Archives of Biochemistry and Biophysics 629 (2017) 54e62

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Archives of Biochemistry and Biophysics

journal homepage: www.elsevier.com/locate/yabbi

The increase in positively charged residues in cecropin D-like Galleria

mellonella favors its interaction with membrane models that imitate
bacterial membranes

On
Jose ~ ate-Garzo
n a, Alessio Ausili b, Marcela Manrique-Moreno a, Alejandro Torrecillas b,
Francisco J. Aranda b, Edwin Patin ~ o a, *, Juan C. Gomez-Ferna

ndez b, **
a
Universidad de Antioquia, Instituto de Química, Facultad de Ciencias Exactas y Naturales, A.A. 1226, Medellín, Colombia
b
Universidad de Murcia, Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Campus of International Excellence Mare Nostrum,
E-30100, Murcia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: A comparative study of three synthetic peptides, namely neutral Cecropin D-like G. mellonella (WT) and
Received 6 April 2017 two cationic peptides derived from its sequence, DM1 (þ5) and DM2 (þ9) is reported in this work. The
Received in revised form influence of charge on the interactions between peptides and membranes and its effect on phase were
16 June 2017
studied by calorimetric assays. Differential scanning calorimetry (DSC) showed that DM2 peptide showed
Accepted 13 July 2017
Available online 15 July 2017
the strongest effect when the membrane contained phosphatidylcholine (PC) and phosphatidylglycerol
(PG), increasing membrane fluidization. Fourier transform infrared spectroscopy (FTIR) was used to
determine lipid segregation in the presence of peptides. When WT and DM1 bound to model membrane
Keywords:
Antimicrobial peptide
containing PG and PC (1:1 molar ratio) a separation of both lipids was observed. Meanwhile, DM2
Cationic charge peptide also induced a demixing of PG-peptide rich domains separated from PC. FTIR experiments also
Galleria mellonella suggested that the presence of DM1 and DM2 peptides increased lipid carbonyl group hydration in DMPG
Peptide-membrane interactions membrane fluid phase, However, hydration at the interface level in fluid phase was notably increased in
Calorimetry the presence of WT and DM1 peptides in DMPC/DMPG. Overall the increase in positively charged resi-
Infrared spectroscopy dues favors the interaction of the peptides with the negatively charged membrane and its perturbation.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction evaluated as potential therapeutic agents [1]. Permeation of the cell

Antimicrobial peptides (AMPs) have been established as
essential molecules for the innate immune defense of different
organisms, a large variety of natural and synthetic AMPs have been
Abbreviations: AMPs, Antimicrobial peptides; DSC, Differential scanning calo-
rimetry; ITC, Isothermal titration calorimetry; FTIR, Fourier transformed infrared
spectroscopy; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; DMPG,
1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; POPC, 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine;
MLV, Multilamellar vesicle; SUV, Small unilamellar vesicle; Tm, Main transition
temperature; Lc phase, Liquid crystalline phase; Tc, Onset transition temperature; Tf,
Final transition temperature; G. mellonella, Galleria mellonella; E. coli ATCC 25922,
Escherichia coli; S. enterica R60, Salmonella enterica; P. mirabilis R45, Proteus
mirabilis.
* Corresponding author.
** Corresponding author.
E-mail addresses: edwin.patino@udea.edu.co (E. Patin ~ o), jcgomez@um.es
(J.C. Gomez-Ferna
ndez).
membrane leading to cell death is a mechanism performed by a
large number of AMPs [2]. AMPs are generally amphipathic,
composed of less than 50 residues and characterized by cationic
properties [3], since the charge is responsible for the initial elec-
trostatic interactions between peptide and anionic microbial
membrane. Furthermore, several studies support that increasing
the charge in a peptide sequence improves the antimicrobial ac-
tivity [4e6].
Cecropins are amply found in the hemolymph of lepidopteran
and dipteran species. Mature cecropins contain 34e39 residues
with a hydrophilic N-terminal helix and a hydrophobic C-terminal
helix joined by a hinge region [7]. Lepidopteran Galleria mellonella
is a source of AMPs including the cecropin D-like (WT), which is a
neutral peptide of 39 amino acid residues with the following
sequence: ENFFKEIERAGQRIRDAIISAAPAVETLAQAQKIIKGGD. This
peptide has activity against some fungi, Gram-negative and Gram-
positive bacteria [8].
Modifications at defined residues of cecropin D-like such as

http://dx.doi.org/10.1016/j.abb.2017.07.008
0003-9861/© 2017 Elsevier Inc. All rights reserved.
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replacing of acidic residues with arginine or lysine residues
increased the positive charge altering its antimicrobial activity
against Gram-negative bacteria and therefore its action mechanism
on model membranes [9]. This work reported a cationic cecropin
analogue (net charge: þ5) from G. mellonella capable of ordering
DMPG membranes unlike wild type peptide which fluidized the
membrane. In addition, exothermic binding interactions between
peptides and lipopolysaccharides (LPS) aggregates from S. enterica
R60 and polymyxin B-resistant P. mirabilis R45 were slightly altered
as a result of residue substitutions.
Biophysical studies on model membranes give valuable insights
into the AMPs mechanism of action, there are enough reports about
the lipid-peptide interaction using tools like calorimetry and
spectroscopy [10e12]. In this work, three synthetic peptides: WT
and modified peptides DM1 and DM2 (net charge: 0, þ5 and þ9,
respectively) were used. It has been recently shown that DM1 and
DM2 showed at least an eightfold increase in their antimicrobial
activity in comparison to WT [13]. These last authors also observed
that the three peptides induced the release of calcein from POPC,
POPG, POPE/POPG and from POPC/POPG liposomes, indicating that
they incorporate into the membranes producing pores and that
they altered the membranes at both the glycerol level and the hy-
drophobic core [13].
These three peptides were used to study the influence of the
charge on the thermotropic behavior on model membranes built
with representative phospholipids of Gram-negative bacteria.
These assays were performed applying differential scanning calo-
rimetry (DSC) and Fourier transform infrared spectroscopy (FTIR).
Finally, by isothermal titration calorimetry (ITC) peptide-lipid in-
teractions and binding selectivity were also studied, since this
method is sensitive to electrostatic interactions which could arise
from the binding between cationic peptides and anionic mem-
branes [14]. It was observed that the increase in positive charges in
the modified peptides favored their capacity to interact with
membranes and to segregate negatively charged phospholipids
from their mixture with amphoteric ones.
2. Materials and methods
2.1. Materials
Lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(POPG), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-
dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and chain per-
deuterated 1,2-dimyristoyl-D54-sn-glycero-3-phosphocholine
(DMPC-d54) were purchased from Avanti Polar Lipids (Alabaster,
AL) and used without further purification. D2O was purchased from
Sigma-Aldrich (St. Louis, Mo).
2.2. Peptide synthesis and purification
Peptides were custom-made in an automatic synthesizer (433 A
Applied Biosystems) in the University of Lausanne, Switzerland.
Wild type peptide (WT) (ENFFKEIERAGQRIRDAIISAA
PAVETLAQAQKIIKGGD) and modified peptides, DM1 (ENFFKRIR-
RAGKRIRDAIISAAPAVETLAQAQKIIKGGD) and DM2 (RNFFKRIR-
RAGKRIRKAIISAAPAVETLAQAQKIIKGGD), were purified applying
Reverse Phase-High Performance Liquid Chromatography (RP-
HPLC) according to (Cruz, Ortiz et al., 2014) [37], to obtain peptides
at 95% purity.
2.3. Sample preparation
Dehydrated anionic and zwitterionic lipids were dissolved in
55
chloroform/methanol (2:1 v/v). Desired amounts of different lipids
were mixed, dried under a gentle current of nitrogen and placed
under vacuum for 3 h to remove any residual solvent. The lipid
films generated were hydrated with Hepes buffer (25 mM Hepes
pH 7.0, 100 mM NaCl, 0.2 mM EDTA) and vigorously vortexed above
the onset transition temperature (Tc) to obtain multilamellar vesi-
cles (MLVs). Peptides were added to the same buffer used to
resuspend the lipids forming the vesicles. According to the applied
technique, the PC/PG molar ratios used were 3:1 for calorimetry
and 1:1 for FTIR.
2.4. Differential scanning calorimetry (DSC)
A MicroCal VP-DSC (MicroCal Inc., Northampton, USA) was used
for DSC experiments. The scanning of the samples was carried out
over a 5e35  C temperature at a heating rate of 1  C min1. MLVs
were prepared using 1 mg of lipid with three peptide dilutions to
give different peptide-lipid ratios: 1:100, 1:50 and 1:25. Vesicle
suspensions at a 2.4 mM concentration of phospholipid, were
degassed and then transferred to the sample cell. Hepes buffer was
used as reference solution. Thermograms were acquired and the
fifth heating scan were analyzed using Origin 5.0 (MicroCal) soft-
ware in order to obtain calorimetric parameters: Tc (temperature of
the transition onset), Tf (temperature of the transition completion),
and DH.
2.5. Isothermal titration calorimetry (ITC)
All measurements were performed with a MicroCal VP-ITC
calorimeter (MicroCal, Northampton, USA). Measurements were
carried out at temperatures above Tc in all the cases. MLVs were
sonicated during 10 min to obtain small unilamellar vesicles SUVs.
300 mL suspensions of vesicles (5 mM) were gradually titrated in
injections of 15 mL for 20 times into a peptide solution (0.025 mM).
Raw data was baseline corrected, integrated and analyzed using
Origin 5.0 software to obtain thermodynamic parameters such as
binding enthalpy (DH), equilibrium binding constant (KA), and
binding entropy (DS). Gibbs free energy (DG) was calculated
applying the following equation DG ¼ DH e TDS. In order to make
calculations with phospholipid concentrations it was assumed that
the effective amount of phospholipids located in the outer mono-
layer of the liposomes was about 60% of the total [15e17].
2.6. Fourier transform infrared spectroscopy
Experiments were performed on samples containing 8 mg of
lipids. MLVs were generated as described above in D2O buffer
(25 mM Hepes pD 7.0, 100 mM NaCl, 0.2 mM EDTA). The lipid
concentration was 140 mM (peptide/lipid ratio 1:50). Samples were
squeezed between two CaF2 windows separated by a 25 mm spacer
and transferred to a Symta cell mount. Once mounted in a Nicolet
6700 FTIR spectrometer (Madison, WI), temperature was settled
using a Peltier device (Proteus system from Nicolet) and the sam-
ples were heated from 8 to 40  C. 256 interferograms were
collected at every temperature with a nominal resolution of 2 cm1.
Spectra were acquired at intervals of 2  C, allowing 5 min equili-
bration between temperatures. Data was processed using Grams
(Galactic Industries, Salem, NH) software as described previously
[18]. To study the thermotropic behavior a sigmoidal curve was
built from the spectra of the vibrations of CH2 and C¼O groups. The
main transition temperature (Tm) obtained, corresponding to the
temperature of the minimal curve slope, was determined
employing the first derivative criterion.
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2.7. Helical wheel projection
Helical wheel projections of WT, DM1 and DM2 were generated
using the online tool “helical wheel projection” created by Don
Armstrong and Raphael Zidovetzki (version ID: wheel.pl,v 1.4
2009-10-20 21:23:36 don Exp). In order to highlight the properties
of the N-domain helix, the first 18 residues of the peptides were
analyzed by this method.
3. Results
3.1. Differential scanning calorimetry studies
DSC measurements were performed to study the effect of pep-
tide binding on the lipid phase transition. In MLVs of pure DMPG
two endothermic peaks that correspond to the pre-transition at
11  C and to the main transition at 22.3  C were observed. A small
shoulder was also observed at the right side of the main transition
peak. When peptides were added at low peptide-lipid ratio (1:100)
to DMPG vesicles, the pre-transition was absent and the endo-
thermic peak corresponding to the main phase transition was
reduced in size and did not shift considerably, but the shoulder at
the right side of the peak was noticeable (Fig. 1).
When the peptide concentration was increased a more pro-
nounced effect on the transition peak height of the DMPG ther-
mograms was observed, overall in the presence of charged peptides
(Fig. 1A, B and C). We monitored the onset (Tc) and the completion
(Tf) of the gel to fluid transition phase. Tc is taken as the temperature
at where the transition curve first inclines from the low-
temperature baseline. Tf is taken as the temperature where the
transition curve returns to the high-temperature baseline. Both
temperatures may be used to build a phase diagram [19,20].
At the maximum evaluated concentration all peptides induced a
decrease of DMPG Tc from 21.9 to 18, 15.7 and 10.5  C for WT, DM1
and DM2, respectively (Fig. 1D), while the Tf were increased, being
the most significant change from 24.4 to 28.3  C for DM1. Therefore
both parameters, Tc and Tf, suggest that the peptides promote a less
stable phase below the gel to fluid transition and more stable phase
above of the transition particularly by DM2 (Fig. 1D) widening the
main transition peak. The change of enthalpy associated to the
transition (DH) of DMPG vesicles decreases when the peptide
concentration increases (Fig. 1E). Comparing the effect of the
different peptides on DH at the peptide/lipid ratio (1:100), it was
seen that DM1 peptide had the strongest one, decreasing DH by
25%, whereas WT and DM2 peptides did not show any effect. When
the comparison was made at the peptide-lipid ratio (1:25) DH
decreased by 29, 81 and 58% in the presence of WT, DM1 and DM2
respectively.
WT peptide had the lowest effect in DMPC/DMPG (3:1) mixture.
At 50:1 peptide-lipid ratio a slight effect on the width of the main
transition peak was observed. Additionally, at the maximum con-
centration evaluated (25:1) the heat capacity modestly decreased
and a subtle shoulder arose towards the final stages of the main
transition (Fig. 2A). Furthermore, slight changes at the onset and
finishing temperature of the transition were observed being the
highest change from 23.1 to 22.2 for Tc and from 24.6 to 25.5  C for
Tf (Fig. 2D). In contrast, cationic peptides induced a decrease of the
area under the transition curve (a decrease in DH) of DMPC/DMPG
mixture even at the lowest concentrations of peptide whereas at
the highest concentration multicomponent melting transitions
were seen (Fig. 2B and C). In the presence of DM2 peptide, the
transition temperatures exhibited the most significant change at
the maximum peptide concentration evaluated, e.g., from 23.1 to
16.2  C for Tc and from 24.6 to 29.4  C for Tf (Fig. 2D) producing a
pronounced widening of the main transition peak. Additionally,
this peptide also had the highest effect on DH of the transition
reducing it by 46%, whereas WT and DM1 peptides decreased it by
11 and 30%, respectively (Fig. 2E).
3.2. Isothermal titration calorimetry
This technique is used to measure binding to the membrane at a
single temperature. For this purpose SUV formed by unsaturated
phospholipids are used. The reasons for using this type of vesicles is
as follows. Using a fluid system helps to better extrapolate these
results with what could happen in a biological membrane. In
addition a membrane based in unsaturated lipids would be always
more similar to a biological membrane. These unilamellar unsatu-
rated vesicles are far more stable than unilamellar saturated vesi-
cles. Finally it is convenient to use unilamellar vesicles since the
purpose is to measure the binding to the membrane of an exter-
nally added molecule (the peptide) and using multilamellar vesi-
cles many bilayers are not exposed to the externally added
molecule making difficult the interpretation of the results. ITC
measurements were carried out at 25  C, a temperature at which all
the membranes used are in a fluid condition, which is convenient to
extrapolate these results to biological membranes.
The heat flow (upper panel) and integrated heats of binding
(lower panel) of the titration of POPC/POPG (3:1 molar ratio) SUVs,
with the three peptides, are shown in Fig. 3. Each injection of lipid
produced an exothermic peak which decreases due to the reduction
of free peptides whereas phospholipid concentration was increased
with subsequent injections. For cationic peptides, the exothermic
binding reaction finished after fifteen injections (Fig. 3). In contrast,
WT peptide did not show a considerable decrease after the seventh
injection (Fig. 3).
KA values were twice as bigger for DM1 (5.163E4 ± 6393 M1)
than for WT (2.473E4 ± 4221) and also nearly double for DM2
(4.561E4 ± 8645) than for WT (Table 1), indicating an increase in
affinity of the liposomes for the peptides as these peptides possess
a higher positive charge. Negative DG was deduced from the
binding of POPC/POPG vesicles to the studied peptides, providing a
quantitative measurement of the strength of the binding. Judging
from these DG values, the highest strengths were observed for both
cationic peptides. It can be seen that DS values are similar for the
three peptides but DH values are more exothermic for the cationic
peptides, especially for DM1 (Table 1). When applying this model n
values were also obtained. However these values have a very
difficult interpretation in our case, since we are studying the
binding of macromolecular structures, as liposomes are, which
present an undetermined number of binding sites, to peptides.
In another set of experiments the binding of pure POPG lipo-
somes to the peptides were studied (Fig. 4). However it was
observed that only the binding of POPG liposomes to WT could be
analyzed in detail, since in the case of the cationic peptides DM1
and DM2 the first addition of liposomes already produced a very
large exothermic release that was much higher than the released
heat produce by the following injections, making impossible to
obtain a good fit. It seems that because these peptides possess such
a high number of positive charges they avidly bind POPG liposomes
even at very low concentrations of vesicles. In the case of the WT
peptide, Table 1 shows that DH was considerably more exothermic
than the interactions of any of the three peptides with POPC/POPG
vesicles, although since DS value was lower, DG was similar to the
values observed for the three petides interacting with POPC/POPG
liposomes.
3.3. Fourier transform infrared spectroscopy results
In order to verify the results obtained by DSC experiments, FTIR
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Fig. 1. DSC measurements using DMPG MLVs. Thermograms for DMPG and DMPG-peptides: WT (A), DM1 (B) and DM2 (C) at different lipid/peptide molar ratio. Temperatures of the
beginning and completion of the main phase transition (D). Changes in phase transition enthalpy (in kJ/mol of phospholipid) under peptides influence at different peptide/lipid
molar ratios (E). WT (C), DM1 (-) and DM2 (:).

Fig. 2. DSC measurements using DMPC/DMPG (3:1) MLVs. Thermograms for DMPC/DMPG: WT (A), DM1 (B) and DM2 (C) at different lipid/peptide molar ratio. Temperatures of the
beginning and completion of the main phase transition (D). Changes in phase transition enthalpy (in kJ/mol of phospholipid) under peptides influence at different peptide:lipid
molar ratios (E). WT (C), DM1 (-) and DM2:.
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Fig. 3. ITC experimental data obtained for the interaction of peptides with POPC/POPG (3:1 molar ratio) vesicles. Upper parts depict mcal evolved per second versus molar ratios of
outer monolayer lipid (60% of total) to peptide. Lower parts correspond to kcal evolved to mol of outer monolayer lipid (60% of total) depicted versus molar ratios of outer monolayer
lipid (60% of total) to peptide. SUVs added into WT, DM1 and DM2 at 25  C. Upper panels: heat flow, lower panels: integrated heats of titration of SUVs into the cell containing
peptide, in at initial concentration of 5 mM and 0.025 mM, respectively. Control negative data (pure buffer) was subtracted in each case.

Table 1
Thermodynamic parameters corresponding to the interaction between peptides and liposomes (SUVs), POPC/POPG (3:1 molar ratio) and pure POPG. Thermodynamic pa-
rameters are given per mol of outer monolayer lipid (60% of total). Mean and standard deviation values from the fitted data of three independent experiments are shown. In the
case of POPG vesicles ND stands for not determined, since it was not possible to fit the resulting data. All measurements were carried out at 25  C. One-site model is used for
fitting, according to the software supplied by Microcal.

SUV Peptide KA (M1) DH (cal/mol) DS (cal/mol K) DG (cal/mol) n

POPC/POPG WT 2.473E4 ± 4221 213.8 ± 24.8 19.4 ± 1.3 5771.8 ± 115.1 3.22 ± 0.46
DM1 5.163E4 ± 6393 440.8 ± 19.6 20.1 ± 1.2 6339.3 ± 237.1 6.15 ± 0.21
DM2 4.561E4 ± 8645 281.9 ± 12.9 20.4 ± 1.0 6263.2 ± 450.8 11.13 ± 0.38
POPG WT 1.540E4 ± 2062 919.7 ± 105.5 16.1 ± 1.7 5982.9 ± 247.4 2.54 ± 0.26
DM1 ND ND ND ND ND
DM2 ND ND ND ND ND

was used to explore the effect of the peptides on the thermotropic
transition of DMPG and DMPC/DMPG MLVs. Additionally, hydration
of the phospholipid carbonyl group was also studied. Monitoring
the symmetric CH2 stretching vibration (yCH2) centered near
2850 cm1 provides information about the influence of peptide
binding on the hydrophobic core region of the bilayer since it is
sensitive to the conformation of the hydrocarbon chains. The band
shift to higher wavenumber observed, upon increasing tempera-
ture, indicates a growth in the proportion of gauche conformers in
the acyl chains corresponding to mobile and conformationally
disordered lipid hydrocarbon chains. WT peptide increased the
wavenumber on DMPG vesicles in gel phase at 1:50 peptide/lipid
molar ratio, whereas cationic peptides increased it moderately at
the start of the transition (Fig. 5A).
One of our main objectives using FTIR was to find out whether
the peptides induced demixing on DMPC/DMPG mixtures. There-
fore, a phospholipid containing perdeuterated acyl chains was used
(DMPC-d54) to be mixed with DMPG at a 1:1 molar ratio in order to
obtain distinct vibrations of the acyl chains of each of these two
phospholipids. Interestingly, DM2 induced a notable fluidization on
DMPG mixed with DMPC-d54 reflected by the band shift to higher
wavenumber both in Lb0 and La phases (Fig. 6B) and by the decrease
of the Tm from 22 to 18  C in comparison with pure DMPG, sug-
gesting a disturbance of the acyl chain packing concomitant with a
fluidization. On the other hand, the presence of DM1 and WT
peptides did not show any change in comparison with the slight
effect exhibited in pure DMPG (Fig. 5A and B). By analyzing the shift
of the band position of symmetric CD2 stretching vibration (y(CD2))
centered near to 2090 cm1 of DMPC-d54 upon increasing tem-
peratures, it emerged that WT and DM1 peptides induced a slight
decrease of the wavenumber in the transition (Fig. 6C) and an in-
crease of the membrane Tm from 20 to 24 and 22  C, respectively,
suggesting an enhancement in the order of the acyl chains. On the
other hand, the binding of DM2 peptide resulted in the lowest
temperature (14  C) of the phase transition onset (Fig. 5C), although
Tm did not change.
Characteristic vibrational bands of the polar head region of
these lipids are the carbonyl stretching region y(C¼O) containing
two subcomponents bands centered near 1741 and 1722 cm1,
which are attributable to non-hydrogen-bonded C¼O groups and to
C¼O groups having one hydrogen bond, respectively [21]. There-
fore, the C¼O stretching band is also a good indicator for the hy-
dration of the membranes in the interfacial region and thus allows
the study of the interfacial interactions between peptides and
phospholipids. It is interesting to note that in DMPG membrane, the
presence of cationic peptides produced a shift of the maximum of
the C¼O band to lower frequencies after phase transition as
compared to both pure phospholipid and in the presence of WT
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Fig. 4. ITC experimental data obtained for the interaction of peptides with POPG vesicles. Upper parts depict mcal evolved per second versus molar ratios of outer monolayer lipid
(60% of total) to peptide. Lower parts correspond to kcal evolved to mol of outer monolayer lipid (60% of total) depicted versus molar ratios of outer monolayer lipid (60% of total) to
peptide. SUVs added into WT, DM1 and DM2 at 25  C. Upper panels: heat flow, lower panels: integrated heats of titration of SUVs into the cell containing peptide, in at initial
concentration of 5 mM and 0.025 mM, respectively. Control negative data (pure buffer) was subtracted in each case.

Fig. 5. Gel to liquid crystalline phase transition of the acyl chains according to CH2 stretching vibration (y(CH2)) displacement upon increase of temperature in pure DMPG (A),
DMPG mixed with DMPC-d54 (B) and DMPC-d54 mixed with DMPG (C), in the absence (,) or in the presence of peptide: WT (A), DM1 (:) and DM2 (C).

Fig. 6. Temperature dependence of the maximum of the y(C¼O) vibrational band in DMPG (A) and DMPC-d54/DMPG MLVs (B) in the absence (,) or in the presence of peptide: WT
(A), DM1 (:) and DM2 (C).

peptide (Fig. 5A).
In DMPC/DMPG vesicles without peptides, the wavenumber of
C¼O groups downshifted by 2 cm1 compared to pure DMPG
(Fig. 6). On the other hand, all peptides increased the wavenumber
on gel phase (Fig. 6B). In fluid phase, the presence of WT peptide
shifted considerably the C¼O peak to higher wavenumber and DM1
had a slight effect on C¼O band position, in contrast to their effect
on pure DMPG vesicles. Meanwhile, DM2 peptide showed the
highest effect on the hydration at C¼O group, which can be
deduced by the wavenumber downshift from about 1739 (gel
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phase) to 1734 cm1 (fluid phase) a similar wavenumber to that
exhibited by pure lipid (Fig. 6B).
4. Discussion
The use of different biophysical techniques leads to the conve-
nience of using different types of lipid vesicles. Each technique has
its own limitations. In the case of techniques such as DSC and FT-IR
multilamellar vesicles are used in most cases and this is due to the
fact that these vesicles show cooperativity in the phase transition.
DSC is a powerful tool to study the interaction between intrinsic
molecules and membranes. Studying the effect of intrinsic mole-
cules (such as peptides or other hydrophobic molecules) it is
possible to extract valuable information about the molecular in-
teractions and the location of these molecules in the bilayer [22,23].
But when trying to discern the effect of these molecules on the
phase transition, saturated membranes are preferred because most
cis-unsaturated membranes show the transition at temperature
below 0  C. In addition multilamellar vesicles possess a membrane
curvature that it is relatively close to biological membranes and this
is not the case of usual unilamellar vesicles such as SUV (sonicated)
or LUV (extruded). But there is still another important reason to use
MLV for DSC measurements and this is their stability. Unilamellar
vesicles made of saturated phospholipids would be prone to fusion
when they are cycled through the phase transition [24]. Since these
vesicles must be prepared in the fluid condition, during DSC mea-
surements they will be subjected to this type of cycling when doing
heating scans. In general when measurements are done on systems
in equilibrium MLV are preferred, for techniques such as DSC, FT-IR,
X-ray diffraction or NMR [24].
On the other hand, when dynamic measurements are carried
out, using other techniques as ITC or the measurement of the
insertion of a molecule from the external phase into the membrane
(by for example fluorescence spectroscopy), then the study is based
on just the outermost membrane. In the last cases unilamellar
vesicles is the obvious choice since the multilamellar ones would
present an undetermined number of lamella not exposed to the
external aqueous phase and this would made extremely difficult to
analyze many of the results. It is obvious that some unilamellar
vesicles, oftenly used in biophysical measurements, have the
problem of differing from biological membranes in the membrane
curvature characteristics and therefore some cautions should be
adopted when interpreting these results.
With respect to membrane composition, for DSC and ITC mea-
surements we are doing here a comparison of pure PG and PC/PG at
a 3:1 molar ratio. This ratio is used to make this composition similar
to that of a biological membrane. However for FT-IR we are using a
PC/PG 1:1 molar ratio because we use a labeled PC and both lipids
can more easily monitored in this way.
In this study, we focused on the possible role of cationic charge
of modified antimicrobial peptides derived from Cecropin D-like
Galleria mellonella on distinct membrane models. It has been
shown before that these modified peptides exhibited an eightfold
increase in their antibacterial activity compared to the WT and that
they were able of releasing calcein contents from liposomes and
that they altered both the glycerol backbone and the hydrophobic
palisade of model membranes [13]. All these observations pointed
to the insertion of the peptides in the membranes forming pores, as
it has been previously described for cecropins [25].
In order to better understand the mechanisms of action of these
peptides and how these mechanisms are altered by the modifica-
tions consisting in increasing their positive charges, we applied
calorimetric and spectroscopic techniques to analyze the in-
teractions between peptides and membrane models which simu-
late the charge surface of Gram-negative bacteria membranes.
Fig. 7 illustrates the consequences of the substitution of residues
in peptides DM1 and DM2. As it was pointed out previously [25,26],
the structure of cecropins consist in two amphipathic a-helices
linked by a Pro hinge. According to the model proposed [25], the N-
terminal helix of cecropin will bind to the membrane in such a way
that the hydrophobic face will be in contact with the part of the
fatty acyl chains of the phospholipids that are close to the polar part
of the membrane whereas the hydrophilic face of this helix will be
in contact with the polar head groups of these phospholipids. As it
can be seen in Fig. 7, the mutations introduced new positively
charged residues that were located in the hydrophilic face of the N-
terminal helix as it is easily visualized through the helical wheels
projections. The increase in negative charges of this helix will
reinforce its interaction with negatively charged phospholipids and
this will explain the stronger effects observed, when comparing
with the WT peptide.
In DSC thermotropic profiles of pure DMPG, the slight shoulder
in the right side of the main transition peak is common at a 100 mM
concentration of NaCl [27] and at this concentration a thermal
behavior similar to that of the lipid DMPC is exhibited, which un-
dergoes a gel to liquid crystalline phase transition at 23  C [27].
Interestingly, all peptides showed a pronounced effect on the main
transition, in spite of the neutral charge of WT peptide. The pres-
ence of three arginines in N-terminal region of WT would
contribute in such interaction since the Arg residues are involved in
the binding of the peptides to anionic membrane surfaces [28].
Additional studies about Arg-PG interactions suggest that Arg side
chain can approach PG head groups to 5 Å [29,30].
At a DMPG lipid-to-peptide molar ratio of 100:1, all peptides
promoted the emerging of a shoulder in the right side of the main
phase transition peak suggesting a membrane separation into do-
mains rich in peptide and a region where the lipid is unperturbed
[31]. The alterations in the phase transition can be explained by the
ability of the peptides to induce a perturbation in the bilayers
which leads to a change in the thermodynamic parameters related
to the phase transition. Additionally the increase in the bilayer
order could be due to the shielding of the polar group charges after
peptide binding avoiding the lateral repulsion between the lipids
and therefore the gel phase is stabilized [10].
On the other hand, all peptides considerably decreased the
onset transition temperature being DM2 the peptide with the
strongest effect. The results showed two separate components at
the highest concentration: one peptide-rich domain highly fluid-
ized and another corresponding to pure lipid. The enthalpy of
transition of DMPG MLVs was decreased by the presence of pep-
tides. This change of enthalpy in the main transition is due to an
alteration at interactions between lipid acyl chains, as a result of the
disruption of the intra and intermolecular van der Waals in-
teractions and trans-gauche isomerization and this interpretation
points out to the insertion of the peptide, or at least a part of it in
the hydrophobic core of the membrane.
We have used pure negatively charged DMPG liposomes as a
simple model system and DMPC/DMPG liposomes as a represen-
tative model of a biological membrane. In the presence of the latter
WT did not induce changes in the pre-transition at the maximum
peptide concentration indicating a very small effect of WT on
DMPC/DMPG mixtures. All peptides have a lower interaction with
pure DMPC vesicles (data not shown), since zwitterionic mem-
branes are inert when confronted with charged peptides [32] and
the presence of DMPC in mixed membranes will cause a decrease in
the binding points exhibited by DMPG polar groups.
The ITC experiments on the binding of POPC/POPG liposomes to
peptides indicated that the introduction of additional positive
charges in the peptides increased KA and the absolute values of DG,
indicating an increase in the binding strength between liposomes
 ~ ate-Garzo
J. On
n et al. / Archives of Biochemistry and Biophysics 629 (2017) 54e62
Fig. 7. Helical wheel projection of WT, DM1 and DM2 N-terminal helix (1e18 residues). The residues hydrophobicity is shown from green (most hydrophobic residues) to yellow
(zero hydrophobicity) color, the potentially charged residues are represented in blue (negatively charged as triangles, and positively charged as pentagons) and the red circle are
hydrophilic residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
that bear the negative charge of POPG and peptides. Unfortunately
in the case of pure POPG liposomes it was not possible to analyze
the data in detail, except for the case of the WT peptide. It seems
that given the great negative charge density present in the surface
of pure POPG vesicles and the increased positive charges in DM1
and DM2 peptides there is a very exothermic binding after the first
injection of liposomes, even with a very low concentration of
vesicles.
ITC titration peak positions and the corresponding DH demon-
strated that membrane-peptide interactions were exothermic,
suggesting the formation of new non-covalent bonding [33]. DG
was calculated from the binding constant and this was obtained
adjusting the data to a one-site binding model. The negative value
of free energy exhibited for peptides suggests a spontaneous
peptide-lipid interaction. However, a relatively small variation of
free energy together with a pronounced variation of enthalpy and
entropy between zwitterionic and cationic peptides as resulted of
the binding peptide-lipid was observed. This could be explained by
enthalpy-entropy compensation, where an enhanced enthalpy
does not reflect an improved affinity since the gain enthalpy is
compensated with a loss of entropy. This is because the amino
groups from basic residues added in the cationic peptides provide
additional interactions with the polar headgroup of phospholipids
contributing to an increase in enthalpy, meanwhile these new in-
teractions reduce the conformational freedom of the phospholipid
thereby decreasing the entropy [34].
FTIR results exhibited a shift to higher wavenumber of the
maxima absorption frequency of CH2 stretching vibrations when
the temperature was increased, due to the enhancement of gauche
isomers and the decrease of van der Waals interactions in the fluid
state of the membrane. The frequency of the CH2 stretching of
DMPG mixed with DMPC-d54 increased considerably in presence of
DM2 peptide compared to the control, suggesting a fluidization of
DMPG domains. However, the wavenumbers of CD2 vibration in
DMPC-d54 were not altered, implying a preferential interaction of
DM2 with DMPG separating it away from the mixture and lefting
the DMPC-d54 component in a purer form, exhibiting a similar
phase transition to that of the membranes of pure zwitterionic lipid
and resulting in a lateral separation of the mixture due to electro-
static interactions between lipids and DM2. Thus, fluctuations and
re-orientations of the acyl chains will increase the fluidization of
DMPG-DM2 complex [35].
The frequency of C¼O stretching band decreases upon increase
of temperature as a result of hydration since this band is sensitive to
hydrogen bonding between C¼O with water or with other
hydrogen bond donor groups [36]. In fluid DMPG MLVs, the
wavenumber of the carbonyl band was decreased considerably
under the effect of cationic peptides, suggesting that both peptides
could either affect bilayer packing, promoting hydration at the
interface level or the formation of hydrogen bonds between side
61
amines of basic residues and C¼O groups. In contrast, peptides
increased the frequency of the carbonyl band on DMPC-d54/DMPG
mixtures in gel state. This lack of hydration could be due to an in-
crease of interface order as a result of the interactions between
peptides and headgroups of lipids where water molecules are
excluded [10].
In conclusion, calorimetric studies provided evidence that
peptides are electrostatically attracted to polar headgroups of the
membrane and that they have a strong effect on the main phase
transition. On the other hand, spectroscopic assays showed that
peptides have the ability of demixing phospholipids from binary
mixtures which simulate the charge surface of Gram-negative
bacterial membranes, leading to the formation of fluid (DM2) or
ordered domains (WT and DM1), and it could be speculated that
this may be connected to the release of intracellular components.
Acknowledgments
This work was supported by Universidad de Antioquia Research
~ ate would like to thank the support of
Project CODI IN618CE. J. On
COLCIENCIAS through their Fellowship Doctoral Program. We also
thank Universidad de Murcia, grant 365, for support.
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