar to magainin 2. However, the
Ala8,13,18-mag2 also disrupted the neutral DMPC membrane
[100] at nearly double the P:L ratio. From the high P:L ratio
reflecting the saturated surface coverage at the point of
membrane disruption, a “carpet mechanism” would be sim-
ply attributed to the mechanisms of action of these frog pep-
tides. However, the drop in birefringence was reversible for
aurein 1.2 after its disruption of both neutral DMPC and ani-
onic DMPC/DMPG membranes [95, 100, 102]. In contrast, a
partially reversible birefringence drop with permanent disor-
dering was obtained for Ala8,13,18-mag2 after disrupting the
membranes. Such partially reversible birefringence changes
with membrane disruption are consistent with peptide inser-
tion and bilayer expansion as evident from AFM studies
[100]. Both fluorescence dye release and AFM results also
showed that aurein 1.2 and magainin 2 and Ala8,13,18-mag2
disrupt the membrane differently [95, 100, 102]. Overall,
these experiments clearly demonstrate that AMP action con-
sists of complex sequential intermediates that define the
steps leading to cell death. The specific models describing
the end-point configuration (eg, barrel stave, toroidal, and
carpet-like) can mislead the interpretation of mechanism

Fig. (6). Birefringence versus mass plots for the interaction of magainin 2 with (A) DMPC and (B) DMPC/DMPG or Ala8,13,18-mag2 with
(C) DMPC and (D) DMPC/DMPG. (E) Interconversion of membrane structure intermediates as exemplified by the interaction of magainin 2
and the analogue Ala8,13,18-Mag2 with lipid bilayers as a template for antimicrobial peptide action. This model describes alternate pathways
of intermediate membrane states that occur in the presence of increasing amounts of bound peptide (PLX) and the prevalence of each state is
dependent on the peptide and the lipid composition. (Adapted from [100]).
Antimicrobial Peptide Structure and Mechanism of Action
without details on membrane structural changes. In particu-
lar, the reversible packing disorder-order behaviour observed
for different bilayers is quite distinctive for various AMPs,
behaviour which is not accessible by other spectroscopic
techniques. The analysis of membrane disordering has there-
fore extended our understanding of how the membrane un-
dergoes a reversible structural change upon exposure to an-
timicrobial peptides and how the bilayer can either recover
or, at a critical peptide concentration, begins to disintegrate.
Table 2. Molar ratio of peptide to lipid determined at the
point where mass loss and membrane disruption.
DMPC DMPC/DMPG (4:1)
Aurein 1.2 1:14 1:23
Magainin 2 - 1:15
8,13,18
Ala -Magainin 2 1:8 1:13
Together with DPI, the experimental design allows both
the acquisition of kinetic data on bilayer formation via vesi-
cle adsorption [93] and subsequent analysis of peptide-
membrane interaction, throughout the entire sequence of
events involving peptide binding, insertion and membrane
destruction. DPI provides simultaneous quantification of real
time changes in the thickness, mass/density and birefrin-
gence of the membrane. While optical birefringence is a
well-known property for aligned bilayer membranes [93, 97,
107-109], changes to these properties during peptide interac-
tions have been less studied [94, 108, 110]. Since the bire-
fringence quantifies the degree of alignment and uniaxial
packing of the lipid molecules on the planar surface, changes
in birefringence as a function of peptide binding to the mem-
brane provide a unique insight into the mechanism of bind-
ing, and the rate and concentration dependent changes in
lipid packing and membrane destabilisation. Thus, emphasis
can now be placed on examining the geometric and dynamic
arrangements within a lipid matrix and the functional role of
lipid molecular ordering in peptide and lipid interactions.
Overall, these studies have shown that DPI analysis of AMP
binding data correlates well with previous biosensor [27, 94,
95, 100] and spectroscopic studies such as NMR[89], neu-
tron reflectometry (NR) [26] and AFM imaging [100]and
provides new data on the dynamic, geometric changes in
membrane structure leading to a more detailed understanding
of the multiple steps associated with peptide-membrane in-
teractions.
5. MEMBRANE PLASTICITY: MEMBRANE ORDER-
ING PROFILES GIVE NEW INSIGHTS INTO MULTI-
STATE KINETIC PROCESSES
Membrane bilayer components alter the physiological
profiles of a vast number of membrane proteins, either
through specific interactions with the protein itself or altera-
tion of membrane physical properties such as curvature, lat-
eral pressure, and bilayer thickness [51, 111]. For a large
number of cases, the available evidence points to an impor-
tant link between physicochemical properties for AMP ac-
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1 35
tion, and the surrounding membrane composition. For exam-
ple, as discussed above, antimicrobial peptide action is con-
trolled by the nature of the bacterial and mammalian mem-
brane [6].
The data that is now available from techniques such as
DPI provides the opportunity to categorise membrane-
mediated events in terms of patterns of membrane structure
changes. In particular mass-birefringence plots, which reveal
the changes in membrane ordering that occur during peptide
binding, now allow very detailed and subtle differences in
the interactions between lipids and peptides to be visualised.
This birefringence analysis provides novel information on
the impact of peptides on the changes in organisation occur-
ring within a lipid bilayer, and assists in describing the be-
haviour of membrane-active peptides when interacting with a
lipid membrane. Overall a number of transitions can be de-
scribed based on the birefringence vs. mass plots (as illus-
trated in Figs. 5 and 6), which can be used to evaluate pep-
tide behaviour and mechanism of action and are the centre-
piece of this new membrane biophysical methodology. The
molecular events corresponding to these four transitions are
schematically drawn in Fig. (5) in the context of aurein 1.2,
citropin 1.1, maculatin 1.1 and caerin 1.1 [89, 95, 96, 100,
102].
Based on the multiple stages of membrane structure
changes evident in Fig. (5), specific models can be defined
for the molecular mechanism of action in terms of a series of
intermediate states, each representing an ensemble of
closely-related bilayer structures. In most cases, three gen-
eral membrane-inserted states are outlined corresponding to
(1) surface-bound peptides (2) partially inserted peptides and
(3) significantly inserted peptides. Different degrees of bi-
layer disorder reflect the lipid perturbation by each of these
states. The understanding of structural changes in a mem-
brane also allow the description of alternate pathways of
intermediate membrane states that occur in the presence of
increasing amounts of bound peptide and the prevalence of
each state which is dependent on the peptide and the lipid
composition. This mass-structural change model, based on
birefringence changes at critical mass loadings, allows the
properties of membrane-active peptides to be analysed in
sequential steps of surface binding, insertion, membrane
opening and bilayer lysis, and in more detail than has been
previously possible in terms of dynamic structural changes.
In particular, the molecular events prior to membrane lysis
can be fully characterised in relation to the physicochemical
properties of membrane and provides new criteria that can
guide the design of selective antibacterial peptides. An im-
portant element to this endeavour will be the ability to derive
affinity and rate constants for the different states and initial
success has been reported for the description of three mem-
brane states [101, 112].
Characterizing a series of intermediate states that corre-
spond to different degrees of bilayer disruption in terms of
antimicrobial peptide action can also provide a new basis for
understanding the membrane lysis in terms of membrane
plasticity and its ability to recover from peptide assault.
Based on the membrane plasticity model of the multiple
stages of membrane structure changes, the ability of a dam-
aged membrane to recover from the effect of peptide binding
36 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 1
may also assist in the understanding of mechanisms of mem-
brane repair. Significantly, these results also demonstrate
that subtle differences in peptide structural properties can
modulate the mechanism of peptide action on membranes
and provides new approaches towards the design of peptides
with selective membrane activity.
A very significant outcome of these studies is therefore
the ability to track the changes in membrane structure disor-
der and correlate these changes with the amount of bound
peptide. In terms of the different structural states adopted by
the bilayer, it has been previously shown that the presence of
DMPG in DMPC leads to the formation of an isotropic state
and that the negative charges on the membrane surface re-
sulted in a greater susceptibility of the bilayer to peptide-
induced disordering [89, 95, 96, 100, 102]. Based on the
multiple stages of membrane structure changes such as those
evident in Figs. (5 and 6), we propose a new membrane plas-
ticity model for antimicrobial peptide action in terms of a
series of intermediate states as follows and depicted sche-
matically in Fig. (6E). This model describes alternate path-
ways of intermediate membrane states that occur in the pres-
ence of increasing amounts of bound peptide (PLX) and the
prevalence of each state is dependent on the peptide and the
lipid composition. For magainin 2 on DMPC, the pathway
comprises PLA – PLB – PLC - PLA′ states corresponding to
reversible changes in membrane order, while on
DMPC/DMPG, magainin 2 binding follows the PLA – PLB –
PLC – PLC’ pathway characterised by reversible changes in
membrane order with a net loss in mass. By comparison, the
binding of Ala8,13,18-Mag2 to DMPC can be described by the
PLA – PLB – PLD – PLE – PLF – PLG pathway and on
DMPC/DMPG Ala8,13,18-Mag2 follows the PLA – PLB – PLD
– PLE – PLF – PLG – PLH pathway characterised by the induc-
tion of irreversible membrane disorder and is associated with
insertion, pore formation, membrane expansion and mass
loss [100].This model, which is based on birefringence
changes, now allows the properties of membrane-active pep-
tides to be analysed in terms of surface binding, insertion,
membrane opening and bilayer lysis and in much more detail
than has been previously possible. In particular, the molecu-
lar events prior to membrane lysis can be fully characterised
in terms of bilayer structural changes and provides new crite-
ria that can guide the design of selective antibacterial pep-
tides. For example, the different pathways are associated
with both reversible and irreversible transitions and the abil-
ity to differentiate and more importantly to enhance one
pathway over the other provides the definition of selectivity
control; ie can a peptide be designed to induce the irreversi-
ble pathway in a target bacterial membrane but induce re-
versible and recoverable changes in the host cell membrane?
CONCLUSION
Society is currently facing a public health crisis with the
increasing resistance to antibiotics. While peptides offer
enormous potential as a new class of antibiotics, we have yet
to see an AMP reach the market for internal use. A major
issue facing researchers is the difficulty in untangling the
factors that govern membrane selectivity of AMPs, which in
turn prevents a clear definition of the mechanism of action of
these peptides. Without this information, the quest to design
highly selective non-toxic AMPs will continue to be unsuc-
Lee et al.
cessful. While a majority of studies focus on peptide struc-
ture and how it changes in response to binding to a mem-
brane, it is now clear that the membrane itself undergoes
substantial structure change during AMP binding, but the
details and extent of this membrane structure change is very
poorly understood.
This review therefore discusses the recent studies which
provide new information on the changes in membrane struc-
ture based on measurements of bilayer birefringence. The
simultaneous measurement of this parameter with bound
mass significantly expands the potential of optical biosensors
to provide new insights into membrane-mediated cellular
events. Compared to reasonably similar overall profiles ob-
tained by SPR, the wide variety of membrane structure
changes evident by DPI could not be anticipated and has
opened up exciting opportunities for more detailed under-
standing of a vast range of membrane–mediated biological
events. The examples described above demonstrate how
knowledge of the membrane structure changes significantly
informs our understanding of a wide range of biological
processes. So the question is what are the specific structural
features of each phospholipid bilayer that influence the rela-
tive binding and disruptive properties by each peptide?
Moreover, what are the specific physical properties of each
bilayer that influence its ability to survive or succumb to a
peptide assault? The answers to these questions will no
doubt emerge as we move forward in the quest to establish
new AMP design criteria to tackle the on-going crisis of an-
tibiotic resistance.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The financial support of the National Health & Medical
Research Council (#1044327) and the Australian Research
Council (DP1110101866) is gratefully acknowledged.
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