Investigation of the interaction of antimicrobial peptides with lipids and lipid membranes 3

141
CHAPTER 7 INTERACTION BETWEEN ANTIMICROBIAL PEPTIDES
AND LIPID MEMBRANE LAYERS

7.1 Introduction
V4 was shown to first bind to the membranes and finally induce membrane permeation
by membrane aggregation and disruption. However, the intermediate process is not clear.
In this chapter, monolayer is mainly used to investigate the process of V4 inserting into
membranes and the comparision of V4 to other antimicrobial peptides is also shown.

7.2 Materials and methods
Materials
POPG, POPC and DPPG were purchased from Avanti. Solvent chloroform (HPLC grade)
and methanol (HPLC grade) and antimicrobial peptide magainin 2 (M2), melittin (ME)
and polymyxin B (PB) were purchased from Sigma-Aldrich. V4 peptide was synthesized
by Genemed. The purity of different peptides has been presented in previous chapters.
All the materials were used without further purification.

Instrumentation
Langmuir film balance (model 601M) (NIMA Technology Ltd. England) was used for
experiments. The instrument includes a 105 cm
2
trough connected with an external
circulator for temperature control, two mechanically coupled barriers, a surface pressure
sensor using Wihelmy plate, a sapphire window and a dipper well (25mm stroke). An
interface unit (IU4) connected the film balance and the computer. An operating software

142
(version 5.16) provided by NIMA Technology Ltd was used to collect data. The
cleanness of the trough was checked by closing and opening of the barriers to ensure that

surface pressure did not vary by more than ±0.1 mN/m.


Monolayer isotherms
POPG and POPC were dissolved in chloroform with a concentration of 0.2 mM. Due to
the low solubility of V4 in chloroform, V4 was first dissolved in the minimal volume of
methanol to prepare a clear solution. Additional chloroform was then added to prepare a
solution with V4 concentration of 0.2mM. Required volume of POPG or POPC was
mixed with V4 to form lipid/V4 mixture with V4 percentage of 0%, 5%, 10%, 20%, 33%,
50% and 100%. A syringe was cleaned completely and an appropriate volume of
individual lipid/V4 mixed solution was drawn and carefully deposited on the water
surface in a drop-wise manner, making sure that the surface pressure did not change after
deposition. The monolayer of lipid in the absence or in the presence of V4 formed
spontaneously on the air-water interface. After solvent evaporation for 10 minutes, the
monolayer was compressed with a rate of 7 cm
2
/min and the isotherm curve was record.
Each curve was repeated at least twice for reproducibility.


Penetration studies
POPG and POPC were dissolved in chloroform and DPPG was dissolved in the mixture
of chloroform and methanol (v/v=3:1) with a final lipid concentration of 0.2 mM. All the
studied antimicrobial peptides were dissolved in water with high concentrations as stock
solution. Required volume of lipid solution (usually 60
µl) was drawn by using a clean

143
syringe and spread on the water surface carefully in a drop-wise manner, making sure
that the surface pressure did not change after lipid deposition. The lipid monolayer

formed spontaneously on the air-water interface. After solvent evaporation for 10 minutes,
the monolayer was compressed with a rate of 7 cm
2
/min to a target surface pressure. The
lipid monolayer was allowed to adjust until a constant molecular area was achieved.
Afterward an appropriate volume of peptide solution was injected underneath the
monolayer into the subphase, generating different peptide concentration in the trough.
The surface pressure change with time with fixed molecular area was record. All the
experiments were done at 37 °C and each penetration experiment was repeated at least
twice for reproducibility. A water subphase but not buffer was used to avoid
crystallization of salt on the sample which would interfere with the imaging of sample in
the AFM experiments.

AFM experiment
A monolayer which was penetrated by antimicrobial peptides was transferred to freshly
cleaved mica (Electron Microscopy Sciences, USA) by vertically placing the mica in the
water subphase before lipid was spread on the water surface. After the penetration
experiment, the surface pressure was kept constant at the surface pressure of complete
penetration. The mica was slowly extracted from the subphase to the air phase with a
constant rate of 3 mm/min. A lipid monolayer was obtained by compressing the lipid
monolayer to a certain surface pressure and extracted from the mica from the subphase at
the constant target surface pressure. The monolayer with or without peptide on the mica
was dried in a desiccator overnight before AFM imaging.

144
AFM experiment was performed in air on the NanoScope IIIa MultiMode Scanning
Probe Microscope manufactured by Digital Instruments Inc. (Santa Barbara, CA 93117,
USA). Topographic images were acquired in tapping mode. The typical scan rates ranged
from 1 to 1.25 Hz depending on the scan size. The monolithic silicon probes (NanoWorld
AG, Switzerland) with a cantilever length of 125

µm and force constant of 42 N/m were
used for measurements. Images was obtained and analyzed by the Nanoscope software
provided by the company. Images from at least two different sample prepared on
different days with several macroscopically separated areas on each sample were
acquired for data reproducibility. Representative images are shown.

Insertion study of V4 into POPG bilayer
Dual polarization interferometry was used to study the insertion of V4 into solid
supported bilayers. This technique allows the opto-geometrical properties (density and
thickness) of adsorbed layers at a solid-liquid interface to be determined
132
. When a
peptide is introduced into the thin lipid bilayer, the changes of average thickness and
average density (through the refractive index) of the lipid bilayer as well as the mass can
yield information of how the peptide interacts with the lipid bilayer. Experiments were
done on an AnaLight
® Bio 200 system. POPG SUVs were prepared and deposited on an
amine modified surface sensor chip. After obtaining a stable POPG bilayer on the surface,
V4 was injected and the density and mass change of the POPG bilayer were recorded.




145
7.3 Results and discussion
7.3.1 Isotherm studies of V4 interacting with POPG and POPC
7.3.1.1 Isotherms of lipid monolayers
The surface pressure (
π
) and molecular area (A) isotherms for POPG and POPC

monolayer at the air-water interface at 37 °C are shown in Fig. 7.1. When the lipid
monolayer was compressed, the isotherm for POPG and POPC began to rise at a
molecular area of 107
Å and 96 Å, respectively. With increasing compression, the surface
pressure increased continuously until the collapse pressure of 45.6 mN/m and 44.5 mN/m
for POPG and POPC, respectively. The shape of isotherms of POPG and POPC
monolayer was similar especially at low molecular area, which indicated that the packing
of POPG and POPC lipid molecules was similar. POPG and POPC are both unsaturated
lipids containing one double bond. They have the same hydrophobic alkyl chains and
differ in the headgroup. The molecular packing of the monolayer is mainly dependent on
the hydrophobic interaction between the alkyl chains of the lipid molecules. The same
alkyl chain of POPG and POPC allowed similar molecular packing so that POPG and
POPC showed similar isotherms. Thus hydrophobic interaction played a significant role
during the compression. Although POPG bears a negative charge, which might impose a
repelling effect between POPG molecules especially when the molecules were close,
there was not much difference between the POPG and POPC isotherms, indicative of the
negligible effect of electrostatic interaction.

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Fig. 7.1 Isotherms of POPG and POPC monolayers

7.3.1.2 Isotherms of mixed lipid/V4 monolayers
Fig. 7.2 shows the surface pressure (
π
) and molecular area (A) isotherms of POPG/V4
and POPC/V4 monolayers at the air-water interface at 37 °C. The pure V4 showed strong
surface activity compared with lipid. At a molecular area of 125
Å, the isotherm of V4
began to rise. With increasing compression, the surface pressure of V4 monolayer

continuously increased to 45.0 mN/m, which was comparable to the collapse pressure of
POPG and POPC. When V4 was incorporated into the lipids, the isotherms of the mixed
POPG/V4 and POPC/V4 monolayer shifted right to the high molecular areas with
increasing percentage of V4. The collapse pressure for all isotherms was similar except
for the POPG/V4 mixture with 50% V4 incorporation, which was an incomplete isotherm
because the two barriers were too close. The shape change of isotherms was complicated.
The isotherms of POPG/V4 with V4 percentage of 5% and 10% were similar to the
isotherm of pure POPG. When V4 percentage increased to 20% or higher, there was a
kink at surface pressure of 30 mN/m, which indicated that V4 peptide might induce a

147
phase transition. Above 30 mN/m, the increase of surface pressure due to the
compression slowed down. The presence of V4 in the POPC monolayer with V4
percentage of 5% did not induce much change in the isotherm. However when the
percentage of V4 increased to 10% and higher, the shape of the mixed POPC/V4
isotherms was similar to pure V4 isotherm. Comparing all the molecular areas at which
the surface pressure began to increase (lift-off area), it was found that with increasing
incorporation of V4, the lift-off areas gradually increased for both lipids, which indicated
that V4 had an area-expanding effect on the lipid monolayer at low surface pressure.

148

Fig. 7.2 Isotherms of mixed POPG/V4 and POPC/V4 monolayers

7.3.1.3 Miscibility analysis of monolayers
When the studied components were mixed to form monolayers on the air-water interface,
it was not easy to determine if the components were really miscible or not from the direct
measurement. Analysis of the monolayer isotherm may provide useful information to

149

determine the miscibility of the studied components in the monolayer. Each pure
component has its own collapse pressure. In a two-component system, if the components
were immiscible, two collapse pressures would be observed at the corresponding collapse
pressure of pure components. However if the two components were miscible, only one
collapse pressure would be obtained
220
. Fig. 7.2 showed that all mixed monolayers had
one collapse pressure for both POPG and POPC, which gave an indication that V4 was
miscible with POPG and POPC.

According to the phase rule of Defay and Crisp
221
, in a two-component system, if the
components are completely miscible for all the ratios, there is only one degree of freedom
at constant temperature and pressure, assuming no externally imposed electrical
potentials. Therefore when the percentage of V4 in the mixed lipid/V4 solution varies,
the surface pressure will vary correspondingly at fixed molecular area. If V4 and lipid
mix ideally, the ideal surface pressure can be calculated by
AAidealA
XX )()(
2211,
π
π
π
+=
(7.1)
π
1
and
π

2
are the surface pressure of the pure lipid and V4 at a given molecular area A,
respectively. X
1
and X
2
imply the percentage of lipid and V4, respectively. If the mixing
is ideal, the surface pressure will vary linearly with the percentage of V4 in the mixed
monolayer. However if the mixing is non-ideal, a deviation from the linear relationship
will be obtained. From the excess surface pressure, which describes the deviation from
the ideal mixing, interaction between V4 and lipid could be deduced. The excess surface
pressure
π
A,ex
can be obtained by the difference between the surface pressure of the real
mixed monolayer
π
A, exp
and the ideal mixed monolayer according to Eq. 7.2.

150
idealAAexA ,exp,,
π
π
π
−= (7.2)
Fig. 7.3 shows that the surface pressure varied with different percentage of V4
incorporating into POPG and POPC monolayer at different molecular areas. An apparent
difference between the different molecular areas was observed for both lipids. At high
molecular area, which the molecules were loosely packed, the surface pressure increased

gradually with the increasing percentage of V4. When there was 50% V4 incorporated
into the lipid monolayer maximal surface pressure was obtained. With increasing
molecular area, the surface pressure difference between the different molecular areas
became smaller. At low molecular area, the curve became flat (POPG) or the maximal
surface pressure shifted to low percentage of V4 incorporation (POPC).

Fig.7.3 Surface pressure of lipid monolayers incorporated with different percentage of V4.
Left: POPG; Right: POPC.

The excess surface pressure, which indicates the deviation from ideal mixing is shown in
Fig. 7.4. For the POPG lipid, except at a low molecular area of 50
Å, the surface pressure
at the other molecular areas all showed positive deviation from linearity. The positive
excess surface pressure indicated that V4 had a surface pressure increasing effect on the
monolayer, which was equivalent to an area expanding effect. The more V4 was
incorporated, the greater the effect was until a maximal value was reached with V4
incorporation of 33% to 50% dependent on different molecular areas. At the molecular

151
area of 50
Å, there was not much excess surface pressure. When the percentage of V4
was smaller than 20%, slight negative deviation from the linearity was observed. This
indicated that at this molecular area, V4 peptide was almost ideally mixed with POPG
with a tiny area condensing effect on the monolayer at a certain percentage of V4. Within
the all studied molecular areas, V4 induced positive deviation from the linearity for the
POPC monolayer. Similar to POPG, with increasing V4 incorporated into POPC
monolayer, the excess surface pressure increased until it reached a maximal value at the
percentage of V4 between 20% and 50%. Simultaneously with decreasing molecular
area, the maximal excess surface pressure shifted to low V4 percentage. In brief, similar
to POPG, V4 also generated an area expanding effect on the POPC monolayer.

Fig. 7.4 Excess surface pressure of lipid monolayers incorporated with different
percentage of V4. Left: POPG; Right: POPC.

7.3.1.4 Stability analysis of monolayers
The stability of a monolayer can be investigated by the evaluation of the excess energy
caused by interaction between V4 and lipids. According to the method developed by
Zhao and Feng
220,222
, the excess Helmholtz energy can be used to determine the energy
change. The excess Helmholtz energy was defined as the deviation from the ideal value
of Helmholtz energy according to Eq. 7.3.

152
dAXXA
A
A
ex
m
)]([
221112
0
πππ
+−∫=∆
(7.3)
A
0
and A are the molecular area where the surface pressure begins to increase from zero
and where the Helmholtz excess energy is calculated, respectively.
π

12
,
π
1
and
π
2
are the
surface pressure of mixed monolayer, pure lipid monolayer and pure V4 monolayer at the
molecular area of A, respectively. X
1
and X
2
imply the percentage of lipid and V4
respectively in the mixed monolayer.

Fig. 7.5 shows the excess Helmholtz energy change at different molecular areas. For both
POPG and POPC, the presence of V4 in the mixed monolayer induced a negative
deviation from ideal mixing. The negative excess Helmholtz energy indicated that there
was interaction between lipid and V4 peptide and less energy was needed for a stable
system. Therefore the stability of a system can be determined by the value of excess
Helmholtz energy. With increasing percentage of V4 incorporated into the lipid
monolayer, the excess Helmholtz energy continuously decreased, which indicated an
increasing interaction between lipid and V4. The minimal excess energy which
corresponded to a stable system was obtained in the presence of 50% V4 in the mixed
monolayer. Therefore when V4 was incorporated into the monolayer with the percentage
from 0% to 50%, V4 increased the system stability. Except the percentage of V4, the
molecular area also affected the change of excess Helmholtz energy. It was shown that at
the biggest studied molecular area of 100
Å, V4 created a relatively small energy

decrease. Decrease in the molecular area induced apparent decrease in excess energy
when the percentage of V4 was fixed. This result indicated that in the presence of a

153
particular percentage of V4, the compression increased the interaction between V4 and
lipid and consequently the system stability.

Fig. 7.5 Excess Helmholtz energy of lipid monolayers incorporated with different
percentage of V4. Left: POPG; Right: POPC.

7.3.1.5 Compressibility analysis of monolayers
Compressibility is a parameter to describe the elastic packing of the monolayer. It is
defined as
)/(
1
π
ddAACs
e
−
−=
(7.4)
where A
e
is the extrapolated area at zero surface pressure
220
. The compressibility of lipid
POPG, POPC, V4 peptide and different mixture is shown in Fig. 7.6. When V4
incorporated into POPG monolayer, the presence of V4 increased the compressibility
with the percentage of V4 lower than 10%, indicating that the monolayer became more
compressible due to V4. The compressibility kept constant between V4 percentage of

10% and 33%. When there were more V4 incorporated into the POPG monolayer, the
compressibility decreased, which indicated a decreasing elastic monolayer packing.
Different from POPG lipid, the presence of V4 slightly increased the compressibility of
POPC monolayer at 5% V4. When the percentage of V4 increased, the compressibility
kept constant until pure V4 monolayer. These results indicated that V4 had a great effect

154
on the packing of POPG monolayer, which can be attributed to the interaction between
V4 and POPG lipid. Compared with POPG, V4 induced much less effect on the POPC
monolayer packing.

Fig. 7.6 Compressibilities of lipid monolayers incorporated with different percentage of
V4.

POPG and POPC showed similar isotherms especially at low molecular area. As
mentioned before, the determining factor for the isotherm is the hydrophobic interaction
between the alkyl chains of lipids and charge nearly has no effect. Compared with some
saturated lipids, POPG and POPC both have high lift-off molecular areas at which the
surface pressure began to rise from zero upon compression
223
. Because of the unsaturated
structure, the kink in the alkyl chains, which is formed by the double bond, makes one
molecule occupy a large area than a saturated lipid did. Therefore unsaturated lipid
monolayers are more compressible than saturated lipid monolayers. This was confirmed
by the result of the compressibility shown in Fig. 7.6 in which both POPG and POPC
monolayers have a relatively high value. The presence of V4 further increased the
compressibility of the monolayer, indicative of the increased molecular area, which was
consistent with the result of excess surface pressure.

155
The incorporation of V4 decreased the system energy as shown in Fig. 7.5. With
increasing percentage of V4, the energy decreased steadily which indicated an increasing
interaction between V4 and lipid. This result was consistent with the previous study that
V4 had affinity for both POPG and POPC. Therefore V4 did increase the system stability.
Simultaneously, the presence of V4 induced a positive excess surface pressure, which
indicated that V4 promoted a surface pressure-increasing effect or an area-expanding
effect on both lipid monolayers. One of the possible reasons might be the conformational
change due to the interaction between V4 and lipid. When there was no V4, the alkyl
chains of the lipid molecules extended upward to the air phase and the headgroup
contacted with water phase. The hydrophobic interaction between the hydrophobic part of
V4 and alkyl chains of the lipid made the alkyl chains tilt to the interface which increased
the area needed for one molecule and led to an area expanding effect. When the area was
fixed, the increased surface pressure was observed as shown in Fig. 7.4. Therefore, V4
changed the conformation of lipid through peptide-lipid interaction and imposed a
surface pressure increase effect on the lipid monolayer.

7.3.2 Penetration studies of antimicrobial peptides interacting with lipid monolayers
7.3.2.1 Penetration of antimicrobial peptides into POPG monolayers
The presence of antimicrobial peptide in the subphase induces a surface pressure change,
which indicates the penetration or insertion of the peptide into the lipid monolayer. The
interaction between magainin 2, melittin, polymyxin B and V4 with POPG monolayers is
shown in Fig. 7.7. Before peptide was injected, the POPG monolayer was compressed to
a target surface pressure (initial pressure) of 15 mN/m. The addition of antimicrobial

156
peptide into the subphase resulted in an increase in the surface pressure for all studied
peptides. Fig. 7.7 shows the penetration kinetics of different peptides into POPG
monolayers. With time increasing, the surface pressure increased continuously. When the
surface pressure showed no significant increase, the monolayer was saturated and the

penetration was thought to reach equilibrium. With increasing peptide concentration, the
penetration apparently sped up. At the lowest studied peptide concentration of 50 nM, the
time needed to achieve complete penetration was over 6000 s. However, when there was
more peptide in the solution, less time was needed to complete penetration. At a peptide
concentration of 150 nM, magainin 2, melittin and polymyxin B led to a fast penetration
of less than 2000 s. V4 took more time with about 4000 s. When the peptide
concentration further increased, the rate of penetration correspondingly increased. This
result indicated that the rate of penetration depended on the peptide concentration and is
diffusion limited. The rate of penetration was related to the peptide concentration, which
was close to the surface of the monolayer in the water phase. Because concentrated
peptide was injected underneath the monolayer in the water phase, the peptide molecules
accumulated in several regions. These peptide molecules diffused and some of them
reached the surface of the monolayer, where peptide molecules might eventually
penetrate the monolayer. At high peptide concentration more peptide molecules reached
the surface of the monolayer and penetrated into the monolayer, which showed a fast
penetration compared with low peptide concentration.

157

Fig. 7.7 Kinetics of antimicrobial peptides with different concentrations penetrating into
POPG monolayers


Fig. 7.8 Comparison of antimicrobial peptides penetrating into POPG monolayers

The peptide concentration affected not only the rate of penetration, but also the extent of
penetration. Fig. 7.8 compares the penetration of antimicrobial peptides into POPG
monolayers. ∆π is used to describe the surface pressure increase from the target surface
pressure (or initial surface pressure). When V4 concentration increased from 0.05
to 1

158
µM, V4 exhibited a gradual increase in ∆π from 4.9 to 19.7 mN/m. Further addition of
V4 into the subphase did not induce much change in ∆π, which indicated that the
monolayer reached saturation at 1
µM. Similar to V4, magainin 2 and melittin generated
increasing ∆π with increasing peptide concentration up to 0.5
µM. More addition of
peptide into the subphase did not induce apparent increase in surface pressure. Thus the
maximal penetration into POPG monolayer due to magainin 2 and melittin was obtained
at 0.5
µM. Polymyxin B was different from the other antimicrobial peptides. From a
peptide concentration of 0.05
µM to 1 µM, polymyxin B generated similar penetration.
Further addition of peptide induced slight increase in ∆π compared with the lower
peptide concentration. This result implied that at 0.05
µM, polymyxin B caused
monolayer saturation. According to the increased surface pressure ∆π at a saturated
peptide concentration, for example 1
µM, the ability of peptide penetrating into the
POPG monolayer was determined. Therefore from Fig. 7.8 the penetration ability of
these antimicrobial peptide was melittin > magainin 2 > V4 > polymyxin B.

7.3.2.2 Penetration of antimicrobial peptides into POPC monolayers
It is known that mammalian cell membranes consist mainly of zwitterionic lipids in
contrast to bacterial membranes. Thus POPC was chosen in this study to investigate the
interaction between antimicrobial peptides and zwitterionic monolayers. The penetration
of antimicrobial peptides into POPC monolayers and comparison with POPG monolayers
are shown in Fig. 7.9. Compared with POPG monolayers, melittin, polymyxin B and V4
all displayed relatively weak penetration ability for POPC. One important reason might

be the charge difference between POPG and POPC. POPG harbors a negative charge,

159
which electrostatically interacts with cationic antimicrobial peptides. Electrostatic
interaction leads to the absorption of higher amount of peptide on the headgroup of
POPG, which increased the opportunity of peptides penetrating into the lipid monolayer.
Once the peptide molecules absorbed on the monolayer, the hydrophobic interaction
drove the peptide molecules to penetrate into the monolayer and induced an increase in
surface pressure. However, due to the lack of electrostatic interactions between the
peptides and POPC, less peptide molecules were absorbed on the monolayer surface,
which reduced the possibility of penetration. Therefore cationic antimicrobial peptides
showed higher penetration ability for POPG than for POPC monolayers, which is the
basis for selectivity.

160

Fig. 7.9 Comparison of melittin, polymyxin B and V4 penetrating into POPG and POPC
monolayers

The experiments showed that the penetration of antimicrobial peptides into POPC
monolayers is also peptide concentration dependent. With increasing melittin
concentration from 0.05
to 1 µM, ∆π increased gradually until monolayer saturation. The
addition of the extra melittin led to a constant surface pressure increase of 6-7 mN/m.
Compared to melittin, polymyxin B induced much less increase in the surface pressure

161
which indicated weak penetration into POPC monolayers. At the peptide concentration of
0.05
and 0.15 µM, nearly no penetration was observed. When the concentration increased

to 0.5
µM, only about 1mN/m surface pressure increase was obtained. Additional peptide
did not generate a higher surface pressure increase up to a peptide concentration of 3
µM.
The low value in ∆π caused by polymyxin B indicated that polymyxin B might only
absorb on the headgroup of the lipid without real penetration into POPC monolayer. V4
displayed relatively strong penetration ability compared with melittin and polymyxin B.
At the lowest studied peptide concentration of 0.05
µM, V4 induced a ∆π of 3.3 mN/m.
Increase in the peptide concentration led to an increase in ∆π. At 0.5
µM, V4 caused
POPC monolayer saturation with ∆π of 10 mN/m. Comparing these three antimicrobial
peptides, it is found that V4 had the highest penetration ability into POPC monolayer
followed by melittin. Polymyxin B showed nearly no penetration ability to this
zwitterionic lipid. The difference in penetration into POPC monolayer can be attributed
to the hydrophobicity of the peptide. V4 is a very hydrophobic antimicrobial peptide,
containing 8 valines out of a total of 19 amino acids. It was shown that even in detergent,
V4 aggregated due to its high hydrophobicity (Chapter 5). The strong hydrophobic
interaction between V4 and the alkyl chains of the lipids led to the highest penetration
among the studied peptides. Correspondingly melittin and polymyxin B are less
hydrophobic which resulted in lower penetration. This result is not consistent with the in
vivo lower hemolytic activity and lower cytotoxic activity of V4 compared to polymyxin
B. One possible reason is that V4 might strongly penetrate into POPC membranes and
interact with hydrophobic alkyl chains. However, due to the high hydrophobicity of V4,
the possibility to translocate into the inner layer of the membrane, which faces the

162
hydrophilic cytosol, is much lower than polymyxin B. Therefore V4 has shown higher
penentration ability into POPC monolayers but less ability to translocate into the
membrane inner layer leading to final cell death.

Fig. 7.10 shows the penetration kinetics of melittin and V4 into POPC monolayers. More
time was needed to achieve complete penetration into POPC than POPG monolayers,
especially at high peptide concentration for both melittin and V4. The slow penetration
into POPC monolayers indicated that charge not only affected the extent of penetration,
but also the rate. Electrostatic interaction allowed peptide molecules to strongly absorb
on the lipid monolayer, not easy to escape, and reach full penetration rapidly. However in
the absence of electrostatic interaction, it was difficult for the monolayer to capture the
peptide molecules, which diffused away easily, and resulted in more time needed for full
penetration. Thus the electrostatic interaction played a significant role in penetration.
Fig. 7.10 Kinetics of melittin and V4 with different concentrations penetrating into POPC
monolayers


163

Fig. 7.11 Comparison of V4 penetrating into different lipid monolayers

7.3.2.3 Penetration of V4 into different lipid monolayers
The interaction of V4 with different lipid monolayers was investigated to examine the
penetration ability of V4 into different membranes. Fig. 7.11 illustrates the comparison of
V4 penetrating into POPG, POPC, DPPG and a mixed monolayer of POPG/POPE (1/2).
With increasing peptide concentration, the penetration of V4 into the monolayers
gradually increased until a saturation at a certain peptide concentration between 0.5
µM
to 1
µM. Similarly according to ∆π at a saturated peptide concentration at which extra
peptide did not induce higher increased surface pressure, the ability of V4 penetrating
into the different monolayer was determined. Thus the sequence of V4 penetrating into
different lipid monolayer was POPG > DPPG > POPG/POPE (1/2) > POPC.

Among the studied lipid or mixed lipid monolayers, all but POPC are negatively charged.
Fig. 7.11 showed that V4 had a higher ability to penetrate into these negatively charged
lipid monolayers than the zwitterionic POPC monolayer, which confirmed the significant
role of electrostatic interaction in the penetration. This result is also consistent with the

164
result that V4 shows less affinity for POPC SUVs compared to negatively charged SUVs
in the binding experiments.

Lipid mixture of POPG/POPE (1/2) bears part negative charges, so that less penetration
was observed than for pure POPG. However, this result is different from the result in the
previous study that V4 has a similar binding affinity for the mixed lipid vesicles and
POPG vesicles. The reason for this difference was that in the binding FCS experiments,
lipid was present in a much higher concentration than V4. Once one V4 molecule bound
to a lipid vesicle, the vesicle was detected. When more peptide molecules bound to same
vesicle, the vesicle number did not change although it was more fluorescent. Thus the
main factor to determine the binding was lipid vesicle without bound peptide compared
to vesicles containing at least one peptide, allowing fluorescent detection. In other words,
it was the first V4 molecule binding to the lipid vesicle that determined the binding.
Therefore the binding of V4 to mixed lipid SUVs and pure POPG SUVs was similar.
However in the monolayer experiments, the absorption of the peptide molecules was
dependent on the charge amount of the lipid monolayer. The more charge the monolayer
harbored, the more V4 molecules absorbed on the monolayer, which led to a higher
penetration. Therefore the amount of charge exerted an important influence on the
penetration.

POPG and DPPG possess the same headgroup, thus the same negative charge, however
V4 showed a slightly higher penetration into POPG than DPPG monolayers. An
explanation would be the difference in alkyl chain of POPG and DPPG. The double bond

165
of POPG increased the fluidity of the monolayer, leading to less dense lipid packing. This
provided peptide molecules better access to interact with the alkyl chains and increased
penetration. DPPG is a saturated phospholipid and lipid molecules are closely packed
which hindered the penetration to some extent. Therefore the saturation of the lipid
molecule contributed to the penetration of V4.

In the previous study of V4 binding to different lipid vesicles, V4 showed higher affinity
for POPG than for DPPG vesicles. However, in this case the difference between these
two anionic lipids was not large. One possible explanation is the curvature of the
membrane. The unsaturated property made the POPG form smaller vesicles than DPPG
as shown by the fast diffusion time of POPG vesicles. This small sized vesicle has a
bigger curvature and allows the head group of the POPG molecule to occupy more space.
Therefore it is easier for V4 to bind to and insert into the POPG vesicle than DPPG
vesicle. However, in the monolayer, there is no difference in the curvature and the
absorption is mainly dependent on the charge. Therefore the different penetration into
POPG and DPPG was primarily due to the different hydrophobic interaction between V4
and alkyl chain of lipid after absorption.

Above results showed that both electrostatic interaction and hydrophobic interaction
contributed to antimicrobial peptide penetration. The injected antimicrobial peptide first
absorbed on the headgroup of the lipid in the water phase through electrostatic interaction
followed by penetration into the alkyl chain region of the monolayer due to hydrophobic
interaction. Especially the electrostatic interaction was of great importance because it