Antimicrobial Properties and Membrane-Active Mechanism of a Potential α-Helical Antimicrobial Derived from Cathelicidin PMAP-36
et al. (2014) Antimicrobial Properties and Membrane-Active Mechanism of a Potential a-Helical Antimicrobial
Derived from Cathelicidin PMAP-36. PLoS ONE 9(1): e86364. doi:10.1371/journal.pone.0086364
Antimicrobial Properties and Membrane-Active Mechanism of a Potential a-Helical Antimicrobial Derived from Cathelicidin PMAP-36
Yinfeng Lv 0
Jiajun Wang 0
He Gao 0
Zeyun Wang 0
Na Dong 0
Qingquan Ma 0
Anshan Shan 0
Rizwan H. Khan, Aligarh Muslim University, India
0 Laboratory of Molecular Nutrition and Immunity, Institute of Animal Nutrition, Northeast Agricultural University , Harbin, Heilongjiang , China
Antimicrobial peptides (AMPs), which present in the non-specific immune system of organism, are amongst the most promising candidates for the development of novel antimicrobials. The modification of naturally occurring AMPs based on their residue composition and distribution is a simple and effective strategy for optimization of known AMPs. In this study, a series of truncated and residue-substituted derivatives of antimicrobial peptide PMAP-36 were designed and synthesized. The 24-residue truncated peptide, GI24, displayed antimicrobial activity comparable to the mother peptide PMAP-36 with MICs ranging from 1 to 4 mM, which is lower than the MICs of bee venom melittin. Although GI24 displayed high antimicrobial activity, its hemolytic activity was much lower than melittin, suggesting that GI24 have optimal cell selectivity. In addition, the crucial site of GI24 was identified through single site-mutation. An amino acid with high hydrophobicity at position 23 played an important role in guaranteeing the high antimicrobial activity of GI24. Then, lipid vesicles and whole bacteria were employed to investigate the membrane-active mechanisms. Membrane-simulating experiments showed that GI24 interacted strongly with negatively charged phospholipids and weakly with zwitterionic phospholipids, which corresponded well with the data of its biological activities. Membrane permeabilization and flow cytometry provide the evidence that GI24 killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. GI24 resulted in greater cell morphological changes and visible pores on cell membrane as determined using scanning electron microscopy (SEM) and transmission electron microscope (TEM). Taken together, the peptide GI24 may provide a promising antimicrobial agent for therapeutic applications against the frequently-encountered bacteria.
Funding: This work was supported by the National Natural Research Foundation of China (31272453), the National Basic Research Program (2012CB124703), and
the China Agriculture Research System (CARS-36). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
The discovery of antibiotics effectively reduces the happening of
infectious diseases and saved countless lives in less than nine
decades. However, the widespread and often indiscriminate use of
antibiotics in recent years has led to the rapid emergence of
multidrug-resistant superbug strains, making infectious diseases
increasingly difficult to control with the existing classes of
antibiotics. Therefore, there is an urgent need to develop new
classes of antimicrobial agents. Data from both the laboratory and
the clinic in the last decade indicate that antimicrobial peptides
(AMPs) are suitable templates for an alternative class of potential
therapeutics . AMPs found in a large number of species
constitute a major component of the innate immune system, and
they have broad-spectrum activities against gram-negative and
gram-positive bacteria , including antibiotic-resistant bacterial
strains  and some fungi , viruses , parasites , and even
cancer cells . Moreover, unlike conventional antibiotics that
inhibit specific biosynthetic pathways such as cell wall or protein
synthesis, the majority of AMPs carry out their respective functions
via the rapid physical disruption of microbial cell membranes to
cause leakage of cell contents leading to cell death . This is
expected to provide an inherent advantage for AMPs in the
clinical setting because it is metabolically costlier for most
microbial to promote resistance by mutating or repairing its
membrane components . Currently, there are at least four
different commonly used models describing possible AMP
membrane-active mechanism that include barrel-stave, carpet,
toroidal-pore, and aggregate channel models [9,10].
Although the primary and secondary structures of AMPs display
a large heterogeneity, comparison of AMPs sequences reveals that
two types of side chains are essential for antimicrobial activity. The
cationic side chains provided electrostatic interactions between
peptides and the negatively charged membranes and/or cell walls
of bacteria, including lipopolysaccharide (LPS) . Nonpolar side
chains presumably provided lipophilic anchors that ultimately
induce membrane disruption . According an updated database
(APD: http://aps.unmc.edu/AP/main.php), AMPs generally
possess 19 positively charged lysine or arginine residues and up to
50% hydrophobic amino acids. Despite the clear potential of
AMPs, only very few AMPs such as polymyxins and gramicidins
are being used clinically. The usage of AMPs is mainly limited by
systemic toxicities, in vivo stability, and high cost for large scale
manufacturing . So, truncation and redesign of nature
antimicrobial peptides are considered to be a simple and effective
approach of developing new antibacterial agents. Recently,
Paulsen et al reported that the N-terminal fragment arasin 1(1
23) was almost equally active to the full length peptide arasin 1(a
37-residue peptide) . We have previously shown that the
truncation of the C-terminal region of linear chicken-defensin-4
(AvBD-4) retained the antimicrobial activity and eliminated its
hemolytic activity [15,16].
Cathelicidins, a prominent family of antimicrobial peptides,
have been identified in many species . Until now, 11 porcine
cathelicidins have been found. Among all the porcine cathelicidins,
porcine myeloid antimicrobial peptide-36 (PMAP-36) has the
highest net positive charge, the proportion of cationic amino acids
reach 36% . The high net charge may be advantageous to
PMAP-36 binding the bacterial via electrostatic interactions
between the positive charged of peptides and the negatively
charged molecules at the surface of the bacterial cell membrane.
The N-terminal of PMAP-36 is charge-rich domain. Furthermore,
structure analyses of this N-terminal region have demonstrated
that the highly cationic sequence adopts a typical amphipathic
ahelical conformation [18,19], which provided a good template for
researching quantitative structure-activity relationships (QSARs).
Previous studies have indicated that the N-terminal a-helical
domain of PMAP-36 was its active region, and derivative
PMAP36 (120) had the ability to interact and penetrate the bacterial
So, in this study, a series of derivatives were designed and
synthesized by truncating of PMAP-36 based on its structural
characteristics and residue distribution. The in vitro antimicrobial
and hemolytic activities of the peptides were determined.
Tryptophan fluorescence experiment was performed for the
Wcontaining peptides in the presence of synthetic lipid vesicles to
preliminary elucidate the peptide-membrane selectivity, and
explained the reason of high bactericidal activity and low toxicity
of peptides. Then, whole bacteria were further employed to
investigate potential membrane destruction mechanisms. Scanning
electron microscopy (SEM) and transmission electron microscopy
(TEM) assays allowed us to directly observe the change of cell
morphology and the integrity of cell membrane after peptide
Materials and Methods
In the present study, written informed consent was obtained
from all participants.
Peptide Design and Sequence Analysis
According to the three-dimensional structure and helical wheel
projection, we designed a series of derivatives by truncating and
residue substituting of PMAP-36 (Figure 1). Firstly, different
peptide fragments derived from PMAP-36 sequence was obtained.
The 24-residue peptide, GI24 contained the entire a-helical region
and all of the cationic amino acids of PMAP-36. The helical wheel
projection for GI24 indicated that it displays amphipathic residue
arrangement with an unusually wide and cationic polar face
(Figure 1). So, secondly, GI24-V3 and GI24-V6 were further
designed by replacing 3 and 6 cationic residues (R or K) of GI24
with V, respectively, and the antimicrobial activity of GI24-V3
and GI24-V6 is expected to enhance with increased nonpolar face.
In addition, the W-substituted analogs of GI24 were designed by
replacing the W with different types of amino acids to investigate
the contribution of W at position 23 of GI24 on the antimicrobial
The mean hydrophobicity of the peptides was calculated online
using CCS scale (http://www.bbcm.univ.trieste.it/,tossi/HydroCalc/
HydroMCalc.html). Primary sequence analysis of the peptides
was performed by bioinformatics programs ProtParam (ExPASy
Proteomics Server: http://www.expasy.org/tools/protparam.html).
The three-dimensional structure projection was predicted online by
ITASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). The
helical wheel projection was calculated online using the Helical Wheel
Projections (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi) .
The peptides listed in Table 1 were synthesized at GL Biochem
Corporation (Shanghai, China) by solid-phase methods using
N(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry. The peptides
were amidated at the C-terminal. Reverse-phase
high-performance liquid chromatography (HPLC) determined the purity of
the peptides to be .95%. Electrospray mass spectrometry was
used to identify the peptides. Peptides were then dissolved in DI
water at a concentration of 2.56 mM and stored at 220uC before
The antimicrobial activity of each peptide was determined
against the following bacteria strains: Escherichia coli ATCC 25922,
Escherichia coli UB1005, Salmonella enterica serovar typhimurium
C77-31, Staphylococcus aureus ATCC 29213, Staphylococcus aureus
ATCC 25923 and Staphylococcus epidermidis ATCC 12228. The
minimal inhibitory concentrations (MICs) of the peptides were
measured according to a modified version of the National
Committee for Clinical Laboratory Standards (NCCLS) broth
microdilution method as described previously . Briefly,
midlogarithmic phase bacteria were cultured in Mueller-Hinton (MH)
broth and then diluted to 16105 CFU/ml. Equal volumes (50 ml)
of microorganism solution and 2-fold serially diluted different
concentrations (0.25128 mM) peptides in 0.01% (v/v) acetic acid
and 0.2% (w/v) bovine serum albumin (BSA, Sigma) were added
to each well of the sterile 96-well plate. After incubation for 24 h at
37uC, MICs were determined as the lowest concentration of
peptides that prevented visible turbidity by visual inspection. The
tests were performed in triplicate. Cultures without peptides and
uninoculated MH broth were employed as positive and negative
controls, respectively. In addition, the salt stability of the peptides
was tested in the MIC assay mentioned above. Different
concentrations of monovalent and divalent cations tested were
listed in Table S1.
Measurement of Hemolytic Activity
The hemolytic activity of the peptides was measured as the
amount of hemoglobin released by the lysis of human erythrocytes
. Briefly, 1 ml of fresh human blood cells (hRBCs) obtained
from a healthy donor (Na Dong, Harbin, China) in a
polycarbonate tube containing heparin was centrifuged at 1,0006g for
5 min at 4uC . The experimental protocol was reviewed and
approved by the ethics committee of the Northeast Agricultural
University Hospital. The erythrocytes obtained were washed three
times with phosphate-buffered saline (PBS) solution (pH 7.2) and
resuspended in PBS. A 50 ml volume of the erythrocyte solution
was incubated with 50 ml of various peptides dissolved in PBS for
1 h at 37uC. Intact erythrocytes were pelleted by centrifugation at
1,0006g for 5 min at 4uC, and the supernatant was transferred to
a new 96-well plate. The release of hemoglobin was monitored by
measuring the absorbance at 570 nm. As negative and positive
Figure 1. Three-dimensional structure projections of PMAP-36 (left) and helical wheel projections of GI24 (right). In the picture of
helical wheel projections of GI24, by default the output presents the hydrophilic residues as circles, hydrophobic residues as diamonds, and
potentially positively charged as pentagons. Hydrophobicity is color coded as well: the most hydrophobic residue is green, and the amount of green
is decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are coded red with pure red being
the most hydrophilic (uncharged) residue, and the amount of red decreasing proportionally to the hydrophilicity. The potentially charged residues
are light blue.
controls, hRBCs in PBS without the peptide and 0.1% Triton
X100 were employed, respectively.
Tryptophan Fluorescence and Quenching
Small unilamellar vesicles (SUVs) were prepared for tryptophan
fluorescence experiments as described previously . The SUVs,
which included egg yolk L-a-phosphatidylethanolamine (PE), egg
yolk L-a-phosphatidyl-DL-glycerol (PG), egg yolk
L-a-phosphatidylcholine (PC), and cholesterol, were obtained from
SigmaAldrich Corporation (St. Louis, MO). PE/PG (7:3, w/w) or PC/
cholesterol (10:1, w/w) lipids were dissolved in chloroform solvents
and dried with a stream of nitrogen to form a thin lipid film. The
lipid films were resuspended in 10 mM Tris-HCl buffer (10 mM
Tris, pH 7.4, 150 mM NaCl, 0.1 mM EDTA) via vortex mixing.
The resultant suspensions were sonicated in an ice bath for about
20 min using an ultrasonic cleaner until the solutions clarified.
Tryptophan fluorescence measurements were obtained using an
F-4500 fluorescence spectrophotometer (Hitachi, Japan). The
fluorescence was excited at 280 nm and emission was scanned at
wavelengths ranging from 300 to 400 nm. Spectra of each peptide
with liposomes were baseline corrected by subtracting blank
spectra of the corresponding solutions without the peptide.
Measurements were performed for each peptide in 10 mM
Tris-HCl buffer (pH 7.4) with 500 mM PE/PG or PC/cholesterol
lipids. The peptide/lipid molar ratio was 1:50.
Fluorescence quenching experiments were conducted by using
an excitation wavelength of 295 nm instead of 280 nm .
Tryptophan fluorescence was quenched by titration with
acrylamide (Sigma) from a 4 M stock solution to a final concentration
of 0.4 M in the absence or presence of liposomes at a peptide/lipid
molar ratio of 1:50. The quenching data were analyzed by the
Stern-Volmer quenching constant (KSV), which was estimated
using the Stern-Volmer equation: F0/F = 1+KSV (Q ), where F0 and
F are the fluorescence values of the peptides in the absence or the
presence of acrylamide, KSV represents the Stern-Volmer
quenching constant, and Q represents the concentration of acrylamide.
Outer Membrane Permeabilization Assay
The outer membrane permeability of the peptides was
determined by using the N-phenyl-1-napthylamine (NPN) uptake
assay as previously described . Briefly, E. coli UB1005 were
washed and resuspended in buffer (5 mM HEPES, 5 mM glucose,
pH 7.4). NPN was added to 2 ml of cells in a quartz cuvette to give
a final concentration of 10 mM, and the background fluorescence
was recorded (excitation l = 350 nm, emission l = 420 nm).
Changes in fluorescence were recorded using an F-4500
fluorescence spectrophotometer (Hitachi, Japan). Peptide samples were
added to the cuvette, and fluorescence was recorded as a function
of time until no further increase in fluorescence was observed. As
the outer membrane permeability increased due to the addition of
peptide, NPN incorporated into the membrane resulted in an
increase in fluorescence. Values were converted to % NPN uptake
using the equation: % NPN uptake = (Fobs2Fo)/(F1002Fo)6100,
where Fobs is the observed fluorescence at a given peptide
concentration, F0 is the initial fluorescence of NPN with E. coli
cells in the absence of peptide, and F100 is the fluorescence of NPN
with E. coli cells upon addition of 10 mg/ml Polymyxin B (Sigma).
Polymyxin B is used as a positive control because of its strong outer
membrane permeabilizing properties.
Inner Membrane Depolarization Assay
The cytoplasmic membrane depolarization activity of the
peptides was measured by using E. coli UB1005 and the membrane
potential sensitive fluorescent dye diSC3(5) as described previously
. Briefly, mid-logarithmic phase E. coli were washed with
5 mM sodium HEPES buffer, pH 7.4, containing 20 mM glucose,
and resuspended to an OD600 of 0.05 in the same buffer. The cell
suspension was incubated with 0.4 mM diSC3(5) until a stable
reduction of fluorescence was achieved (approximately 1 h). Then
KCl was added to a final concentration of 0.1 M to equilibrate the
cytoplasmic and external K+. Two milliliters of the cell suspension
were placed in a 1 cm cuvette, and the peptides were added to
achieve the desired concentrations. Changes in fluorescence were
recorded using an F-4500 fluorescence spectrophotometer
(Hitachi, Japan) with an excitation wavelength of 622 nm and an
emission wavelength of 670 nm. 0.1% Triton X-100 was
employed as positive controls .
The integrity of the bacterial cell membranes after peptide
treatment was determined using a previously described method
. In brief, mid-logarithmic phase E. coli ATCC 25922 were
harvested by centrifugation and washed three times with 10 mM
PBS solution. The peptides (16MIC and 26MIC) were added and
incubated for 30 min at 28uC with constant shaking at 140 rpm,
and then, propidium iodide (PI, 10 mg/ml final concentration,
Sigma) was added and incubated for a further 30 min at 4uC.
After incubation, the unbound dye was removed via excessive
washing of cells with PBS. Flow cytometry analysis was conducted
using a FACScan instrument (BectonDickinson, San Jose, CA).
E. coli cells were incubated with PI without peptide treated as
Scanning Electron Microscopy (SEM)
E. coli ATCC 25922 were cultured in Mueller-Hinton (MH)
broth to mid-log phase and harvested by centrifugation at 1,0006g
for 10 min. Cells pellets were washed twice with 10 mM PBS and
resuspended to an OD600 of 0.2. The cell suspension was
incubated at 37uC for 60 min with different peptides at a
concentration of 16MIC. After incubation, the cells were
centrifuged and washed 3 times at 5,0006g for 5 min with PBS.
Bacterial pellets were then fixed in 500 ml of 2.5% (v/v)
glutaraldehyde in PBS at 4uC overnight. Thereafter, the bacteria
were washed twice with PBS and dehydrated through a graded
ethanol series (50%, 70%, 90%, and 100%), for 15 min in each.
The samples were then transferred to a mixture (1:1, v/v) of
ethanol and tertiary butanol, and pure tertiary butanol, for 20 min
in each. After lyophilization and gold coating, the specimens were
observed using a scanning electron microscope (Hitachi S-4800,
Transmission Electron Microscope (TEM)
Preparation of the bacteria samples was conducted in the same
manner as for SEM treatment. After fixing with 2.5%
glutaraldehyde overnight, the bacterial pellets were washed three times
with PBS and post-fixed with 1% osmium tetroxide in PBS for 2 h.
The fixed bacterial cells were washed three times with PBS, and
followed by dehydration for 15 min in a graded ethanol series
(50%, 70%, 90%, and 100%). After placing in absolute acetone for
20 min, these samples were transferred to 1:1 and 1:3 mixture of
absolute acetone and epoxy resin for 1 h in each, followed by
transferring to pure epoxy resin for overnight. Ultrathin sections
obtained by using ultramicrotome, were post-stained with uranyl
acetate and lead citrate. Specimens were observed by transmission
electron microscope (Hitachi H-7650, Japan).
Circular dichroism spectra of the peptides in different
environments were obtained at 25uC by using a J-820 spectropolarimeter
(Jasco, Tokyo, Japan). The peptide samples were recorded at a
final concentration of 150 mM in 10 mM sodium phosphate
buffer, pH 7.4 (mimicking the aqueous environment), 30 mM
SDS micelles (mimicking the negatively charged prokaryotic
membrane comparable environment, Sigma), and 50% TFE
(mimicking the hydrophobic environment of the microbial
membrane, Sigma). The samples were loaded in a rectangular
quartz cell (0.1 cm path length), and the spectra were recorded at
a scanning speed of 10 nm/min in the wavelength range of 190 to
Antimicrobial Activities of the Peptides
MICs of the synthetic peptides against gram-negative and
grampositive bacteria are presented in Table 2. GI24 corresponding to
the N-terminal of PMAP-36 displayed activity comparable to the
full length peptide with MICs ranging from 1 to 4 mM, while no
antimicrobial activity of PG12 (12-residue C-terminal fragment of
PMAP-36) was observed for tested microorganisms. Further
truncating from GI24, resulted in a significantly decrease or loss
Table 2. Antimicrobial activity of the peptides.
aMinimum inhibitory concentrations (MICs) were determined as the lowest concentration of the peptides that inhibited bacteria growth.
bThe geometric mean (GM) of the MICs of the peptides against all four bacterial strains was calculated. When no antimicrobial activity was observed at 128 mM, a value
of 256 mM was used to calculate the geometric mean.
of antimicrobial activity, as demonstrated by GK12 and RI12,
The antimicrobial activity of the derivatives of GI24 were
further determined and compared to GI24. As shown in Table 2,
increasing the hydrophobicity of GI24 by substituting cationic
residues (R or K) with hydrophobic residues (V) did not improved
the antimicrobial activity of GI24-V3 or GI24-V6 with MICs
against tested bacteria for 2 or 4 mM. The single site-substituting of
W at position 23 of GI24 with A or K to give GI24-W23A or
GI24-W23K resulted in a notable decrease in antimicrobial
activities, especially in the case of GI24-W23A. The GM value of
GI24-W23A was about 40 times higher than that of GI24. When
the W was replaced by L, the antimicrobial activity of GI24-W23L
recovered to the original level of inhibition observed for GI24.
Hemolytic Activity of the Peptides
The hemolytic activity of the peptides against the highly
sensitive human erythrocytes was determined as a measure of their
toxicity to mammalian cells (Figure 2). GK12, RI12, PG12,
GI24W23K, and GI24-W23A displayed no hemolytic activity even at
the maximum concentration of 128 mM. PMAP-36, GI24, and
GI24-W23L showed slightly hemolytic activity in a
dose-dependent manner. GI24-V3 and GI24-V6 demonstrated relatively high
hemolytic activity at each concentration when compared to GI24.
In contrast, melittin used as a control peptide caused complete
hemolysis at a low concentration of 4 mM.
Binding of Peptides to Model Membranes
The binding of peptides to lipid vesicles composed of PE/PG
(7:3, w/w) or PC/cholesterol (10:1, w/w) was measured by
monitoring the emission spectra of a tryptophan fluorophore
(Table 3 and Figure S1). In Tris-HCl buffer, the maximum
wavelengths of the peptides were approximately 350 nm,
indicating the exposure of the tryptophan fluorophore to aqueous
environment . When the peptides were titrated to PE/PG
phospholipid vesicles, a large blue shift was observed. However,
little or no shift in wavelength was observed in PC/cholesterol
phospholipid vesicles for PMAP-36 and GI24. These results
suggested that the degree of insertion of the W into the zwitterionic
vesicles was significantly less than that for vesicles with net negative
charge. As a reference, melittin induced larger blue shifts in both
PE/PG and PC/cholesterol phospholipid vesicles.
Tryptophan Fluorescence Quenching by Acrylamide
To further investigate the extent of the burial of the W into
phospholipids, a fluorescence quenching experiment was
performed using a neutral quencher acrylamide . A smaller KSV
value reflects a more protected W residue. The results are
presented in Table 3 and Figure S2. PMAP-36 and GI24
displayed same KSV values in the presence of PE/PG and PC/
cholesterol phospholipid vesicles. In addition, the KSV values of
PMAP-36 and GI24 in PE/PG vesicles were less than those in
PC/cholesterol, demonstrating that the peptides were more
protected in bacteria-mimicking cell membranes. The KSV value
of melittin was smaller than PMAP-36 and GI24 in both PE/PG
and PC/cholesterol phospholipid vesicles.
Permeabilization of Outer Membranes
The outer membrane permeabilization of E. coli was determined
by using the NPN uptake assay. NPN, a neutral hydrophobic
fluorescent probe, is normally excluded by the outer membrane
but exhibits increased fluorescence intensity when it partitions into
the outer membrane. As shown in Figure 3, PMAP-36 and GI24
rapidly permeabilized the outer membrane of E. coli in a
concentration-dependent manner as observed by an increase in
NPN fluorescence. The peptides were able to permeabilize the
outer membrane even at the concentrations lower than their
MICs. The increase in fluorescence of PMAP-36 and GI24 was
similar with that of melittin at the same concentrations.
Depolarization of Inner Membranes
The membrane potential-sensitive dye diSC3(5) was used to
evaluate the depolarization of the peptides on E. coli cytoplasmic
membranes. Depolarization of the peptides was monitored over a
period of 360 s (Figure 4). The results showed that three peptides
PMAP-36, GI24, and melittin induced dose-dependent increases
in diSC3(5) fluorescence, reflecting cytoplasmic membrane
The DNA intercalating dye propidium iodide (PI) was used to
evaluate cell membrane integrity by flow cytometry. The
fluorescence intensity indicates that the peptides induced the
influx of PI into the cells. As shown in Figure 5, in control bacterial
cells (no peptide), only 3.0% of the bacterial cells had PI
fluorescence (Figure 5A). The majority of the bacterial cells
(.90%) were labeled fluorescently after treatment with PMAP-36
and GI14 at MIC or 2MIC. However, melittin resulted in only
40.7% cells stained at MIC. A significant increase in staining ratio
of melittin was observed when the peptide concentration increased
from MIC to 2MIC.
To directly observe cell morphological changes after peptides
treatment, a SEM study was conducted. E. coli cells treated for
60 min with PMAP-36, GI24, and melittin at 16MIC were
visualized. As shown in Figure 6, treatment with all peptides
induced membrane surface disruption in comparison to the
control, which exhibited a bright and smooth surface. Bacterial
cells treated with PMAP-36 and GI24 became surface roughening
and corrugating, similar to the cells treated with melittin.
depolarization. PMAP-36 and GI24 were more effective and
rapidly at permeabilizing the inner membrane than melittin at the
same molar concentrations, especially when the peptide
concentration is greater than their MIC. An immediate increase in
fluorescence intensity was detected in 60 s after the addition of the
peptides to E. coli suspensions.
Table 3. Fluorescence spectroscopy parameters measured for the peptides in the presence and absence of PE/PG and PC/
Fluorescence emission maxima (nm)
aBlue shift of emission maximum compared to Tris buffer.
bStern-Vollmer constant KSV were calculated by the Stern-Vollmer equation: F0/F = 1+KSV (Q), where Q is the concentration of the quencher (acrylamide). Concentrations
of the quencher were increased from 0.01 to 0.40 M. A smaller KSV value reflects a more protected W residue.
In addition to SEM, TEM was employed to study the
membrane integrity and intracellular alteration of E. coli cells
before and after treatment with the peptides. As shown in Figure 7,
complete cell membrane and full intracellular contents were
observed in the untreated E. coli cells. It can be seen that the
treated bacteria cells by PMAP-36, GI24, and melittin, exhibited
obvious cytoplasmic clear zones, and the integrity of the E. coli
membrane was disrupted with visible pores.
Circular dichroism spectroscopy was performed for the peptides
in sodium phosphate buffer, 30 mM SDS, and 50% TFE
(Figure 8). All of the peptides formed random coil structures in
aqueous solution as indicated by the presence of a strong
minimum peak near 200 nm. When 30 mM SDS and 50%
TFE were mixed with PMAP-36 and GI24, an increase in the
mean residue ellipticity at 208 and 222 nm was observed,
consistent with the formation of an a-helix . The conformation
of PG12 in the presence of SDS or TFE was the same as that in
buffer, with a random coil structure.
The truncation and residue-substitution of natural antimicrobial
peptide is considered an effective method for developing a
candidate of antimicrobial peptides with fewer residues. Several
studies have demonstrated that it is possible to truncate the
naturally occurring peptide to less than half its length and retain
antimicrobial and other functions [31,32]. Studies on cathelicidin
BMAP-27 and its truncated derivative indicated that the a-helical
fragment BMAP-18 was almost equally active to the full length
peptide [33,34]. Similar results were found for the peptide in this
study. The deletion of C-terminal hydrophobic tail did not affect
the antimicrobial activity of GI24, the N-terminus truncated
derivative of PMAP-36. GI24 displayed high antimicrobial activity
that was comparable to that of parental PMAP-36. However, the
antimicrobial activity of GK12 and RI12 further truncated
peptides from PMAP-36 was significantly reduced as compared
to parental peptide. PG12, the C-terminal sequence of PMAP-36
with 12 residues, displayed no antimicrobial activity against all
tested bacteria, which may be due to the absence of cationic amino
acids. These results indicated that truncated derivative (GI24) may
retain complete antimicrobial activity of parental peptide
Furthermore, according to the helical wheel projection, we
noticed that GI24 displayed an unusually wide cationic polar face
(Figure 1). Previous studies have shown that increasing
hydrophobicity in a certain range contributed to improve antimicrobial
activity . For this purpose, two analogs of GI24 were
synthesized by substituting R or K with V. The net positive
charge decreased from +14 (GI24) to +11 (GI24-V3) and +8
(GI24-V6), and the hydrophobicity increased from 22.81 (GI24)
to 21.05 (GI24-V3) and 0.69 (GI24-V6). Antimicrobial assay
showed that the hydrophobic increase did not improve the
antimicrobial activity as we expected. On the contrary, the
antimicrobial activity of GI24-V3 and GI24-V6 was slightly
reduced as compared to GI24. This may be due to the low
solubility of high hydrophobic peptides. In addition, compared
with PMAP-36 (120), a truncated N-terminal derivative with 20
residues reported by Storici et al , we noticed that GI24
displayed approximately 324 fold higher antimicrobial activity
across the bacterial species. Actually, we also synthesized the
Nterminal sequence of PMAP-36 with 20 residues. Antimicrobial
assay showed that the peptide GRFRRLRKKTRKRLKKIGKV
displayed extremely low antimicrobial activity against six bacterial
strains tested (data not shown). The difference of antimicrobial
activity indicated that residues 2124 (LKWI) are important for
the antimicrobial activity of GI24. Among the four residues
(LKWI), the W attracted our attention. It was reported that W
with bulky side chain ensured more efficient interaction of peptides
with the bacterial membrane [36,37]. We conjectured that the W
at position 23 of GI24 may play a crucial role in antimicrobial
activity, and the lost of W may be an important reason for the
reduced antimicrobial activity of PMAP-36 (120). So, to
investigate the contribution of W at position 23 of GI24 on the
antimicrobial activity, a series of W-substituted mutants were
developed by substituting W with A, K, and L. Antimicrobial assay
showed that the antimicrobial activity of GI24-W23A and
GI24W23K against gram-negative and gram-positive bacterial was
significantly reduced. When the W of GI24 was replaced with L,
the antimicrobial activity of GI24-W23L was recovered to a level
Figure 5. Flow cytometric analysis. The DNA intercalating dye propidium iodide (PI) was used to evaluate the cell membrane integrity via flow
cytometry. The fluorescence intensity was monitored after treating with the peptides. Flow cytometry was performed using a FACScan instrument.
(A) control, no peptide; (B) PMAP-36 (MIC, 1 mM); (C) PMAP-36 (2MIC, 2 mM); (D) GI24 (MIC, 1 mM); (E) GI24 (2MIC, 2 mM); (F) melittin (MIC, 2 mM); (G)
melittin (2MIC, 4 mM).
similar to GI24. We noticed that the antimicrobial activity of these
peptides seem to be well correlated with their mean
hydrophobicity (Table 1). GI24 and GI24-W23L shared the same mean
hydrophobicity (22.81), which was higher than that of
GI24W23A (23.26) and GI24-W23K (23.63). These results suggested
that the hydrophobicity of an amino acid at position 23 played an
important role in determining the level of antimicrobial activity of
GI24 rather than the bulky side chain of W. In addition, both
PMAP-36 and GI24 displayed strong resistance to salts (Table S1).
The hemolytic activity of the peptides against highly sensitive
human erythrocytes is an important indicator of the toxicity to
mammalian cells. All 12-residue peptides displayed no hemolysis
at the tested concentrations. The hemolytic activity of the
24residue derivatives was related to the hydrophobicity, especially for
high hydrophobic GI24-V3 and GI24-V6 which caused 50%
hemolysis at 128 mM and 8 mM, respectively. Chou et al reported
that high hydrophobicity and amphipathicity (hydrophobic
moment) are more correlated with increased hemolytic activity
rather than antimicrobial activity . Our results indicated that
both size and hydrophobicity modulated hemolytic activity, and
short peptides represented more performance committed to
decrease the hemolytic activity. GI24 did not cause 50% hemolysis
even at the highest determination concentration (128 mM), which
was at least 60 times larger than its MICs.
The CD spectra exhibited that all peptides displayed typical
random coil structure in aqueous solution. The addition of TFE
and SDS induced a conformational change in PMAP-36 and GI24
consistent with the formation of a-helical structure, which was
consistent with our hypothesis and confirmed the previous reports
[18,19]. After the initial electrostatic adsorption, AMPs aggregate
on the surface of bacterial cell and correct orientation according to
the plane of binding, following with the partitioning of the peptide
to the membrane and the a-helical amphipathic structure
transition. This conformational transformation is the key feature
for AMPs to partition in bacterial cell membranes, which
ultimately leads to bacterial cell death .
To elucidate the interaction of the peptides with phospholipid
membranes, the fluorescence emission and quenching of W
residue were monitored. Blue shift of emission maximum and
Stern-Vollmer constant KSV value reflect the different cell
selectivity of the peptides. PE/PG (7:3, w/w) or PC/cholesterol
(10:1, w/w) SUVs were prepared to simulate negatively charged or
zwitterionic membranes. The shift of the maximum emission in
the presence of the lipid vesicles is because of the sensitivity of the
W to the polarity of its environment . Both PMAP-36 and
GI24 displayed larger blue shifts and smaller KSV values in the
negatively charged PE/PG vesicles than in the zwitterionic PC/
cholesterol vesicles, suggesting that the tryptophan of the peptides
insert or penetrate bacteria-mimicking membranes more deeply
than eukaryote-mimicking membranes . The selective
membrane interaction of PMAP-36 and GI-24 corresponded well with
the high antimicrobial activity and weak hemolytic activity.
Results of the peptide-lipid interactions suggested that
membrane permeation may play a key role in the antimicrobial activity
of PMAP-36 and GI24. Thus, the outer and cytoplasmic
membrane permeability assays were performed to detect the
target site of the peptides. An increase in the NPN fluorescence of
the peptides indicated that all of the peptides mediated NPN
uptake across the outer membrane in a dose-dependent manner.
Then, the ability of the peptides to depolarize cytoplasmic
membrane was assayed by using the membrane potential-sensitive
dye diSC3(5), which concentrates in the cytoplasmic membrane
under the influence of the membrane potential resulting in a
selfquenching of fluorescence. If the cytoplasmic membrane is
disrupted or forms channels, the fluorescent probe will be released
into the medium, causing an increase in fluorescence .
PMAP36 and GI24 induced different extent of the depolarization to
cytoplasmic membrane in a dose-dependent manner. Consistent
with previous study , the membrane permeability experiments
suggested that PMAP-36 and GI24 targeted the cell membrane,
similar to melittin, a membrane-active peptide. PMAP-36, GI24,
and melittin caused similar NPN uptake (Figure 3), and the
increased fluorescence intensity of GI24 and melittin in the inner
membrane permeabilization (Figure 4) was slight less than that of
PMAP-36. However, PMAP-36, GI24, and melittin displayed
same MIC against E. coli UB1005, indicating that the difference in
the antimicrobial activity of the peptides is likely due to the
difficulties in permeabilizing the outer membrane, and not the
inner membrane. The outer membrane of gram-negative bacteria
forms an effective permeability barrier composed of a stable
complex of lipopolysaccharide (LPS) and divalent cations. The
initial process of peptide-membrane interaction was considered the
displacement of divalent cations that stabilize adjacent polyanionic
LPS molecules . With 14 net positive charges, PMAP-36 and
GI24 can associate readily with anionic membranes via
electrostatic interactions. Following initial attachment, the peptides
aggregate and reach a threshold concentration, which enables
productive action. At this concentration, peptides begin to
rearrange and alter pathogen permeability via generally accepted
mechanisms . Then, antimicrobial peptides insert into the
hydrophobic core of the membrane and promote the formation of
transmembrane pore-like structures [44,45].
To further assess whether the peptides damaged the bacterial
cell membrane, we determined PI staining of nucleic acids as an
indicator of cell death. In addition, scanning electron microscopy
and transmission electron microscope assays were employed to
directly observe the cell morphological changes as well as the
integrity of the E. coli membrane. FACScan analysis indicated that
treatment of E. coli cells with the peptides enhanced uptake of PI,
suggesting that the bacterial cell membrane were disrupted. The
surface roughening and significant rupture of cell membranes and
the release of cellular contents of E. coli treated with both
PMAP36 and GI24 were observed by SEM and TEM. These data
further confirmed that both PMAP-36 and GI24 kill bacteria via
membrane disruption, and roughening and corrugating observed
on the cell membrane surface is probably due to the leakage of
Previous studies have shown that melittin kills bacterial via a
toroidal-pore mechanism . Furthermore, increased
accumulation of certain peptides such as melittin may lead to a
detergent-like disintegration of membrane via a carpet mechanism
[50,51]. Sato and Feix also pointed out that different
membraneactive mechanism need not be mutually exclusive, one process
may represent an initial or intermediate step and another may be
its consequence . In this study, the membrane-active
experiments revealed that PMAP-36 and GI24 damaged the cell
membrane in a manner similar to melittin. The peptides rapidly
permeabilized the out and inner membrane of E. coli even at the
concentrations lower than their MICs (Figure 3 and 4). This
membrane-permeabilizing behavior was estimated by the torodal
pore-formation. However, visible pores with large diameter were
observed in the E. coli membrane treated with the peptides by
TEM (Figure 7). The diameter of the pore in the membrane
treated with the peptides was larger than the pore sizes of melittin
(1.5 to 5 nm) reported in toroidal-pore mechanism . So,
we conjectured that PMAP-36 and GI24 damaged the cell
membrane by a detergent-like carpet mechanism at a high peptide
concentration similar to melittin.
In summary, the results presented here demonstrate that the
antimicrobial activity of the porcine myeloid antimicrobial peptide
PMAP-36 was not affected by the truncation of the 12-residue
Cterminal tail. GI24 displayed strong antimicrobial activity and
weak hemolytic activity. The hemolysis assay showed that the
hemolytic activity of long peptides was more sensitive to the
increase of hydrophobic as compared to short peptides. In
addition, the crucial site of GI24 was identified through single
site-mutation. Then, the membrane-active mechanisms were
determined by using lipid vesicles and whole bacteria. The
preference of PMAP-36 and GI24 for binding to negatively
charged phospholipids over zwitterionic phospholipids, which led
to greater cell selectivity. The screened peptide GI24 was shown to
permeabilize the outer membrane of bacterial cells and depolarize
the cytoplasmic membrane, thus damaging the membrane
integrity and causing intracellular content leakage, which leads
to cell death. The findings reported in this study supported that
truncation is an effective strategy for developing novel
antimicrobial peptides, and GI24 could be a promising antimicrobial agent
for clinical application.
Figure S1 Tryptophan fluorescence emission spectra of
PMAP-36 (A), GI24 (B), and melittin (C) in the buffer or
in the presence of PE/PG or PC/cholesterol liposomes.
Figure S2 SternVolmer plots for the quenching of Trp
fluorescence of PMAP-36 (A), GI24 (B), and melittin (C)
by acrylamide in the buffer or in the presence of PE/PG
or PC/cholesterol liposomes.
Conceived and designed the experiments: YFL ND QQM ASS. Performed
the experiments: YFL JJW ZYW. Analyzed the data: YFL HG.
Contributed reagents/materials/analysis tools: HG ASS. Wrote the paper:
YFL. Critically revised the manuscript: ND QQM ASS.
1. Teixeira V , Feio MJ , Bastos M ( 2012 ) Role of lipids in the interaction of antimicrobial peptides with membranes . Prog Lipid Res 51 : 149 - 177 .
2. Vila-Farres X , Garcia de la Maria C , Lopez-Rojas R , Pachon J , Giralt E , et al. ( 2012 ) In vitro activity of several antimicrobial peptides against colistinsusceptible and colistin-resistant Acinetobacter baumannii . Clin Microbiol Infect 18 : 383 - 387 .
3. Gopal R , Seo CH , Song PI , Park Y ( 2013 ) Effect of repetitive lysine-tryptophan motifs on the bactericidal activity of antimicrobial peptides . Amino Acids 44 : 645 - 660 .
4. Baek JH , Lee SH ( 2010 ) Isolation and molecular cloning of venom peptides from Orancistrocerus drewseni (Hymenoptera: Eumenidae) . Toxicon 55 : 711 - 718 .
5. Carballar-Lejarazu R, Rodrguez MH , de la Cruz Hernandez-Hernandez F, Ramos-Casta neda J , Possani LD , et al. ( 2008 ) Recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens . Cell Mol Life Sci 65 : 3081 - 3092 .
6. Haines LR , Thomas JM , Jackson AM , Eyford BA , Razavi M , et al. ( 2009 ) Killing of trypanosomatid parasites by a modified bovine host defense peptide , BMAP-18. PLoS Negl Trop Dis 3 : e373 .
7. Dobrzynska I , Szachowicz-Petelska B , Sulkowski S , Figaszewski Z ( 2005 ) Changes in electric charge and phospholipids composition in human colorectal cancer cells . Mol Cell Biochem 276 : 113 - 119 .
8. Zasloff M ( 2002 ) Antimicrobial peptides of multicellular organism . Nature 415 : 389 - 395 .
9. Li Y , Xiang Q , Zhang Q , Huang Y , Su Z ( 2012 ) Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application . Peptides 37 : 207 - 215 .
10. Ahmad A , Ahmad E , Rabbani G , Haque S , Arshad M , et al. ( 2012 ) Identification and design of antimicrobial peptides for therapeutic applications . Curr Protein Pept Sci 13 : 211 - 223 .
11. Brown KL , Hancock REW ( 2006 ) Cationic host defense (antimicrobial) peptides . Curr Opin Immunol 18 : 24 - 30 .
12. Tam JP , Lu YA , Yang JL ( 2002 ) Antimicrobial dendrimeric peptides . Eur J Biochem 269 : 923 - 932 .
13. Brogden NK , Brogden KA ( 2011 ) Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? Int J Antimicrob Agents 38 : 217 - 225 .
14. Paulsen VS , Blencke HM , Benincasa M , Haug T , Eksteen JJ , et al. ( 2013 ) Structure-activity relationships of the antimicrobial peptide arasin 1 - and mode of action studies of the N-terminal, proline-rich region . PLoS One 8 : e53326 .
15. Dong N , Ma QQ , Shan AS , Lv YF , Hu WN , et al. ( 2012 ) Novel design of short antimicrobial peptides derived from the bactericidal domain of avian b-defensin4 . Protein Pept Lett 19 : 1212 - 1219 .
16. Dong N , Ma QQ , Shan AS , Wang L , Sun WY , et al. ( 2012 ) Influence of truncation of avian b-defensin-4 on biological activity and peptide-membrane interaction . Protein Pept Lett 19 : 430 - 438 .
17. Sang Y , Blecha F ( 2009 ) Porcine host defense peptides: expanding repertoire and functions . Dev Comp Immunol 33 : 334 - 343 .
18. Storici P , Scocchi M , Tossi A , Gennaro R , Zanetti M ( 1994 ) Chemical synthesis and biological activity of a novel antibacterial peptide deduced from a pig myeloid cDNA . FEBS Lett 337 : 303 - 307 .
19. Scocchi M , Zelezetsky I , Benincasa M , Gennaro R , Mazzoli A , et al. ( 2005 ) Structural aspects and biological properties of the cathelicidin PMAP-36 . FEBS J 272: 4398 - 4406 .
20. Liu YF , Xia X , Xu L , Wang YZ ( 2013 ) Design of hybrid b-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity . Biomaterials 34 : 237 - 250 .
21. Steinberg DA , Hurst MA , Fujii CA , Kung AHC , Ho JF , et al. ( 1997 ) Protegrin1: a broad spectrum, rapidly microbicidal peptide with in vivo activity . Antimicrob Agents Chemother 41 : 1738 - 1742 .
22. Stark M , Liu LP , Deber CM ( 2002 ) Cationic hydrophobic peptides with antimicrobial activity . Antimicrob Agents Chemother 46 : 3585 - 3590 .
23. Lee KH , Lee DG , Park Y , Kang DI , Shin SY , et al. ( 2006 ) Interactions between the plasma membrane and the antimicrobial peptide HP (2-20) and its analogues derived from Helicobacter pylori . Biochem J 394 : 105 - 114 .
24. Zhu WL , Lan H , Park IS , Kim JI , Jin HZ , et al. ( 2006 ) Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cellpenetrating peptide Pep-1 . Biochem Biophys Res Commun 349 : 769 - 774 .
25. Lee DL , Powers JPS , Pflegerl K , Vasil ML , Hancock REW , et al. ( 2004 ) Effects of single D-amino acid substitutions on disruption of b-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S . J Pept Res 63 : 69 - 84 .
26. Friedrich CL , Rozek A , Patrzykat A , Hancock REW ( 2001 ) Structure and mechanism of action of an indolicidin peptide derivative with improved activity against gram-positive bacteria . J Biol Chem 276 : 24015 - 24022 .
27. Park Y , Kim HJ , Hahm KS ( 2004 ) Antibacterial synergism of novel antibiotic peptides with chloramphenicol . Biochem Biophys Res Commun 321 : 109 - 115 .
28. Rabbani G , Ahmad E , Zaidi N , Fatima S , Khan RH ( 2012 ) pH-Induced molten globule state of Rhizopus niveus lipase is more resistant against thermal and chemical denaturation than its native state . Cell Biochem Biophys 62 : 487 - 499 .
29. Rabbani G , Ahmad E , Zaidi N , Khan RH ( 2011 ) pH-Dependent conformational transitions in conalbumin (ovotransferrin), a metalloproteinase from hen egg white . Cell Biochem Biophys 61 : 551 - 560 .
30. Rabbani G , Kaur J , Ahmad E , Khan RH , Jain SK ( 2013 ) Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi . Appl Microbiol Biotechnol In press.
31. Durr UH , Sudheendra US , Ramamoorthy A ( 2006 ) LL-37, the only human member of the cathelicidin family of antimicrobial peptides . Biochim Biophys Acta 1758 : 1408 - 1425 .
32. Stromstedt AA , Pasupuleti M , Schmidtchen A , Malmsten M ( 2009 ) Evaluation of strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, aninternal segment of LL-37 . Antimicrob Agents Chemother 53 : 593 - 602 .
33. Skerlavaj B , Gennaro R , Bagella L , Merluzzi L , Risso A , et al. ( 1996 ) Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements of their antimicrobial and cell lytic activities . J Biol Chem 271 : 28375 - 28381 .
34. Lee EK , Kim YC , Nan YH , Shin SY ( 2011 ) Cell selectivity, mechanism of action and LPS-neutralizing activity of bovine myeloid antimicrobial peptide-18 (BMAP-18) and its analogs . Peptides 32 : 1123 - 1130 .
35. Chen Y , Guarnieri MT , Vasil AI , Vasil ML , Mant CT , et al. ( 2007 ) Role of peptide hydrophobicity in the mechanism of action of a-helical antimicrobial peptides . Antimicrob Agents Chemother 51 : 1398 - 1406 .
36. Wimley WC , White SH ( 1996 ) Experimentally determined hydrophobicity scale for proteins at membrane interfaces . Nat Struct Biol 3 : 842 - 848 .
37. Strom MB , Haug BE , Rekdal O , Skar ML , Stensen W , et al. ( 2002 ) Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity . Biochem Cell Biol 80 : 65 - 74 .
38. Chou HT , Kuo TY , Chiang JC , Pei MJ , Yang WT , et al. ( 2008 ) Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp . Int J Antimicrob Agents 32 : 130 - 138 .
39. Dong N , Ma QQ , Shan AS , Lv YF , Gu Y , et al. ( 2012 ) Strand-length-dependent antimicrobial activity and membrane-active mechanism of arginine- and valinerich b-hairpin-like antimicrobial peptides . Antimicrob Agents Chemother 56 : 2994 - 3003 .
40. Kiyota T , Lee S , Sugihara G ( 1996 ) Design and synthesis of amphiphilic alphahelical model peptides with systematically varied hydrophobic-hydrophilic balance and their interaction with lipid- and bio-membranes . Biochemistry 35 : 13196 - 13204 .
41. Oren Z , Shai Y ( 2000 ) Cyclization of a cytolytic amphipathic alpha-helical peptide and its diastereomer: effect on structure, interaction with model membranes, and biological function . Biochemistry 39 : 6103 - 6114 .
42. Wu M , Maier E , Benz R , Hancock REW ( 1999 ) Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli . Biochemistry 38 : 7235 - 7242 .
43. Piers KL , Brown MH , Hancock REW ( 1994 ) Improvement of outer membranepermeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification . Antimicrob Agents Chemother 38 : 2311 - 2316 .
44. Herce HD , Garcia AE ( 2007 ) Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes . Proc Natl Acad Sci U S A 104 : 20805 - 20810 .
45. Vedovato N , Rispoli G ( 2007 ) A novel technique to study pore-forming peptides in a natural membrane . Eur Biophys J 36 : 771 - 778 .
46. Matsuzaki K , Yoneyama S , Miyajima K ( 1997 ) Pore formation and translocation of melittin . Biophys J 73 : 831 - 838 .
47. Yang L , Harroun TA , Weiss TM , Ding L , Huang HW ( 2001 ) Barrel-stave model or toroidal model? A case study on melittin pores . Biophys J 81 : 1475 - 1485 .
48. Park SC , Kim JY , Shin SO , Jeong CY , Kim MH , et al. ( 2006 ) Investigation of toroidal pore and oligomerization by melittin using transmission electron microscopy . Biochem Biophys Res Commun 343 : 222 - 228 .
49. Allende D , Simon SA , McIntosh TJ ( 2005 ) Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores . Biophys J 88 : 1828 - 1837 .
50. Naito A , Nagao T , Norisada K , Mizuno T , Tuzi S , et al. ( 2000 ) Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solidstate 31P and 13C NMR spectroscopy . Biophys J 78 : 2405 - 2417 .
51. Ladokhin AS , White SH ( 2001 ) 'Detergent-like' permeabilization of anionic lipid vesicles by melittin . Biochim Biophys Acta 1514 : 253 - 260 .
52. Sato H , Feix JB ( 2006 ) Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic a-helical antimicrobial peptides . Biochim Biophys Acta 1758 : 1245 - 1256 .