Article

Selective Membrane Disruption Mechanism of an

Antibacterial g-AApeptide Defined by EPR
Spectroscopy
Pavanjeet Kaur,1 Yaqiong Li,2 Jianfeng Cai,2,* and Likai Song1,*
1
National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida; and 2Department of Chemistry, University of South
Florida, Tampa, Florida

ABSTRACT g-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are
highly resistant to protease degradation. It is not clear how g-AApeptides interact with bacterial membranes and alter lipid as-
sembly, but such information is essential to understanding their antimicrobial activities and guiding future design of more potent
and specific antimicrobial agents. Using electron paramagnetic resonance techniques, we characterized the membrane inter-
action and destabilizing mechanism of a lipo-cyclic-g-AApeptide (AA1), which has broad-spectrum antibacterial activities.
The analyses revealed that AA1 binding increases the membrane permeability of POPC/POPG liposomes, which mimic nega-
tively charged bacterial membranes. AA1 binding also inhibits membrane fluidity and reduces solvent accessibility around the
lipid headgroup region. Moreover, AA1 interacts strongly with POPC/POPG liposomes, inducing significant lipid lateral-ordering
and membrane thinning. In contrast, minimal membrane property changes were observed upon AA1 binding for liposomes
mimicking mammalian cell membranes, which consist of neutral lipids and cholesterol. Our findings suggest that AA1 interacts
and disrupts bacterial membranes through a carpet-like mechanism. The results showed that the intrinsic features of g-AApep-
tides are important for their ability to disrupt bacterial membranes selectively, the implications of which extend to developing new
antibacterial biomaterials.

INTRODUCTION
Antibiotic resistance is one of the major threats to public
health; therefore, it is essential to develop more effective
antibacterial reagents with minimum microbial resistance.
Antimicrobial peptides (AMPs), both natural and synthetic,
have shown antimicrobial activities without inducing resis-
tance in bacteria (1). This has generated interest in the utility
of AMPs to curtail bacterial infection (2). One major differ-
ence between conventional antibiotics and AMPs lies in
their distinct mechanisms of antimicrobial activities. Antibi-
otics exert their antibacterial abilities by interfering with the
pathways in protein synthesis, cell wall synthesis, or DNA/
RNA replication (3). These pathways are prone to be
avoided by genetic mutations of bacteria and thereby induce
antibiotic resistance due to their defined targets (4). In
contrast, the antimicrobial activities of AMPs are attained
by the nonspecific interactions of AMPs with bacterial
Submitted October 26, 2015, and accepted for publication February 19,
2016.
*Correspondence: jianfengcai@usf.edu or song@magnet.fsu.edu
Editor: David Cafiso.
http://dx.doi.org/10.1016/j.bpj.2016.02.038
Ó 2016 Biophysical Society
membranes, which lead to membrane disruption. In partic-
ular, AMPs are capable of selectively interacting with bac-
terial membranes versus mammalian cell membranes. The
former contains ~25% negatively charged lipids, whereas
the latter is composed of mostly neutral lipids (zwitterionic)
with high cholesterol content in the outer leaflets (5–7). For
bacterial membranes, the interaction of cationic AMPs with
anionic lipids contributes to peptide-induced membrane
permeability changes and cell death, as shown in many
AMPs, including magainins (8–11). In contrast, for
mammalian membranes, the presence of a large amount of
cholesterol inhibits the membrane disruption activities of
AMPs (5,12–14).
Several mechanisms have been proposed for the antibac-
terial activities of AMPs, including carpet, toroidal-pore,
barrel-stave, lipid-clustering, and interfacial activity (15–
18). In the carpet model, AMPs accumulate on the mem-
brane surface; the lipid bilayers are destabilized after a
critical concentration has been reached (19,20). In this
model, membrane disruption may include the formation of
transient pores or a global collapse of the membrane
through detergent-like behaviors of some AMPs (19,20).

Biophysical Journal 110, 1789–1799, April 26, 2016 1789

Kaur et al.
The barrel-stave mechanism is initiated by the penetration
of AMPs into lipid bilayers, leading to hydrophilic pores
formed by peptide–peptide interactions (21). In the
toroidal-pore model, on the other hand, pores are formed
by both AMPs and lipid molecules (22). In the lipid-clus-
tering model, AMPs binding to the membrane surface cause
charged lipid clustering and the phase separation of lipids
(23). The interfacial-activity model proposes that AMP ac-
tivity is initiated through the binding of peptides to the
membrane interfacial region. This leads to disruption of
the segregation of the interfacial and hydrophobic regions
of the membrane, which results in membrane leakage
accompanied by translocation of lipid molecules, peptides,
and solutes (24). These models are able to explain the
AMP activities, but the detailed molecular mechanisms
and the changes in lipid organization on AMP binding are
still not completely understood.
Despite exhibiting broad spectrum activities against
Gram-positive and Gram-negative bacteria, the applications
of natural AMPs remain limited due to their susceptibility to
proteolytic degradation (25). Synthetic peptidomimetics
become an attractive alternative by mimicking the antibac-
terial activities of AMPs with no protease vulnerability
(26). Recently, we developed a new family (to our knowl-
edge) of synthetic peptidomimetics: g-AApeptides (Fig. 1)
(27). They are termed ‘‘g-AApeptides’’ as they are oligo-
mers of n-acylated-n-aminoethyl amino acids. g-AApepti-
des can project the same number of side chains as
a-peptides with the same lengths. In addition, g-AApepti-
des are known to resist proteolytic degradation, and are
amendable for derivatization with diverse functional groups.
They have shown promise in biological applications, one of
which is the development of antimicrobial agents that mimic
the mechanisms and functions of AMPs (28–30). Among a
few types of antimicrobial g-AApeptides, lipo-cyclic-g-
AApeptides display intriguing antimicrobial activity (29).
In the structures of lipo-cyclic-g-AApeptides, lipid tails
were introduced to ring structures to increase the lipophilic-
FIGURE 1 Structure of lipo-cyclic-g-AApeptide 1 (A) and comparison
of an a-peptide (B) and a g-AApeptide (C). A g-AApeptide is comparable
to an a-peptide in unit length and half of the side chains of a g-AApeptide
are linked to the amide groups. The lipo-cyclic-g-AApeptide 1 contains a
cyclic g-AApeptide and a lipid tail.
1790 Biophysical Journal 110, 1789–1799, April 26, 2016
ity of the molecules so as to enhance their interactions with
bacterial membranes (Fig. 1). The lead compound 1 (AA1)
shows potent and broad-spectrum activity against multi-
drug-resistant Gram-positive and Gram-negative bacteria
(29). In addition, it can mimic AMPs to modulate immune
response. Fluorescence studies have suggested that lipo-cy-
clic-g-AApeptides might be able to selectively depolarize
and disrupt both Gram-positive and Gram-negative bacterial
membranes, eventually causing bacterial cell death (29).
However, the molecular bases for the selectivity, changes
in lipid organization, and the disruption mechanism are
not known.
Electron paramagnetic resonance (EPR) has been shown
to be a robust tool for characterizing membrane properties,
including dynamics and lipid ordering (31–36). Moreover,
EPR has been used to determine the membrane interactions
of CM15 (37), Cecropins (18,38), and Melittin (39). In this
work, we applied and further developed, to our knowledge,
novel EPR techniques to characterize biological membranes
and protein–lipid interactions. These techniques include: (1)
membrane permeability analysis to assess membrane
disruption by proteins, (2) lipid lateral ordering induced
by protein binding defined using EPR at 94 GHz, and (3)
a new (to our knowledge) application of EPR power satura-
tion methods to determine membrane thinning. To obtain an
EPR signal, EPR spin probes are attached to different posi-
tions of phospholipid molecules (Fig. S1 in the Supporting
Material). We expect these spin probes at varied positions
will reveal a full picture of membrane properties across
the bilayer.
In this study, we employed EPR techniques to elucidate:
(1) the molecular basis of the membrane-disruptive activ-
ities of AA1 on bacterial membranes; and (2) AA1-induced
membrane property changes, including membrane perme-
ability, fluidity, solvent accessibility, and lateral order. The
negatively charged bacterial membranes were mimicked
by POPC/POPG (4:1 wt/wt) liposomes (40–42). Because
the mammalian cell membranes contain mostly neutral
lipids with high cholesterol content ranging from 20 to
50 mol % (43,44), the corresponding membrane mimic
was used with POPC and 30–50 mol % cholesterol. We
found that AA1 selectively interacts and disrupts the bacte-
rial-mimic membranes over the mammalian-mimic mem-
branes, possibly through carpet-like interactions. These
results will give insight into the mechanism of the action
of lipo-cyclic-g-AApeptides, and therefore provide a basis
for the design of more potent and selective antimicrobial
agents.
MATERIALS AND METHODS
Materials
Phospholipids POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine),
POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]), T-PC

(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho -tempocholine), N-TP (N-tem-
poyl palmitamide), 5-PC (1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-
phosphocholine), 7-PC (1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-phos-
phocho-line), 10-PC (1-palmitoyl-2-stearoyl-(10-doxyl)-sn-glycero-3-
phosphocholine), 12-PC (1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3-
phosphocholine), and CHOL (cholesterol) were purchased from
Avanti Polar Lipids (Alabaster, AL). 5-SASL (2-(3-carboxypropyl)-4,
4-dimethyl-2-tridecyl-3-oxazolidinyloxy), 5-MeSL (2-(4-methoxy-4-oxobu-
tyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy), 4-PT (4-phosphonooxy-
TEMPO), and VC (vitamin C) were purchased from Sigma-Aldrich
(St. Louis, MO).
Synthesis of AA1
The peptide was synthesized in the solid phase following the reported pro-
tocol (27). Briefly, synthesis and assembling blocks of lipo-g-AApeptides
followed a standard protocol of solid-phase synthesis using Fmoc-chemis-
try on a Rink amide resin (Sigma-Aldrich). The N-terminus of AA1 was
lipidated by reactions with lauric acid, palmitic acid, or oleic acid using
DIC (diisopropylcarbodiimide)/DhBtOH (oxohydroxybenzotriazole) as
activation agents. After the desired sequences were assembled, the peptides
were cleaved from the solid support using 50:45:5 TFA/CH2Cl2/triisopro-
pylsilane overnight. The solvent was evaporated and the residues were
analyzed and purified on an HPLC instrument (Waters, Milford, MA).
The desired fractions (>95% purity) were collected and lyophilized. The
molecular weights of the peptides were determined using an AutoFlex
MALDI-TOF mass spectrometer (Bruker, Billerica, MA).
Liposome preparation
Large unilamellar vesicles (LUVs) were prepared as described in Hope
et al. (45) and Szoka et al. (46). Lipids in chloroform were mixed in a glass
tube and dried as thin films under a stream of nitrogen gas. To obtain EPR
signals, 1–2 mol % of spin-labeled lipid analogs (Fig. S1) were added to the
lipid mixture. To remove residual organic solvent, the lipid films were
further dried using a vacuum pump for ~16 h. The lipids were resuspended
in an HK buffer (20 mM HEPES, 150 mM KCl, pH 7) by vortexing for
1–2 min and then subjected to 10–15 freeze-and-thaw cycles. This was
followed by extruding the lipid suspension 10–15 times through a mini-
extruder with a 100 nm polycarbonate membrane (Avanti Polar Lipids).
 1

EPR spectroscopy
Membrane fluidity and permeability measurements
The measurements were carried out on a model No. E680 X-/W-band contin-
uous-wave and pulsed EPR spectrometer (Bruker) with a high-sensitivity
resonator (model No. 4119HS; Bruker) at the National High Magnetic Field
Laboratory (NHMFL). Spectra were collected at 2 mW microwave power
with a field modulation frequency of 100 kHz and a modulation amplitude
of 1–2 Gauss (G) at room temperature. Samples were loaded in 0.6 mm inner
diameter glass capillary tubes. For membrane fluidity measurements, LUVs
with 1 mol % of spin-labeled lipid analogs (Fig. S1) were prepared in the HK
buffer as described earlier with a 10 mM lipid concentration. The AA1 pep-
tides were added to LUVs at a lipid/peptide ratio (L/P) of 10–80:1.
A modified 4-PT/VC quenching assay was performed to determine mem-
brane permeability (47,48). Briefly, a stock of water-soluble 4-PT spin la-
bels was prepared in the HK buffer. 10 mM LUVs containing 4-PT were
prepared using the extrusion method described earlier. 4-PT-loaded LUVs
were dialyzed against the HK buffer for 48 h to remove the untrapped
4-PT and were subsequently equilibrated with 1 mM VC for 30 min. The
AA1 peptides were added to the liposome samples at desired L/Ps. EPR
signal reduction as a result of membrane penetration and 4-PT/VC mixing
was recorded for 60 min after adding the peptides. Control experiments in
Membrane Disruption by a g-AApeptide
the absence of AA1 showed no significant 4-PT signal reduction for the li-
posomes used in this study during this timescale (Fig. S2).
Accessibility measurements
To determine solvent accessibility, EPR power saturation experiments were
performed on a model No. E680 spectrometer (Bruker) at 9.5 GHz using a
loop gap resonator (Molecular Specialties, Milwaukee, WI). LUV samples
were loaded in gas-permeable TPX capillary tubes (Molecular Specialties)
and purged using either a stream of air or N2 gas. EPR spectra were
collected at microwave powers ranging from 0.4 to 100 mW with a modu-
lation field of 2 G and a modulating frequency of 100 kHz. The accessibility
measurements were performed either in the presence of a nonpolar reagent
O2 or an uncharged polar reagent NiEDDA (nickel(II) ethylenediaminedia-
cetate, 50 mM). NiEDDA accessibility measurements were carried out in a
degassed environment, i.e., purged with N2 gas. The accessibility of both
reagents was quantified using an accessibility parameter (P) (49,50) using
DPPH (Bruker) as a standard. The depth parameter, F (50), was calculated
from the ratio of the accessibility value P(O2) to P(NiEDDA).
Lipid lateral ordering measurements
EPR spectra at 94 GHz or higher are sensitive to lipid lateral ordering (36).
High-frequency high-field EPR improves spectral resolution through
increased g-factor sensitivity, enabling the determination of the motionally
averaged gxx-gyy anisotropy, which reflects lipid lateral ordering. A brief
description of the calculation of the lateral order parameter is as follows:
Eqs. 1 and 2 define the axial and azimuthal anisotropy of the g-tensor, respec-
tively. The motionally averaged g-anisotropy is given by Eqs. 3 and 4, where
qA is the tilting angle of the z axis of a spin label with regard to the membrane
normal (Fig. S3). Angle 4 (with maximum amplitude 5 40 ) represents the
rotation of the nitroxide x-z plane with regard to the Z (magnetic field
axis)-z plane (Fig. S3). The nitroxide mean x axis is tilted at angle 4, which
is the mean value of 4. The angular brackets represent angular averages:
1 
Dg ¼ gzz -- gxx þ gyy ; (1)
2
1 
dg ¼ gxx --gyy ; (2)
2
hDgi ¼ hP2 ðcosqA ÞiDg; (3)
dg ¼ 2 þ 3 cosqA þ P2 ðcosqA Þ cos2ð4--4Þ dg:
6
(4)
The lateral ordering is represented by cos2ð4--4Þ and the conven-
tional order parameter is defined by hP2 ðcosqA Þi, where P2 ðcosqA Þ ¼
(1/2) (3cos2qA – 1).To calculate the lateral ordering, the spin-label motion
is constrained in a cone with a half-angle b, in which case hP2 ðcosqA Þi ¼
ð1=2Þcosbð1 þ cosbÞ and hcosqA i ¼ ð1=2Þð1 þ cosbÞ (51). The value b
can be calculated using Eq. 3 and hcos2ð4--4Þi can be obtained from Eq. 4.
The motionally averaged hgxxi and hgzzi values are determined from the
peak maxima in the low-field and high-field regions of the EPR spectra,
respectively. The value hgxxi can also be resolved using the derivative of
the EPR spectrum, i.e., the second derivative of the absorption spectrum.
The value hgyyi is obtained from the maximum slope in the intermediate re-
gion of the EPR spectrum or the peak maximum in the integrated EPR spec-
trum (absorption-like). The g-tensor components gxx, gyy, and gzz were
determined from the rigid limit spectra at 150 K. The values are 2.0090,
2.0062, and 2.0023, respectively, for POPC/POPG liposomes with 5-SASL.
To examine the lateral ordering of lipids, EPR spectra were recorded on a
W-band 94 GHz continuous-wave and pulsed EPR spectrometer (52),
with a quasi-optical transmission design, at the NHMFL. The EPR
Biophysical Journal 110, 1789–1799, April 26, 2016 1791
Kaur et al.

measurements were performed using a nonresonant sample holder contain-

ing a thin-cylindrical layer of sample of 20-mm diameter, 0.17-mm thick-
ness, and 53-mL volume (53). The sample holder is operating in
induction mode, i.e., samples are irradiated with linearly polarized micro-
waves and signals are detected as the orthogonal mode of the circularly
polarized radiation that is emitted from the sample. The sample holder
was fabricated in-house by the NHMFL machine shop. Spectra were
collected with 300-G sweep width, 4-G modulation amplitude, and 600–
800-Hz modulation frequency at room temperature. LUVs containing
2 mol % 5-SASL were prepared as described before. LUVs were mixed
with the AA1 peptides at an L/P at 10:1 before measurements.

RESULTS
AA1 binding increases membrane permeability
To study the permeability changes of lipid vesicles upon
binding AA1, we carried out an EPR-based quenching
assay. Briefly, liposomes with encapsulated 4-PT were
used to give an EPR signal. Membrane permeability was de-
tected using the signal reduction upon adding VC and AA1.
Permeability change % is defined as the percentage of EPR
signal (4-PT) changes relative to the liposomes without
AA1. One-hundred-percent permeability changes indicate
complete 4-PT and VC mixing as a result of membrane
penetration. Fig. 2 A shows the peptide-induced membrane
permeability changes of liposomes with the following lipid
FIGURE 2 Membrane permeability changes induced by AA1 binding.
compositions: POPC/POPG (4:1 wt/wt), POPC, and POPC
(A) Comparison of the permeability changes of liposomes with different
with varied cholesterol content. POPC/POPG liposomes lipid compositions at an L/P of 10. The following lipids were compared:
show the highest permeability changes of 67 5 2% in POPC/POPG, POPC, and POPC with 10, 30, and 50% CHOL. (B) Perme-
60 min, after peptide binding. In contrast, reduced mem- ability changes of POPC/POPG liposomes with L/Ps ranging from 10 to 80.
brane permeability changes were found for the neutral Note: A 100% permeability change indicates complete 4-PT and VC mixing
and 4-PT signal reduction due to membrane penetration.
POPC liposomes. Moreover, the presence of cholesterol
further diminished the peptide-induced permeability
changes of the lipid membranes. When cholesterol concen- and subsequently reduces the permeability changes of the
trations were increased from 10 to 50 mol %, the perme- loaded liposomes. Moreover, these results are consistent
ability changes at 60 min dropped from 42 5 2% to with the biexponential rates shown in Fig. 2. Specifically,
12 5 2%. In addition, with higher peptide/lipid ratios, the permeability rates may be defined by a fast membrane
increased permeability changes were observed (Fig. 2 B). leakage rate and a slower rate caused by the fluctuation of
To be noted, the permeability data can be fitted with the in- peptides between liposomes (54).
verted biexponential decay curves represented by solid lines
in Fig. 2. Each curve includes a fast component with a decay
AA1 binding reduces membrane fluidity
constant of 4–5 min and a slow component with a constant
ranging from one to several hours. Taken together, these re- The lipid fluidity or mobility in a bilayer can be perturbed by
sults illustrated the selective membrane-disruptive activity the binding of peptides to the membrane. Mobility changes in
of AA1 on negatively charged bacterial-mimic liposomes, lipid bilayers on peptide binding have been observed for
whereas the mammalian-mimic liposomes with large AMPs in previous biophysical studies (55,56). EPR spectral
amounts of cholesterol are resistant to its perturbation. line shapes represent molecular motion, which is suitable for
It has been proposed that AMPs are able to fluctuate or probing lipid fluidity (31,57). Greater spectral peak-to-peak
transfer between cell membranes (54). To ascertain whether splitting and/or broader spectral line shapes indicate slower
this behavior exists for AA1, we compared the permeability molecular motion. Here, to determine the mobility changes
changes of peptide-loaded liposomes both with and without across the bilayer on AA1 binding, EPR spin probes were
adding unloaded bare liposomes. As illustrated in Fig. S4, attached to different positions of the lipid molecules
decreased permeability changes were observed after adding (Fig. S1). The AA1-induced mobility changes on the mem-
the unloaded bare liposomes to the vesicles loaded with brane surface were reflected in the EPR spectra of T-PC.
4-PT and AA1. This confirms that AA1 moves from the The mobility of lipid headgroups was accessed using N-TP.
loaded liposome surfaces to the unloaded lipid surfaces In addition, mobility changes in the acyl-chain region were

1792 Biophysical Journal 110, 1789–1799, April 26, 2016


determined using 5-SASL, 5-PC, 7-PC, 10-PC, and 12-PC.
We expect these spin probes at varied positions will reveal
a full picture of membrane fluidity and peptide-induced
changes across the bilayer. Fig. 3 shows the motional param-
eters of 5-SASL-labeled lipid membranes with different
compositions. Lipid mobility from the EPR spectra of
5-SASL was quantified in terms of a motional parameter
2Tjj, i.e., the peak-to-peak splitting. For POPC/POPG lipo-
somes, the peak-to-peak splitting of the 5-SASL spectrum
is increased on the addition of AA1, and this can be inter-
preted as reduced mobility of lipid chains. Overall, the bacte-
rial-membrane mimic, POPC/POPG liposomes, shows the
greatest change in mobility upon binding of AA1 followed
by neutral POPC lipids. Minimal changes were observed
for liposomes containing 30% cholesterol.
Like 5-SASL, slower lipid motion was also observed for
T-PC, N-TP, 5-PC, 7-PC, 10-PC, and 12-PC for POPC/
POPG liposomes upon AA1 binding (Figs. S5, S6, S7,
and S8). To account for parameter sensitivity for different
EPR line shapes, three different EPR parameters were
used to analyze mobility changes. The line widths of the
central peaks in the EPR spectra (DH0) were compared for
all spin labels (Fig. S9 A). A0/A–1 ratios were compared
for T-PC, N-TP, and 12-PC, where A0 is the intensity of
the central peak and A–1 is the intensity of the high-field
FIGURE 3 Membrane fluidity changes in the presence of AA1. Room
temperature EPR spectra and mobility changes upon AA1 binding of lipo-
somes labeled with 5-SASL for (A) POPC/POPG, (B) POPC, and (C)
POPC/30% CHOL liposomes at an L/P of 10. EPR spectra of bare lipo-
somes are overlaid with the spectra in the presence of AA1. The 2Tjj
changes (D2Tjj) on AA1 binding are shown on the upper-right side of the
spectra. To see this figure in color, go online.
Membrane Disruption by a g-AApeptide
peak of a spectrum (Fig. S9 B (58)). Parameter 2Tjj was
used to quantify the mobility of 5-PC, 7-PC, and 10-PC
(Fig. S9 C). EPR spectra were collected with L/Ps ranging
from 10 to 80 for liposomes containing different spin-
labeled lipids. The largest mobility changes were observed
at an L/P of 10 (Figs. S5, S6, and S7). Overall, the results
show that AA1 induces more mobility changes on N-TP
and 5-PC than on 7-PC, 10-PC, and 12-PC (Fig. S9).
Because N-TP represents the lipid headgroup region and
the spin probes of 5-SASL and 5-PC are adjacent to lipid
headgroups, the data suggest larger mobility changes around
the headgroup region than in the acyl-chain region deep in-
side the membrane.
In an effort to compare AA1 interactions with neutral and
negatively charged lipid molecules, EPR spectra of lipo-
somes containing 5-MeSL and 5-PC were compared with
that of 5-SASL (Fig. S10). These three spin-labeled lipids
were chosen because at neutral pH, 5-SASL is negatively
charged, whereas there is no charge for 5-MeSL and
5-PC. Interestingly, for POPC/POPG, 5-MeSL- and 5-PC-
labeled lipid bilayers show smaller mobility changes upon
AA1 binding when compared with 5-SASL (Fig. S10).
These data suggest electrostatic interactions between the
cationic AA1 and negatively charged lipids. Similar differ-
ences between 5-SASL and 5-PC were also observed on
the binding of melittin peptides to lipid bilayers (39). On
the other hand, for neutral and cholesterol-containing lipids,
5-PC-labeled liposomes showed no changes in EPR spectra
similar to 5-SASL (Figs. S6 and S7). Taken together, these
data argue that AA1 interacts more strongly with the anionic
lipid molecules on the membrane.
AA1 increases lipid lateral ordering
Next, EPR W-band (94 GHz) spectra were used to identify
lipid lateral ordering induced by AA1 binding. Lateral
order reflects the lateral packing or the lateral pressure of
the lipids in a bilayer that can be perturbed by peptide and
AMP binding (59–61). The changes in lateral order can be
measured using EPR spectra in W-band or higher fre-
quencies with increased g-factor resolution (51,62). Fig. 4
shows W-band EPR spectra of POPC and POPC/POPG lipo-
somes with 5-SASL in the absence and presence of AA1.
For POPC, no significant changes were observed in the pres-
ence of AA1 (Fig. 4 B). In contrast, the EPR spectra of
POPC/POPG with AA1 show visible separation of hgxxi
and hgyyi components, as illustrated in Fig. 4 A. POPC/
30% CHOL liposomes were not included in the analysis
because cholesterol was found to induce significant lateral
ordering due to a lateral condensing effect (51). The appear-
ance of g-factor anisotropy (motionally averaged) in the
EPR spectra indicates that AA1 increases the lateral
ordering of POPC/POPG lipid bilayers. This g anisotropy
can be quantified in terms of a lateral-order parameter,
which is defined on the basis of the equations described in
Biophysical Journal 110, 1789–1799, April 26, 2016 1793
Kaur et al.
FIGURE 4 Lipid lateral ordering. 94 GHz EPR spectra of POPC/POPG
(A) and POPC (B) liposomes with 5-SASL in the absence and
presence of AA1. Lateral ordering was detected for (A) with arrows
showing the hgxxi, hgyyi, and hgzzi components. The L/P is 10. No sign of
lateral ordering was observed for (B). (C) Membrane structure showing
transverse and lateral order and the corresponding g-factor anisotropy, indi-
cated by gxx, gyy, and gzz. The averaged principle axes of a nitroxide spin
label (5-SASL) aligned with regard to a bilayer are shown. Changes in lipid
lateral order are reflected in the x and y components of the g tensors of the
spin label. To see this figure in color, go online.
the Materials and Methods (51). Similar to the conventional
axial-order parameter, the lateral-order values vary from
zero (disordered) to one (ordered). For POPC/POPG lipo-
somes in the presence of AA1, the hgxxi, hgyyi, and hgzzi
values determined from the EPR spectra are 2.0075,
2.0062, and 2.0029, respectively. The calculated lateral or-
der is 0.52, which indicates significant lateral ordering. No
apparent lateral ordering was observed in the absence of
AA1. Interestingly, the conventional axial-order parameter
only increases from 0.63 to 0.70 upon AA1 binding. This
suggests that AA1 binding mainly inhibits lipid lateral mo-
tion and induces lipid lateral pressure.
Accessibility changes upon AA1 binding
Lastly, AA1-induced accessibility changes were determined
by EPR power saturation techniques (50). Because of their
amphiphilic nature, an intrinsic aspect of lipid membranes
is a polarity gradient established across the bilayer with a
largely nonpolar environment inside the bilayer and a polar
1794 Biophysical Journal 110, 1789–1799, April 26, 2016
environment on the membrane surface. As a result, the lipid
acyl chains are more accessible to nonpolar reagents than to
polar reagents. This polarity gradient and accessibility pro-
file can be altered by the binding of proteins depending on
the extent and nature of protein–lipid interactions (37).
This accessibility profile can be measured by EPR spectros-
copy using a microwave power saturation technique (50).
Briefly, with increasing microwave power, the EPR signal
becomes saturated, which is described in terms of a power
saturation parameter P1/2. The presence of polar or nonpolar
relaxing reagents in the vicinity of spin probes changes the
P1/2 values due to the collision between the relaxing re-
agents and spin probes. Based on the P1/2 values, the acces-
sibility of spin probes to relaxing reagents can be estimated
in terms of an accessibility parameter, P.
To study the effect of AA1 on the accessibility profile of
lipids, P-parameters in the presence of O2 (nonpolar) and
NiEDDA (polar) reagents were determined. The results of
5-SASL-labeled POPC/POPG liposomes are shown in
Fig. 5. The data revealed that 5-SASL becomes less acces-
sible to both O2 and NiEDDA in the presence of AA1.
This indicates that peptide binding reduces the collision
rate between the spin probes and the relaxing reagents,
most likely by extensive surface interactions. On the other
hand, minimal accessibility changes of 5-SASL were found
for POPC and POPC/30% CHOL liposomes upon AA1
binding, shown in Fig. 5 C. Specifically, at an L/P of 10,
POPC/POPG liposomes show ~30% accessibility changes
upon peptide binding, while POPC and cholesterol-contain-
ing membranes show <5% changes. Therefore, AA1 affects
greater accessibility changes of 5-SASL for bacterial-mimic
membranes versus mammalian-mimic membranes. Consid-
ering that 5-SASL is negatively charged at neutral pH, an
uncharged spin-labeled lipid analog (5-PC) was also used
to determine the peptide-induced accessibility changes of
POPC/POPG liposomes. Similar to 5-SASL, the accessi-
bility of both O2 and NiEDDA to 5-PC were reduced for
POPC/POPG liposomes in the presence of the peptides
(Fig. S11). Moreover, reduced accessibility was also
observed for POPC/POPG liposomes containing N-TP
(Fig. S11). Taken together, similar to the fluidity data, the
above results suggest that AA1 binding reduces solvent
accessibility around the headgroup region of POPC/POPG
liposomes.
In addition, to investigate the effect of AA1 binding on
the hydrophobic core of lipid bilayers, we performed acces-
sibility measurements of POPC/POPG liposomes using
7-PC, 10-PC, and 12-PC. AA1 induces similar changes in
O2 accessibility to these spin-labeled lipids as in 5-PC
(i.e., decreased P-values upon peptide binding). However,
AA1 binding increases the P-values of NiEDDA for these
three spin-labeled lipids (Fig. S11). The P-parameters
were used to calculate the membrane depth parameter,
F (Fig. 6). As expected, in the absence of peptides, F-values
are increased from 7-PC to 12-PC due to the polar gradient

across the bilayer. Remarkably, the F-values appear to
remain constant for 7-PC, 10-PC, and 12-PC after peptide
binding (Fig. 6). This indicates membrane thinning and
acyl-chain overlay, as elaborated below in the Discussion.
DISCUSSION
AA1 selectively interacts and disrupts anionic
bacterial membranes
Membrane permeability
The antibacterial mechanisms of AMPs have been linked to
their capabilities to increase membrane permeability,
thereby causing the leakage of cellular content and eventu-
ally cell death. Here, AA1 was found selectively to induce
large permeability changes in anionic POPC/POPG lipo-
somes mimicking bacterial membranes (Fig. 2), which is
consistent with the peptide’s efficacious antimicrobial activ-
ities against diverse bacterial strains. Notably, the data show
incomplete leakage phenomena (<100%) with a higher per-
centage of permeability changes when decreasing L/Ps from
80:1 to 10:1 (Fig. 2). These ratios seem to be high, but it is
not surprising. Similar L/Ps have been observed in previous
studies on AMPs. For example, membrane permeability
changes induced by LF11, magainin 2, and LL37 were
observed at L/Ps ranging from 10:1 to 50:1 (61,63,64).
FIGURE 6 Depth parameter changes of 7-PC, 10-PC, and 12-PC upon
AA1 binding to POPC/POPG liposomes when compared to bare liposomes.
Membrane Disruption by a g-AApeptide
FIGURE 5 Accessibility changes upon the
binding of AA1. Changes in O2 (A) and NiEDDA
(B) accessibility of POPC/POPG liposomes with
5-SASL on the addition of AA1 are shown. (C)
Comparison of the percentage of the O2 accessi-
bility changes of POPC/POPG, POPC, and
POPC/30% CHOL liposomes upon AA1 binding.
Moreover, geometrical studies have demonstrated that the
effective AMP concentration in vivo could be two orders-
of-magnitude higher than the bacterial lipid concentration
(reviewed in Melo and Castanho (65)). To be noted, the
AA1-induced permeability changes include a fast phase
(several minutes) followed by a much slower phase (a few
hours). Incomplete leakage along with a fast rate may be
due to transient pore formation or temporary failure of the
bilayer structure upon peptide binding (66). The slow phase
might be a result of peptide fluctuation between liposomes,
as shown in Fig. S4. Interestingly, the permeability changes
were reduced for POPC liposomes in the presence of choles-
terol. These results argue that lipo-cyclic-g-AApeptides
have the ability to preferentially disrupt bacterial membrane
versus mammalian membrane.
Membrane fluidity and lateral motion
Upon AA1 binding, the EPR spectra show decreased motion
of lipids across the bilayer, with the greatest mobility
changes being around the headgroup region (Fig. 3 and
Figs. S5, S6, S7, S8, and S9). This rigidifying effect on lipid
bilayers has been observed in the studies of other AMPs
such as CM15 (37), and cecropins B1 and B3 (38). EPR
spectra at 94 GHz suggest that AA1 induces significant
lateral ordering of lipid molecules in POPC/POPG mem-
branes, suggesting reduced lipid motion in the lateral direc-
tion perpendicular to the membrane normal. The reduced
lateral motion may be an indicator of tighter lipid lateral
packing and lateral expansion of lipid bilayers (Fig. 7).
AMPs such as Gramicidin S and PGLa have been suggested
to cause lateral pressure and lateral expansion of membranes
by embedding in the lipids (59). This lateral expansion ef-
fect is accompanied by the increasing lateral area of one
side of a bilayer, which has been related to the thinning of
lipid membranes (59). Taken together, AA1 binds strongly
to anionic membranes, decreases membrane fluidity, re-
duces lipid lateral motion, and possibly induces lipid lateral
expansion.
Solvent accessibility and membrane thinning
EPR power saturation data revealed that AA1 binding
significantly decreases the accessibility of POPC/POPG
membranes to both polar (NiEDDA) and nonpolar (O2)
reagents for N-TP, 5-SASL, and 5-PC (Figs. 5 and S11).
Biophysical Journal 110, 1789–1799, April 26, 2016 1795
Kaur et al.
The decreased accessibility is a result of the reduced con-
centrations of relaxing reagents (O2 and NiEDDA) in the vi-
cinity of spin probes, which lead to a decreased collision
rate between the relaxing reagents and the spin-labeled lipid
chains. Most likely, the extensive interactions of AA1 on the
surface and headgroups of membranes block the diffusion of
the relaxation reagents to the lipids, thereby reducing sol-
vent accessibility to headgroups (N-TP) and atoms adjacent
to headgroups (5-SASL and 5-PC). An interesting observa-
tion was the different trend of accessibility changes in the
hydrophobic core of the POPC/POPG liposomes as deter-
mined by 7-PC, 10-PC, and 12-PC. The P-values of O2
are decreased while the values of NiEDDA are increased
for these positions upon AA1 binding, which indicates
increased polarity of these positions. Moreover, in the pres-
ence of AA1, the polarity gradient between 7-PC, 10-PC,
and 12-PC is diminished and the depth parameters (F) of
these three spin labels become comparable (Fig. 6). A plau-
sible explanation for these observations is membrane thin-
ning after AA1 peptides are inserted into the lipid
membranes (Fig. 7). Intercalation of the peptides into the
membrane leads to an increase in the lateral area of one
leaflet of a bilayer, providing more space for the lipids of
the other leaflet to penetrate into the acyl chains, subse-
quently causing the thinning of the membrane (Fig. 7).
Because of membrane thinning and more acyl-chain over-
lays, the depth parameters of 7-PC, 10-PC, and 12-PC are
averaged and become similar to each other. The thinning
of membranes is also accompanied by decreased lipid mo-
tion because of the higher density of lipid packing in
the acyl-chain region (61). This agrees well with the
decreased lipid mobility of 7-, 10-, and 12-PC upon AA1
binding (Fig. S8).
Preventive effect of cholesterol on mammalian
cells
Our results argue that the large quantity of cholesterol in
mammalian cell membranes might contribute to their resis-
tance to the membrane perturbation caused by AA1. This is
supported by the reduced membrane permeability, mobility,
1796 Biophysical Journal 110, 1789–1799, April 26, 2016
FIGURE 7 Membrane interaction and disruption
mechanism of AA1. (A) AA1 binds to the mem-
brane through electrostatic and hydrophobic inter-
actions. (B) Insertion of the bulky hydrophobic
groups of AA1 into the membrane results in lateral
expansion of the upper leaflet of a lipid bilayer. (C)
Lipid lateral expansion leads to membrane thin-
ning. Moreover, peptide insertion causes transient
membrane disruption or local bilayer disorder,
subsequently leading to permeability changes. To
see this figure in color, go online.
and accessibility changes in the presence of cholesterol.
These data also agree with the results that AA1 is able to
target bacterial cells selectively with low hemolytic activity.
The presence of cholesterol stabilizes the membrane struc-
ture by increasing membrane order and lateral organization.
Cholesterol might inhibit the interaction of AA1 with the
membrane, prevent correct docking of the peptides to the
membrane, and subsequently affect the membrane-disrup-
tive functions of the peptides. For instance, the role of
cholesterol in protecting erythrocytes from antimicrobial ac-
tivity has also been found in studies on magainin 2 and Me-
littin (67,68). In addition, AMPs have been found binding
preferably to disordered domains lacking cholesterol, as
shown in the studies of MSI peptides and d-lysin
(66,69,70). In summary, our results show that cholesterol
has a preventive effect for mammalian cell membranes
and plays an important role in the selective membrane-
disruptive activities of lipo-cyclic-g-AApeptides.
Mechanism of bacterial membrane disruption
by AA1
Based on the EPR data in this study, the membrane-disrup-
tion mechanism of AA1 can be explained using the carpet
model (15). The carpet model of AA1 is supported by the
following EPR observations: (1) Lipid fluidity and
ordering—as shown in Figs. 3 and 4, reduced lipid fluidity
and increased lipid ordering were observed upon peptide
binding, which indicates that AA1 is able to rigidify the
lipid molecules. Moreover, this immobilization effect is
greater around headgroup regions than in the acyl-chain re-
gion (7-PC, 10-PC, and 12-PC), suggesting that the peptides
are not deeply inserted into the bilayer; this is consistent
with the carpet model. (2) Solvent accessibility—extensive
surface interaction and peptide accumulation can also lead
to decreased accessibility of the lipid molecules to both po-
lar and nonpolar reagents in the solvent, as illustrated in
Figs. 5 and S11. (3) Membrane thinning—in the carpet
model, the high density of peptides binding on the mem-
brane surface may lead to thinning of the membrane, as
shown for CM15 (37) and magainin 2 (71). This is

confirmed by the EPR depth data (Fig. 6), which indicates
reduced thickness of the POPC/POPG membranes. (4)
Permeability—the permeability data show an incomplete
and fast leakage event upon AA1 binding. This indicates a
transient disruption of the bilayer, which has been sug-
gested for the carpet model (66). On the other hand, a
long-lasting transmembrane pore (such as a barrel-stave or
toroidal-pore) is unlikely to be formed because the short
hydrophobic groups of AA1 are not long enough to span
the bilayer. To be noted, a complete disruption of the mem-
brane or a detergent effect may occur in the carpet mecha-
nism (66). However, this is not consistent with the
immobilization of lipid chains across the bilayer (Fig. 3
and Figs. S5, S6, S7, S8, and S9) and the decreased solvent
accessibility to 5-PC and N-TP upon AA1 binding
(Fig. S11). In the case of the interfacial-activity model, pep-
tide binding would not lead to rigidification of the lipid
headgroups and steric hindrance of O2 and NiEDDA as
shown upon AA1 binding. Therefore, the mobility and
accessibility changes induced by AA1 are not consistent
with the interfacial-activity model. In addition, the lipid-
clustering model is not supported by the EPR data. No
significant spin-spin interaction or lipid clustering was
observed for POPC/POPG liposomes upon AA1 binding,
as detected by 5-PC spectra at 200 K (not shown). Taken
together, the membrane disruption mechanism of AA1 can
be explained using the carpet model.
Specifically, the binding of AA1 is initiated by the elec-
trostatic interactions between the cationic side chains of
the peptides and the anionic groups in lipid vesicles, which
is evident in the stronger interaction of AA1 with POPC/
POPG liposomes when compared with POPC liposomes
(Fig. 3). This is also confirmed by the greater mobility
changes of the 5-SASL containing liposomes than its un-
charged analog 5-MeSL (Fig. S10). This step is followed
by the insertion of the long carbon tail and the hydrophobic
side chains on the cyclic ring of the peptide into the lipid
bilayer. Moreover, the strong electrostatic and hydrophobic
interactions decrease the membrane fluidity and increase the
ordering of the lipid molecules, resulting in reduced acces-
sibility of lipid headgroups to both polar and nonpolar mol-
ecules in the solvent. In addition, the insertion of the bulky
hydrophobic groups of the peptide into the membrane may
lead to increased lateral pressure, thereby inducing lateral
expansion and membrane thinning (Fig. 7). Furthermore,
peptide penetration also creates transient membrane disrup-
tion or local disorder, subsequently causing leakage of
cellular contents and cell death.
In summary, AA1 features that are important for its anti-
microbial activities have been revealed, and such informa-
tion offers insight for designing new antibiotic agents. Our
structural and biophysical data suggest that the hydrophobic
side chains of AA1 function as the major membrane-disrup-
tive groups. Bulky hydrophobic groups might be more effec-
tive to induce lateral expansion and membrane thinning,
Membrane Disruption by a g-AApeptide
such as the adamantyl group, the naphthyl group, etc. In
addition, a longer tail might be able to insert the aromatic
rings deeper into the lipid acyl chains and more efficiently
promote lateral expansion and membrane thinning. As
such, a C18 alkyl tail may be employed in a future design.
Moreover, the data show that the cationic groups of AA1 ac-
count for its selective bacterial-membrane targeting capabil-
ities. Therefore, it is important to maintain the amphipathic
building blocks with balanced polar and nonpolar groups,
i.e., one cationic and one hydrophobic side chain. Further-
more, the data demonstrate that lipo-cyclic peptides can
be effective AMPs. With its more rigid ring structure, a
cyclic peptide contains a more stable amphiphilic structure
than does a linear peptide. The size of the ring might affect a
peptide’s capability to induce membrane property changes
and this can be further fine-tuned. For instance, a bicyclic
ring may be used to increase the area interacting with bac-
terial membranes, while its rigidity can be retained due to
bicyclization. Our results demonstrate that understanding
the detailed AMP mechanism at a molecular level will facil-
itate new antibacterial biomaterial designs.
CONCLUSIONS
With an aim to understand the molecular basis of the antimi-
crobial activity of lipo-cyclic-g-AApeptides, we investi-
gated the permeability and membrane property changes
induced by our lead peptide—AA1. The peptide selectively
permeates and structurally modifies negatively charged bac-
terial-mimic membranes. In contrast, cholesterol-containing
neutral membranes mimicking mammalian cells were mini-
mally affected by the molecule. Based on combined ana-
lyses of membrane permeability, dynamics, membrane
thinning, and accessibility, we proposed a carpet-like mech-
anism for the antimicrobial activities of AA1. The results
provide implications for the development of effective
AMPs with robust antibacterial activities against antibi-
otic-resistant microbes.
SUPPORTING MATERIAL
Eleven figures are available at http://www.biophysj.org/biophysj/
supplemental/S0006-3495(16)30064-9.
AUTHOR CONTRIBUTIONS
P.K. performed research, analyzed data, and wrote the article; Y.L. synthe-
sized material; J.C. designed research and wrote the article; and L.S. de-
signed research, analyzed data, and wrote the article.
ACKNOWLEDGMENTS
The EPR experiments were performed at the National High Magnetic Field
Laboratory (NHMFL), which is supported by National Science Foundation
grant No. DMR-1157490 and the State of Florida. L.S. acknowledges the
Biophysical Journal 110, 1789–1799, April 26, 2016 1797
Kaur et al.
support of NHMFL User Collaboration Grants Program Award No. 5080.
J.C. acknowledges support from National Science Foundation grant No.
1351265 and National Institutes of Health grant No. GM112652-01A1.
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