The effects of ring-size analogs of the antimicrobial peptide
gramicidin S on phospholipid bilayer model membranes
and on the growth of
Acholeplasma laidlawii
B
Monika Kiricsi
1
, Elmar J. Prenner
1,2
, Masood Jelokhani-Niaraki
1,2,
*, Ruthven N. A. H. Lewis
1
,
Robert S. Hodges
1,2,†
and Ronald N. McElhaney
1,2
1
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada;
2
Protein Engineering Network of Centers of
Excellence, University of Alberta, Edmonton, Alberta, Canada
We have examined the effects of three ring-size analogs of the
cyclic b-sheet antimicrobial peptide gramicidin S (GS) on the
thermotropic phase behavior and permeability of phos-
pholipid model membranes and on the growth of the cell
wall-less Gram-positive bacteria Acholeplasma laidlawii B.
These three analogs have ring sizes of 10 (GS10), 12 (GS12)
or 14 (GS14) amino acids, respectively. Our high-sensitivity
differential scanning calorimetric studies indicate that all

three ring-size GS analogs, on phospholipid bilayer organi-
zation and cell growth correlate relatively well with the
effective hydrophobicities and amphiphilicities of these
peptides but less well with their relative charge density,
intrinsic hydrophobicities or conformational flexibilities.
Nevertheless, all of these parameters, as well as others, may
influence the antimicrobial potency and hemolytic activity of
GS analogs.
Keywords: antimicrobial peptides; gramicidin S; phospholi-
pid bilayers; membranes.
Gramicidin S (GS) is a cyclic decapeptide of primary
structure [cyclo-(Val-Orn-Leu-
D
-Phe-Pro)
2
] first isolated
from Bacillus brevis [1] and is one of a series of
antimicrobial peptides produced by this microorganism
[2,3]. GS exhibits potent antibiotic activity against a broad
spectrum of both Gram-negative and Gram-positive
bacteria, as well as against several pathogenic fungi [4–
7]. Unfortunately, GS is rather nonspecific in its actions
Correspondence to R. N. McElhaney, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Fax: +1 780 4920095, E-mail:
Abbreviations: GS, gramicidin S; Myr
2
Gro-PCho, dimyristoylglycerophosphocholine; Myr
2
Gro-PEtn, dimyristoylglycerophosphoethanolamine;
Myr

be used as potent oral or injectable broad-spectrum
antibiotics [4–7].
GS has been extensively studied by a wide range of
physical techniques [2,3] and its 3D structure is well
determined. In this minimum energy conformation, the
two tripeptide sequences Val-Orn-Leu form an antiparallel
b-sheet terminated on each side by a type II¢ b-turn formed
by the two
D
-Phe-Pro sequences. Four intramolecular
hydrogen bonds, involving the amide protons and carbonyl
groups of the two Leu and two Val residues, stabilize this
rather rigid structure. The GS molecule is amphiphilic, with
the two somewhat polar and positively charged Orn
sidechains and the two
D
-Phe rings projecting from one
side of this molecule, and the four hydrophobic Leu and Val
sidechains projecting from the other side. Moreover, the
amphiphilic nature of GS is required for its antimicrobial
activity [2,3]. A number of studies have shown that this
conformation of the GS molecule is maintained in water, in
protic and aprotic organic solvents of widely varying
polarity, and in detergent micelles and phospholipid bilay-
ers, even at high temperatures and in the presence of agents
which often alter protein conformation.
There is good evidence from studies of the interaction of
GS and its analogs with bacterial cells that the destruction
of the integrity of the lipid bilayer of the inner membrane
is the primary mode of antimicrobial action of this peptide

acyl chains, and that GS penetrates more deeply into
anionic and more fluid phospholipid bilayers [14]. Finally,
using solid-state
19
F-NMR spectroscopy and a
19
F-labeled
GS analog, we showed that GS is aligned with its cyclic
b-sheet ring lying flat in the plane of the bilayer, consistent
with its amphiphilic character, although the peptide
molecules rotate rapidly and wobble in liquid-crystalline
PtdCho bilayers [15].
We have recently shown that there are several structural
variations of the GS molecule which can lead to a
dissociation of antimicrobial and hemolytic activities [4–7],
including variations in ring size [5]. The secondary
structures of these ring-size analogs exhibit a definite
periodicity in b-sheet structure, with rings containing six,
10 and 14 residues having the conventional antiparallel
b-sheet structure of GS, and those containing eight or 12
residues having largely distorted structures [5,16].
Although GS analogs containing fewer than 10 residues
exhibit no significant antimicrobial or hemolytic activities,
the 12-residue peptide (GS12) retains appreciable activity
against Gram-negative bacteria and fungi, exhibits consid-
erably reduced activity against Gram-positive bacteria, but
most importantly also displays a significantly reduced
hemolytic activity, resulting in a significant improvement
in microbial specificity (therapeutic index) for Gram-
negative bacteria. In contrast, the 14-residue analog

disrupt lipid membranes. We chose to study the zwitter-
ionic, bilayer-preferring phospholipid PtdCho as it is
abundant in the outer monolayer of the lipid bilayers of
mammalian plasma membranes [17], while the zwitter-
ionic, nonbilayer phase-preferring PtdEtn and the anionic,
bilayer phase-preferring PtdGro are thought to be
common components of the outer monolayer of the
lipid bilayer of bacterial membranes [18,19]. Finally, we
investigated the relative abilities of GS10, GS12 and
GS14 to inhibit the growth of A. laidlawii B, a cell wall-
less Gram-positive bacteria. The goal of this work is to
understand the relationship between the structure of
these GS ring-size analogs, their interactions with
phospholipid bilayer model membranes, and their anti-
microbial and hemolytic activities.
5912 M. Kiricsi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
MATERIALS AND METHODS
The three ring-size analogs of GS studied here were
synthesized and purified as described previously [4–6]. The
phospholipids utilized in this study were purchased from
Avanti Polar Lipids (Alabaster, AL, USA) and the calcein
from Molecular Probes (Eugene, OR, USA) and all were
used without further purification. All of the other chemicals
utilized here were highest purity reagent grade purchased
from BDH Inc. (Toronto, ON, Canada) and were used as
received.
The lipid/peptide mixtures for the DSC studies were
prepared by mixing the appropriate amounts of phospho-
lipid and peptide dissolved in methanol and ethanol,
respectively, removing the solvent under a stream of N

mixtures, exhibit no detectable thermal events over the
temperature range 0–90 °C. This indicates that these
peptides do not undergo any cooperative thermal denatur-
ation over this temperature range and thus that the
endothermic events observed by DSC arise exclusively from
phase transitions of the phospholipid.
The calcein leakage experiments were performed essen-
tially as previously described [20]. Briefly, the phospholipid
vesicles were prepared by drying chloroform solutions of
PamOleGro-PCho or PamOleGro-PEtn/PamOleGro-
PGro (7 : 3 molar ratio) under N
2
in a round-bottomed
flask and removing traces of the solvent by overnight
vacuum. The dry lipid film was then hydrated by the same
buffer used for the DSC experiments, but in this case also
containing a high concentration (70 m
M
)ofcalcein,by
vortexing at room temperature. The resulting MLVs were
then freeze-thawed several times and extruded through a
100-mm filter using a LipoFast apparatus (Avestin Inc.,
Ottawa, ON, Canada). The resulting LUVs were then
passed through a Sephadex G-50 column to remove calcein
not trapped inside the phospholipid vesicles. The peptide-
induced leakage of the self-quenched calcein from the LUVs
was then monitored by measuring the fluorescence of
calcein released into the aqueous buffer as a function of time
at 25 °C. The fluorescence intensity was measured with a
Perkin-Elmer LS50B spectrophotometer (Beaconsfield,

and antimicrobial and hemolytic activities, of GS and GS10
are similar, although GS is slightly more active against both
Gram-positive and Gram-negative bacteria than is GS10
[5].
Perturbation of phospholipid thermotropic phase
behavior by GS ring-size analogs
We studied the effects of concentrations of these GS ring-
size analogs ranging from 1 to 4 mole percent on the
thermotropic phase behavior of aqueous dispersions of two
zwitterionic phospholipids and one anionic phospholipid by
DSC. In each case the result of progressively increasing the
Fig. 1. The amino acid sequence, structure and conformation of the ring-
size analogs of GS studied here (GS10, GS12 and GS14) in aqueous
solution.
Ó FEBS 2002 Gramicidin S analog–membrane interactions (Eur. J. Biochem. 269) 5913
peptide concentration was simply to progressively increase
the magnitude of the characteristic effects of each particular
analog on the thermotropic phase behavior of each
phospholipid system examined. We have thus chosen to
present DSC thermograms for each GS ring-size analog and
each phospholipid system at only the highest peptide
concentration tested, as the characteristic differences in
their effects are most clear under these circumstances. We
also point out that a peptide concentration of 4 mole
percent (phospholipid/peptide molar ratio of 25 : 1) is well
within the physiologically relevant concentration range for
GS itself [2,3,8].
The initial DSC heating thermograms, illustrating the
thermotropic phase behavior of large MLVs composed of
Myr

series of linear disaturated PCs, the reader is referred to
Lewis et al. [22].
The effect of the incorporation of 4 mole percent
(peptide/phospholipid molar ratio 1 : 25) of the three GS
ring-size analogs studied here on the thermotropic phase
behavior of the host Myr
2
Gro-PCho bilayer varies greatly,
as illustrated in Fig. 2. For GS12, a single DSC endotherm
is observed whose temperature and enthalpy are essentially
unchanged from that of Myr
2
Gro-PCho alone and whose
cooperativity is only moderately reduced. In contrast, the
incorporation of both GS10 and GS14 produce much
broader, lower enthalpy DSC endotherms, particularly in
the case of the latter peptide. In fact in both instances, these
two peptides produce two-component DSC traces consist-
ing of a relatively more cooperative, higher enthalpy
component centered at a lower temperature than the main
phase transition temperature of Myr
2
Gro-PCho alone, and
a much less cooperative, more enthalpic component
centered at a higher temperature (see Fig. 3). Moreover,
the magnitude in the downward shift in the temperature of
the sharp component, and of the upward shift in the
temperature of the broad component, is greater for GS14
than for GS10. Also, the relative enthalpy of the higher
temperature DSC component is considerably greater and

Gro-PCho MLVs con-
taining 4 mole percent GS14 (–
–
—) and its deconvolution into sharp and
broad components (- - - -).
5914 M. Kiricsi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Interestingly, GS14 and GS10 alter the phase behavior of
Myr
2
Gro-PChoMLVstoagreaterextentthandoesGS
itself [9].
DSC heating scans of MLVs composed of Myr
2
Gro-
PEtn alone, or of Myr
2
Gro-PEtn containing 4 mole percent
of one of the three ring-size analogs of GS, are presented in
Fig. 4. Aqueous dispersions of Myr
2
Gro-PEtn alone, which
have not been extensively incubated at low temperatures
prior to calorimetric analysis, exhibit a single fairly cooper-
ative, relatively energetic L
b
/L
a
phase transition centered
near 50 °C (see [23] for a more complete description of the
thermotropic phase behavior of Myr

Fig. 4B). Interestingly, however, GS14 and GS10 again
exhibit larger effects on the thermotropic phase behavior of
Myr
2
Gro-PEtn than does GS12. However, in all cases the
presence of these peptides produces only a slight destabi-
lization of the L
b
phase relative to the L
a
phase of Myr
2
Gro-
PEtn bilayers, as also observed previously with GS itself [9].
Also, repeated recycling through the phase transition
temperature actually increases the magnitude of the effect
of these peptides on Myr
2
Gro-PEtn phase behavior, in
contrast to the situation with Myr
2
Gro-PCho MLVs. This
effect, which was also observed to a lesser extent with GS
itself [9], suggests that repeated exposure to high tempera-
tures facilitates peptide incorporation into Myr
2
Gro-PEtn
bilayers.
The initial DSC heating scans of MLVs of Myr
2

Gro-PGro and other members of
the homologous series of linear saturated PGs, see Zhang
et al. [24].
The addition of 4 mol percent of one of the three GS
ring-size analogs of GS studied has a relatively modest effect
on the thermotropic phase behavior of Myr
2
Gro-PGro
MLVs. In all cases the presence of peptide decreases the
cooperativity of the main transition. Also, each peptide
induces the presence of a second, less enthalpic, broad
component of the DSC endotherm which occurs at a higher
temperature than does the more enthalpic sharp compo-
nent. As well, in the case of GS14 only, additional
endothermic events are noted at temperatures near 31 and
39 °C. As before, the magnitude of the effect of these
peptides on the cooperativity of the main phase transition
decreases in the order GS14 > GS10 > GS12. Moreover,
GS14 decreases the enthalpy of the main phase transition of
Myr
2
Gro-PGro substantially whereas GS12 and GS10
actually appear to slightly increase the total enthalpy of the
two-component main phase transition. We note that GS
itself, however, has a greater effect on the thermotropic
phase of Myr
2
Gro-PGro MLVs than do any of the three
Fig. 4. Initial high-sensitivity DSC heating scans illustrating the effect of
the addition of 4 mol percent GS10, GS12 or GS14 on the thermotropic

ring-size analogs
In order to determine the relative abilities of these three ring-
size analogs of GS to permeabilize phospholipid bilayers, we
determined the amount of entrapped calcein dye released by
the addition of 4 mole percent peptide to LUVs composed
of either PamOleGro-PCho or a mixture of PamOleGro-
PEtn/PamOleGro-PGro (7 : 3 molar ratio). PamOleGro-
PCho was selected to mimic the phospholipid composition
of the outer monolayer of eukaryotic plasma membranes
[17] and the PamOleGro-PEtn/PamOleGro-PGro mixture
tomimicthephospholipid composition of the Escherichia coli
inner membrane [18]. Although we intended to study vesicles
composed of PamOleGro-PEtn or PamOleGro-PGro
alone, the former did not form well defined LUVs under
our experimental conditions [25] and PamOleGro-PGro
formed only small unilamellar vesicles [26]; as antimicrobial
peptide binding can be influenced by the degree of curvature
strain in phospholipid [27], such a size difference would have
made comparisons between the three individual phosphol-
ipids difficult. However, the PamOleGro-PEtn/PamOle-
Gro-PGro mixture formed well-behaved LUVs.
As illustrated in Fig. 6, the addition of each of the three
ring-size analogs of GS cause considerable fluorescence dye
leakage when added to PamOleGro-PCho LUVs at a final
peptide/phospholipid molar ratio of 1 : 25, with the extent
of dye leakage decreasing in the order GS14 >
GS10 > GS12 [28]. Moreover, at lower peptide concentra-
tions, GS14 is perhaps 10-fold more potent at releasing
calcein than is GS10 or GS12 [28]. Interestingly, the
addition of the same amount of these three peptides to

molecular interpretation of any results obtained.
We present in Fig. 7 growth curves for A. laidlawii in the
presence or absence of various concentrations of the three
GS ring-size analogs studied here. It is clear from these
curves that a considerable difference in the growth inhib-
itory potency of these three peptides exists. For example
GS10 is a fairly potent antimicrobial agent, inhibiting
A. laidlawii B growth slightly at the lowest concentration
tested (0.25 l
M
), strongly at the next highest peptide
Fig. 6. A bar graph illustrating the percentage of entrapped calcein dye
leakage at equilibrium from LUVs composed of either PamOleGro-
PCho (white bar) or PamOleGro-PEtn/PamOleGro-PGro(7:3molar
ratio) (hatched bar) upon the addition of 4 mol percent GS10, GS12 or
GS14.
Fig. 7. Growth curves at 37 °CofA. laidlawii B in the absence or
presence of various concentrations of GS10, GS12 or GS14. The sym-
bols utilized are: (·), absence of peptide, and (h), (s), (n), (,), and
(e), peptide concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 l
M
,
respectively, in the growth medium.
5916 M. Kiricsi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
concentrated (0.50 l
M
), and completely suppressingly
growth at concentrations of 1.0 l
M
and higher. In con-

DISCUSSION
We showed previously that the effect of GS on the
thermotropic phase behavior of phospholipid bilayers
depends markedly on both the structure and charge of the
lipid polar headgroup [9,10]. Specifically, the presence of GS
has only a very small effect on the thermotropic phase
behavior of Myr
2
Gro-PEtn bilayers, even at very high
peptide concentrations and after multiple cycling through
the gel/liquid-crystalline phase transition. Only upon expo-
sure of the Myr
2
Gro-PEtn bilayers to high temperature is a
small decrease in the temperature, enthalpy and coopera-
tivity of the main phase transition and the induction of a
minor lower temperature shoulder on this endotherm
observed. The addition of similar amounts of GS to
Myr
2
Gro-PCho bilayers results in a somewhat greater but
still rather small decreases in the temperature, enthalpy and
cooperativity of the main phase transition and induces a
new broad component of the DSC endotherm centered at a
slightly higher temperature. In contrast, the addition of GS
to Myr
2
Gro-PGro bilayer produces a considerably larger
decrease in the temperature, enthalpy and cooperativity of
the main phase transition and induces the presence of a

of the phospholipids studied here depends on the specific
phospholipid vesicle system being studied. Specifically, the
order of decreasing perturbation of phospholipid phase
behavior by GS itself, and by the three ring-sized analogs
studied here, is GS14 > GS10 > GS > GS12 in
Myr
2
Gro-PCho MLVs, GS14 @ GS10 > GS @ GS12 in
Myr
2
Gro-PEtn MLVs, and GS > GS14 >GS10 > GS12
in MLVs of Myr
2
Gro-PGro. Thus, although GS12 has the
weakest effect in all three vesicles systems, the relative order
of effectiveness varies with polar headgroup structure for
the other three peptides, with GS14 and GS10 exhibiting a
greater effect than GS itself in the two zwitterionic
phospholipid bilayers studied here but a smaller effect in
the anionic phospholipid bilayer system.
The potency of the three ring-size analogs of GS in
inducing the leakage of calcein dye entrapped in PamOle-
Gro-PChoLUVsalsodecreasesintheorderGS14>
GS10 > GS12, which is the same decreasing order as
exhibited by these three peptides in perturbing the thermo-
tropic phase behavior of Myr
2
Gro-PCho MLVs. Interest-
ingly, the three GS ring-size analogs are generally less potent
at releasing entrapped calcein from PamOleGro-PEtn/

Ó FEBS 2002 Gramicidin S analog–membrane interactions (Eur. J. Biochem. 269) 5917
good correlation overall between the relative perturbation
of host bilayer organization as measured by DSC, the
permeabilization of phospholipid vesicles as measured by
calcein leakage, and the inhibition of the growth of
A. laidlawii B. We note also that GS is less potent at
inhibiting the growth of this organism than is GS14 but
more potent than GS10 and especially GS12 (data not
presented). Overall, then, these results indicate that studies
of the interactions of other analogs of GS with phospholipid
vesicles may be useful for predicting the antimicrobial
potency of these analogs and possibly also for understand-
ing the molecular basis for their differential antimicrobial
potencies again different classes and species of bacteria,
many of which may differ considerably in the lipid
compositions of their membranes [18,19].
It is instructive to compare the relative antimicrobial
potencies of these three GS ring-size analogs against various
Gram-positive and Gram-negative bacteria and against
A. laidlawii, as the former types of bacteria possess either a
lipopeptidoglycan outer barrier or a lipopolysaccharide-
containing cell wall or outer membrane, respectively, which
is lacking in the latter organism. Against conventional
bacteria, the order of decreasing antimicrobial potency is
GS10 > GS12 > GS14 [5], whereas against A. laidlawii B
the order is GS14 > GS10 > GS12. This result would
appear to confirm our previous suggestion that the low
effective antimicrobial activity of GS14, particularly against
Gram-negative bacteria, is due to its strong binding to the
lipopolysaccharide component of the bacterial cell wall [5],

water decrease in the order GS12 > GS10 > GS14. The
lower solubility of GS14 as compared to GS10 in water
appears to be related to its slightly greater amphiphilicity
and to its significantly greater exposed hydrophobic surface
area, which results in GS14 forming aggregates in aqueous
solution above a concentration of about 50–60 m
M
,
whereas GS10 and GS12 remain monomeric even at much
higher concentrations [22]. Note also that GS14 and GS10
are considerably more amphiphilic than is GS12, as in the
former two peptides the more polar charged Lys and the less
polar Val and Leu residues project on opposite sides of the
ring, whereas this is not the case for GS12.
We can ask whether or not the observed order of
biophysical or biological potencies of GS itself and of the
three ring-size analogs of GS studied here, namely GS
@ GS14 > GS10 > GS12, correlates well with any of the
physical properties of these peptides which we have previ-
ously measured. In terms of the relative conformational
flexibility of this series of peptides, there is not a particularly
good correlation with the observed results, as conforma-
tional rigidity decreases in the order GS > GS10 >
GS14 > GS12. Similarly, an even poorer correlation is
observed between the intrinsic hydrophobicities of these
peptides, which decrease in the order GS > GS10 >
GS14 > GS12, and their biophysical and biological activ-
ities or with the ratio of positively charged Orn or Leu
residues to the total number of residues (GS12 > GS14 >
GS10 ¼ GS). However, a reasonably good correlation is

of their perturbation of phospholipid bilayer membranes
and the growth of A. laidlawii, generally agrees well with the
results of previous studies of the antimicrobial activity of
these [5] and other [4–7] ring-size analogs of GS. This finding
is important in that it adds further support to the hypothesis
that GS and its analogs kill bacteria primarily through their
disruption of the lipid bilayer of the cell membrane.
In the absence of complications arising from the differ-
ential interactions of GS and its ring-size analogs with the
5918 M. Kiricsi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
outer membrane or cell wall of conventional bacteria, we
can identify at least three factors which can determine the
degree to which a particular antimicrobial peptide will
perturb the organization and integrity of phospholipid
bilayer membranes and inhibit the growth of A. laidlawii B.
These are the phospholipid bilayer/water partition coeffi-
cient, the localization and orientation of the peptide within
the phospholipid bilayer, and the degree to which the
peptide disturbs phospholipid packing once inserted into
the bilayer. The fact that the relative order of both
decreasing effectiveness in perturbing the thermotropic
phase behavior and compromising the integrity of phosp-
holipid model membranes, as well as inhibiting the growth
of A. laidlawii B (GS @ GS14 > GS10 > GS12) correlates
well with the increasing water solubility of these peptides,
can be explained in part by the fact that the phospholipid
bilayer/water coefficient should also decrease in the above
order, so that the effective concentration of peptide in the
target membrane also progressively decreases. Similarly, the
good correlation observed between the biophysical and

ACKNOWLEDGEMENTS
This work was supported by operating grants from the Protein
Engineering Network of Centers of Excellence and the Canadian
Institutes of Health Research, and by major equipment grants from the
Alberta Heritage Foundation for Medical Research. MK was sup-
ported in part by a Hungarian Eo
¨
tvo
¨
s Fellowship.
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