Membrane Insertion and Bilayer Perturbation by Antimicrobial
Sara Pistolesi,yRebecca Pogni,yand Jimmy B. Feix*
*Department of Biophysics and National Biomedical Electron Paramagnetic Resonance Center, Medical College of Wisconsin, Milwaukee,
Wisconsin 53226; andyDepartment of Chemistry, Universita ` di Siena, 53100 Siena, Italy
interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane
interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target
membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and
residues 2–9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an
a-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2–3.8 nm pores in its
bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior
of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing
peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on
the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the
N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the
rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface.
However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the
polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar
solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.
Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable
Antimicrobial peptides (AMPs) are an essential part of innate
immune defense against microbial infection. Naturally oc-
curring AMPs are basic peptides composed of 12–50 amino
acids that are ubiquitously distributed throughout all king-
doms of life (1–5) with over 800 peptides now listed in AMP
databases (6). As a group, AMPs display extensive sequence
heterogeneity; however they do share a number of common
characteristics, including a net positive charge of $12 (with
14 to 16 being most common), ;50%–70% hydrophobic
amino acids, and a propensity to fold into amphipathic con-
formations in the presence of membranes (2,7,8). AMPs
display a broad spectrum of antimicrobial activity against
both Gram-negative and Gram-positive bacteria, fungi, and
enveloped viruses (5). Importantly, they retain activity against
antibiotic-resistant strains and do not readily elicit resistance
Although potential intracellular targets are receiving in-
creasing attention, the primary target for most AMPs appears
to be the bacterial cytoplasmic membrane (4,5). AMPs bind
strongly to membranes, dissipate transmembrane ionic po-
tentials, and cause leakage of liposome-entrapped solutes.
Peptides synthesized with D-amino acids retain activity
comparable to their corresponding L-amino acid enantiomer,
indicating that the interaction with their biological target is
nonstereospecific (10–13). The bacterial cell surface has a
highly negative surface charge density due to the presence of
lipopolysaccharides and lipoteichoic acids in Gram-negative
and Gram-positive organisms, respectively, and it is believed
that electrostatic interactions make a significant contribution
to AMP selectivity.
A large and important subclass of AMPs is linear peptides
that fold into amphipathic a-helices upon membrane bind-
ing. For several of these peptides it has now been demon-
strated that initial binding occurs with the helical axis parallel
to the membrane surface (14–19) followed by insertion into
the bilayer and disruption of the membrane permeability
barrier as the amount of bound peptide exceeds a critical
concentration (20–22). Several models have been proposed
to describe the molecular events involved in AMP-mediated
membrane disruption, including the formation of barrel-
stave peptide channels, induction of peptide-lipid toroidal
pores, ‘‘sinking-raft’’ and ‘‘micellar aggregate’’ models, and
a detergent-like carpet mechanism (2,4,5,19,20,23–25). It is
likely that the precise mechanism of membrane disruption
depends on the specific peptide as well as the composition of
the target membrane (22,26,27).
Our studies have focused on a linear, synthetic hybrid
AMP composed of the first seven residues of cecropin A and
residues 2–9 of the bee venom peptide mellitin. This 15-
residue peptide, designated CM15, retains the two-domain
structure of native cecropins (28,29), with a highly cationic
N-terminal region and a mostly hydrophobic C-terminal
Submitted January 5, 2007, and accepted for publication May 9, 2007.
Address reprint requests to Jimmy B. Feix, Tel.: 414-456-4037; Fax: 414-
456-6512; E-mail: firstname.lastname@example.org.
Editor: Lukas K. Tamm.
? 2007 by the Biophysical Society
Biophysical JournalVolume 93 September 20071651–16601651
region. CM15 displays potent, broad-spectrum antimicrobial
activity yet lacks the strong hemolytic activity of mellitin
(30). We have previously shown that CM15 folds into an
a-helix upon membrane binding (16,19) and that at low
peptide/lipid (P/L) ratios (i.e., under initial binding condi-
tions) the helical axis is positioned ;5 A˚below the hydro-
phobic interface of the membrane and aligned parallel to
the bilayer surface (16). Osmoprotection studies with live
bacteria indicate that cell killing by CM15 is mediated by the
formation of membrane pores with a diameter of 2.2–3.8 nm
(31); however, nothing is known about the intermediate
stages between initial binding and pore formation.
In this study we have used site-directed spin labeling
(SDSL) electron paramagnetic resonance (EPR) spectros-
copy to investigate the behavior of a spin-labeled analog of
CM15 as a function of increasing peptide concentration and
utilizedphospholipid-analogspinlabelstoexamine the effects
of CM15 binding and accumulation on physical properties
of membrane lipids. We find that as the concentration of
membrane-bound CM15 is increased, the N-terminal domain
of the peptide becomes more deeply immersed in the lipid
bilayer. Changes in the rotational dynamics of membrane
lipids are minimal and confined primarily to near the mem-
brane surface. However, peptide binding dramatically in-
creases interaction of the lipid-analog spin labels with the
polar relaxation agent NiEDDA (nickel (II) ethylenediamine-
diacetate), indicating that there are significant changes in the
physical state of the lipid bilayer that are not readily detected
by methods that examine motional dynamics. These results
are discussed in relation to the molecular mechanism of
membrane disruption by CM15.
MATERIALS AND METHODS
Phospholipids POPE (1-palmitoyl-2-oleoylphosphatidylethanolamine), POPG
(1-palmitoyl-2-oleoylphosphatidylglycerol), tetraoleoyl-cardiolipin (CL), and
n-PCSL (1-(n-doxylpalmitoyl)-2-stearoylphosphatidylcholine; n ¼ 5, 7, 12)
spin labels were obtained fromAvanti Polar Lipids (Alabaster, AL).NiEDDA
was synthesized according to a protocol provided by Dr. Christian Altenbach
(Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA).
The methanethiosulfonate spin label, MTSL (1-oxy-2,2,5,5-tetramethylpyr-
roline-3-methyl methanethiosulfonate), and its brominated derivative, BrMTSL
(Fig. 1), were obtained from Toronto Research Chemicals (North York, ON,
Peptide synthesis and spin labeling
Peptides used in this study included the ‘‘wild-type’’ cecropin-mellitin
hybrid peptide CM15 with N-terminal acetylation and C-terminal amidation
(Ac-KWKLFKKIGAVLKVL-amide) andan analogcontainingaleucine-to-
cysteine substitution at position 4 (Ac-KWKCFKKIGAVLKVL-amide,
designated C4). Peptides were synthesized by standard n-(9-fluororenyl)-
methoxycarbonyl (Fmoc) solid phase synthesis methods on Rink amide
p-methylbenzhydrylamine (MBHA) resin as previously described (16). Crude
peptides were purified by reverse-phase semipreparative high performance
liquid chromatography (HPLC) on a 10-mm, 1.0 3 2.5-cm C8 column
(Vydac, Hesperia, CA) using a linear gradient of 10%–80% acetonitrile/
0.1% trifluoroacetic acid (TFA) in water/0.1% TFA over 56 min. Upon
elution the peptides were lyophilized and stored at ?20?C until use. For
spin labeling, the C4 peptide was resuspended in 50 mM MOPS (3-
(N-morpholino)propanesulfonic acid) pH 6.8 and reacted with a fivefold
molar excess of spin label (MTSL or BrMTSL) for 3 h at room temperature.
To remove excess spin label the peptide was bound to a 2-ml column of SP
Sepharose(Amersham,Buckinghamshire,UK),washed with50 mlof50 mM
MOPS, pH 6.8, and eluted with 6 M guanidine hydrochloride in the same
buffer. The spin labeled peptide was then repurified by HPLC as described
above, lyophilized, and stored at ?20?C. Final purity was confirmed by
matrix-assisted laser desorption ionization-time of flight mass spectrometry at
the Medical College of Wisconsin Protein and Nucleic Acid facility. Peptides
were rehydrated in 50 mM MOPS, pH 6.8 at time of use, and peptide concen-
trations were determined based on the absorbance of the single tryptophan
residue using an extinction coefficient of 5.69 mM?1cm?1(32).
Liposomes were composed of POPE/POPG/CL (molar ratio of 70:25:5), in
agreement with the composition of bacterial inner membrane lipids (33).
Lipids in the desired molar ratio were dried down from chloroform stock
solutions under a stream of nitrogen gas and then dried overnight under
labels and the resulting side chains produced by reaction
with the peptide cysteine residue.
Structures of the MTSL and BrMTSL spin
1652Pistolesi et al.
Biophysical Journal 93(5) 1651–1660
vacuum. The resulting lipid film was hydrated by the addition of 50 mM
MOPS, pH 6.8 to give a concentration of 50 mM phospholipid. Large
unilamellar vesicles (LUVs) were prepared by freeze-thawing this lipid
suspension five times, followed by extrusion through 200-nm polycarbonate
membrane filters using a miniextruder syringe device (Avanti Polar Lipids).
Final lipid concentration was measured by the method of Stewart (34).
LUVs containing 1 mol % of 5-, 7-, or 12PCSL were prepared as described
Far ultraviolet circular dichroism (CD) spectra were obtained at room tem-
perature on a Jasco (Tokyo, Japan) J-710 spectropolarimeter at a scan rate
of50 nm/min,0.5 sresponsetime, 1nm pitch,and1nm bandwidth.Peptides
were at a concentration of 0.1 mM (;0.2 mg/ml) in 5 mM phosphate buffer
pH 7 (5P7), 50% TFE in 5P7, or in the presence of 10 mM LUVs in 5P7.
Spectra taken in the presence of liposomes were truncated at 200 nm due to
high background scattering and saturation of the detector below this wave-
length. Secondary structure content was estimated via the DICHROWEB
server (35) using the Selcon3 and K2D analysis programs.
Conventional continuous-wave (CW) EPR spectra were recorded on a
Bruker (Billerica, MA) Elexsys E-500 X-band spectrometer equipped with a
super-high-Q cavity. Room temperature spectra were recorded at a micro-
wave power of 10 mW, using a 100 kHz, 1.0 G field modulation. Spectra
taken at 200 K were recorded at a microwave power of 2 mW. For peptide-
binding assays a constant amount of C4MTSL or C4BrMTSL peptide was
mixed with various concentrations of LUVs to give the desired lipid/peptide
(L/P) ratios and incubated at room temperature overnight. Final peptide
concentration was ;50 mM, and the final sample volume was 30 ml. The
fraction of peptide remaining free in solution was calculated based on the
peak-to-peak amplitude of the high-field (MI¼ ?1) line as previously
described (16). From the fraction of bound peptide and known total peptide
concentration, the concentrations of membrane-bound peptide (Cb), peptide
remaining free in solution (Cf), and the molar ratio of bound peptide/lipid
(Cb/L)werecalculated.Aplot of Cb/Lagainst Cfyieldsthe apparentpartition
coefficient, Kp, where Kp[L] ¼ Cb/Cf.
To assess the rotational mobility of 12PCSL, the apparent rotational cor-
relation time, tc, was determined according to (36):
tcðsecÞ ¼ ð0:65310?9ÞDH0½ðA0=A?1Þ1=2? 1?;
where DH0is the peak-to-peak width of the center line in gauss, A0is the
amplitude of the center line, and A?1is the amplitude of the high field line
(see Fig. 7). The rotational correlation time is inversely related to the
motional rate, i.e., an increase in t indicates slower motion.
The accessibility of spin-labeled peptides and PCSLs to the relaxation
agents O2and NiEDDA was determined by CW power saturation. Li-
posomes containing either 5-, 7-, or 12PCSL and native CM15 or spin
labeled C4 and unlabeled LUVs were mixed to obtain the desired L/P ratios.
Samples (final volume 10 ml) with or without 20 mM NiEDDA were
incubated at room temperature overnightand then placed into gas-permeable
TPX capillaries (Molecular Specialties, Milwaukee, WI). EPR spectra for
power saturation studies were obtained on a Varian E-102 Century series
spectrometer equipped with an X-band two-loop one-gap resonator (Molec-
ular Specialties). The saturation parameter P1/2was determined under vari-
ous conditions (under N2, saturated with air (20% O2) and under N2with
samples containing 20 mM NiEDDA) by measuring the amplitude of the
center line at a series of microwave powers from 0.25 to 64 mW as described
previously (37,38). The change in the saturation parameter, DP1/2, in the
presence of O2or NiEDDA is directly proportional to the bimolecular
collision rate with the respective paramagnetic relaxation agent (37). The
depth parameter, F, where
F ¼ ln½DP1=2ðO2Þ=DP1=2ðNiEDDAÞ?
was calculated as a measure of the bilayer immersion depth of the spin label
side chain (16,38,39).
Secondary structure of the spin-labeled peptides
One conserved characteristic of linear cationic AMPs is their
ability to fold into amphipathic secondary structures upon
membrane binding (1–5). We used CD to evaluate secondary
structure formation by C4MTSL and C4BrMTSL. Previous
CD studies have shown that wild-type CM15 adopts an
a-helical secondary structure upon membrane binding (19),
and a nitroxide-scanning study of CM15 was also consistent
with the formation of a continuous a-helix in the membrane-
bound state (16). CD spectra of C4BrMTSL in aqueous solu-
tion, in the helix-promoting solvent trifluoroethanol (TFE),
and in the presence of LUVs are shown in Fig. 2. C4BrMTSL
exhibits little or no secondary structure in solution but adopts
a significant degree of a-helical structure in the presence
of 50% (v/v) TFE or when bound to LUVs (helix content
estimated at 86% and 58%, respectively). CD spectra of
C4MTSL were quite similar in appearance (data not shown)
although estimates of a-helical content were consistently
lower (68% and 52% in TFE and bound to LUVs, respec-
tively). Helix content for wild-type CM15 in 50% TFE and
bound to LUVs was 65% and 58%, respectively (H. Sato and
J. B. Feix, unpublished data). These results indicate that the
spin-labeled C4 peptides adopt an a-helical secondary struc-
ture upon membrane binding that is similar to wild-type
CM15 and suggest that the BrMTSL side chain may facili-
tate or stabilize helix formation.
in 50% TFE (squares) and in the presence of LUVs at an L/P ratio of 100:1
(triangles). The peptide concentration was 0.1 mM (;0.2 mg/ml) in each
Membrane Perturbation by CM151653
Biophysical Journal 93(5) 1651–1660
Binding and mobility of the spin-labeled peptides
To further examine peptide-membrane interactions by EPR
spectroscopy, we utilized the C4MTSL and C4BrMTSL an-
alogs of CM15. Since the spin label side chain is relatively
hydrophobic (40), spin labeling at this site (normally a leu-
cine) is a conservative substitution. EPR spectra of C4MTSL
in aqueous solution, in 50% TFE, and bound to liposomes at
various L/P ratios are shown in Fig. 3. In solution, the EPR
spectrum of C4MTSL consists of three narrow lines, typical
of an unstructured peptide with few constraints on the
motion of the spin label side chain and consistent with the
observed random coil CD spectrum. Addition of 50% TFE
to induce a-helix formation produced only a slight decrease
in spin label motion (Fig. 3 B). However, upon addition of
liposomes a dramatic broadening of the spectrum was ob-
served (Fig. 3, C–E), indicating a significant reduction in
spin label mobility upon membrane binding. Similar experi-
In aqueous solution the EPR spectrum of C4BrMTSL was
quite similar to that observed for C4MTSL, with only a
slightly longer rotational correlation time, tc(0.41 ns for
C4MTSL and 0.56 ns for C4BrMTSL). Induced a-helix
formation by the addition of 50% TFE moderately decreased
the motional freedom of the spin label side chain (Fig. 4 B)
and to a somewhat greater degree than for the nonbrominated
spin label (tcvalues 0.77 ns and 1.17 ns for C4MTSL and
C4BrMTSL, respectively). As with C4MTSL, binding of
C4BrMTSL to LUVs resulted in a significant reduction in
spin label mobility (Fig. 4, C–E). Previous studies have
shown that for labeling sites in a-helices, reduced mobility
of MTSL and its analogs results from interaction between the
substituent at the 49 position of the nitroxide ring and a Ca
hydrogen of the peptide backbone (41,42). Replacing the 49-
hydrogen of MTSL with the large bromine atom substan-
tially increases this interaction (43). Thus, even in these
small peptides, BrMTSL can be used to reduce the flexibility
of the spin label side chain, tethering it more closely to the
peptide backbone relative to MTSL under the same condi-
tions (compare Figs. 3 and 4). The relatively low mobility of
C4BrMTSL in the presence of LUVs also indicates that the
peptide itself has little flexibility when membrane bound.
Both C4MTSL and C4BrMTSL bound to POPE/POPG/
CL (70:25:5) liposomes with high affinity. Binding iso-
therms, determined as described previously (16), yielded
partition coefficients (Kp) in 50 mM MOPS buffer of 1.9 3
105M?1and 2.3 3 105M?1for C4MTSL and C4BrMTSL,
respectively. These values are significantly higher than
previously reported for POPE/POPG (80:20) liposomes in
50 mM MOPS, 0.1 M KCl (16), consistent with the higher
content of anionic lipids and lower ionic strength in this
system. As indicated by the high Kp, the peptide was essen-
tially fully bound even at high P/L ratios (e.g., at 2.5 mM
lipid and 100 mM peptide, L/P ¼ 25:1, the calculated ratio of
bound/free peptide is 575:1).
in 50% TFE, and in the presence of PE/PG/CL (70:25:5) LUVs at L/P ratios
of (C) 250:1, (D) 50:1, and (E) 25:1. Scan width 100 G. Spectra for the mem-
brane-bound peptide are presented at a 10-fold higher gain. Binding to the
LUVs results in a pronounced reduction in spin label mobility, and the
MTSL side chain in the membrane-bound state (compare with Fig. 3).
EPR spectra of C4-BrMTSL in (A) 50 mM MOPS buffer, (B)
50% TFE, and in the presence of PE/PG/CL (70:25:5) LUVs at L/P ratios of
(C) 250:1, (D) 50:1, and (E) 25:1. Spectra for the membrane-bound peptide
are presented at a 10-fold higher gain. The scan width is 100 G.
EPR spectra of C4-MTSL in (A) 50 mM MOPS buffer, (B) in
1654 Pistolesi et al.
Biophysical Journal 93(5) 1651–1660
There were no evident changes in line width or normalized
amplitude for either C4MTSL or C4BrMTSL as the concen-
tration of membrane-bound peptide increased (decreasing
the L/P ratio from 250:1 to 25:1, Figs. 3 and 4 and data not
shown), indicating an absence of spin-spin interactions. As
noted above, both C4MTSL and C4BrMTSL were essen-
tially fully bound even at an L/P of 25:1, as evidenced by the
lack of a sharp component in the EPR spectrum due to un-
bound peptide (Figs. 3 and 4). We also examined EPR
spectra for the membrane-bound peptides as a function of
L/P ratio in frozen samples. Freezing should eliminate
residual rotational motion as well as stabilize the association
of any peptide oligomers that might exist, enhancing the
ability to observe dipole-dipole interactions. However, no
changes in line shape or relative line widths were observed
over a wide range of L/P ratios (Fig. 5). The lack of observed
dipolar coupling indicates that spin labels are separated by at
least 20 A˚at all L/P ratios examined. Alternatively, a dy-
namic equilibrium between oligomeric channels and mono-
mers could limit our ability to detect spin-spin interactions,
especially if the oligomeric species represents a minor popu-
lation of the total peptide. Motional parameters (i.e., central
line width, outer hyperfine splittings) also remained un-
changed as a function of bound peptide concentration (data
not shown), indicating that rotational motion was not altered
by the accumulation of bound peptide. The absence of line
broadening and lack of motional restriction at increasing
peptide concentrations argue against direct peptide-peptide
contact, at least in the vicinity of the spin label side chain.
Accessibility of the spin labeled peptide
To determine the accessibility of the spin labeled side chain
of the peptides, we used power saturation EPR to examine
interaction with the paramagnetic relaxation agents O2and
NiEDDA.NiEDDA is an uncharged, polar reagent that pene-
trates only weakly into phospholipid bilayers, with dimin-
ishing concentrations at increasing bilayer depths (39). The
NiEDDA accessibility parameter, DP1/2(NiEDDA), is pro-
portional to the bimolecular collision rate between the spin
label and the relaxation agent and, therefore, a direct re-
flection of the local NiEDDA concentration. Conversely,
molecular oxygen is nonpolar and partitions favorably into
lipid bilayers, with O2concentration increasing as a function
of bilayer depth (39,44). As seen in Fig. 6 A, interaction with
NiEDDA gradually diminished with increasing concentra-
tions of bound peptide for both C4-MTSL and C4-BrMTSL.
The decrease in DP1/2(NiEDDA) was approximately linear
up to an L/P ratio of 30:1, followed by a sharp decrease at
L/P ¼ 25:1. Both spin-labeled peptides also showed a trend
of increasing O2accessibility with increasing concentrations
of bound peptide (Fig. 6 B). Interaction with O2was much
greater than with NiEDDA under all conditions (note the
different vertical scales in Fig. 6, A and B), confirming the
hydrophobic localization of the MTSL and BrMTSL side
chains (16). The ratio of accessibility parameters for NiEDDA
and O2can be used to calculate an EPR depth parameter, F
(see Materials and Methods). Although significant changes
in the accessibilities of standard lipid-analog spin labels
(discussed below) precluded actual depth calculations, depth
parameters for both spin-labeled peptide analogs indicated a
gradually increasing immersion depth at increasing peptide
concentrations, again with a sharp transition between L/P
ratios of 30:1 and 25:1 (Fig. 6 C). Except at the highest con-
centration of bound peptide, C4BrMTSL consistently exhibited
lower accessibility to NiEDDA, greater O2accessibility, and
an increased depth parameter compared to C4MTSL, consis-
tent with the greater hydrophobicity of the brominated MTSL
Effect of CM15 binding on membrane lipids
To assess the effects of CM15 binding on the lipid phase of
the bilayer, we examined the motion and accessibility param-
eters for phosphatidylcholine spin labels (PCSLs) with the
nitroxide moiety positioned at various depths along the alkyl
chain. CW-EPR spectra for 5-, 7-, and 12PCSL in POPE/
POPG/CL (70:25:5) LUVs with and without bound wild-
type CM15 peptide are shown in Fig. 7. For 5PCSL, a plot of
the motional parameter 2T//as a function of the L/P ratio
suggests a slight decrease in motion (i.e., an increase in 2T//)
with increasing concentration of bound peptide (Fig. 8, Table
1). A similar trend is observed for 7PCSL, although changes
undergoes more rapid motion than 5- or 7PCSL, the 2T//
state. C4MTSL was mixed with LUVs at final L/P ratios of (A) 160:1, (B)
80:1, (C) 40:1, and (D) 20:1 and allowed to equilibrate at room temperature
for 1 h. The samples were then placed in the EPR cavity and brought to 200
K. Scan widths are 160 G. No changes in line widths or relative amplitudes
were observed as a function of the L/P ratio.
EPR spectra of C4MTSL bound to membranes in the frozen
Membrane Perturbation by CM151655
Biophysical Journal 93(5) 1651–1660
parameter is less sensitive to changes in mobility than the
width of the center line (DH0) or relative line amplitudes,
which can be used to calculate an empirical rotational cor-
relation time, tc(see Materials and Methods). For 12PCSL,
increasing concentrations of bound peptide produced no
significant changes in either DH0or tc(Table 2). These re-
sults indicate that perturbation of bilayer lipid motional dy-
namics upon accumulation of bound CM15 is observed only
(triangles) as a function of the L/P ratio. (A) The change in the EPR satura-
tion parameter, P1/2, in the presence of 20 mM NiEDDA, (B) the change in
P1/2upon equilibration with air (20% O2), and (C) the EPR depth parameter,
calculated as described in the text. Error bars indicate standard deviations
from at least three separate measurements.
Accessibilities of C4-MTSL (squares) and C4-BrMTSL
PE/PG/CL liposomes. In each set of spectra the upper spectrum is in the
absence of peptide and the lower spectrum is in the presence of wild-type
CM15 at an L/P ratio of 25:1. The increase in 2T//observed for 5- and
7PCSL indicate a decrease in rotational mobility. For 12PCSL there is no
measurable difference in the width of the center line (DH0) or in the relative
line amplitudes (A0and A?1). Scan widths are 100 G for 5- and 7PCSL, and
80 G for 12PCSL.
EPR spectra of (A) 5PCSL, (B) 7PCSL, and (C) 12PCSL in
CM15 was added to PE/PG/CL (70:25:5) LUVs containing 1 mol % PCSL
and equilibrated at room temperature overnight before recording the EPR
spectrum. The motion parameter 2T//for 5PCSL (squares) and 7PCSL
(triangles) is plotted as a function of the lipid ratio (with N corresponding to
control values in the absence of peptide). An increase in 2T//indicates de-
creased mobility of the spin label. Error bars indicate the standard deviation
from at least three separate experiments.
Effect of wild-type CM15 binding on the motion of PCSLs.
1656 Pistolesi et al.
Biophysical Journal 93(5) 1651–1660
near the membrane surface, and even those effects are min-
The binding of CM15 induced far more pronounced
changes in PCSL accessibilities, significantly altering the
accessibility of all three PCSLs to NiEDDA (Fig. 9). In the
absence of peptide, NiEDDA accessibility parameters ex-
hibit the expected profile, with the accessibility of 5PCSL .
7PCSL . 12PCSL (Fig. 9). At concentrations of CM15
giving P/L ratios in the range 0–0.02 (L/P ¼ N to 50:1), an
increase in NiEDDA interaction is observed for all three of
the PCSLs, indicating an increase in NiEDDA concentration
at all depths of the bilayer. At L/P ratios in the range 40:1–
30:1, interactions between NiEDDA and the PCSLs plateau,
with no difference observed in the accessibilities of 5- and
7PCSL. These results are consistent with a pronounced
disruption of the membrane permeability barrier, particularly
near the membrane surface, and may also indicate a thinning
of the bilayer (see Discussion). Remarkably, as the peptide
concentration is increased even further, to an L/P of 25:1,
NiEDDA accessibility of the PCSLs reverts to a profile
similar to that observed in the absence of peptide.
Peptide binding had little effect on oxygen accessibility
parameters for the PCSLs (data not shown), so that a plot of
F against the L/P ratio is approximately the inverse of the
dependence seen with NiEDDA (Fig. 10). In the absence of
peptide we observed the expected gradient in the depth
parameter, with 12PCSL . 7PCSL . 5PCSL and significant
differences between the F values of each of the labels. Depth
parameters decreased (reflecting increased accessibility to
NiEDDA) for all three of the PCSLs up to an L/P ratio of
50:1 and then plateaued at L/P ratios between 50:1 and 30:1.
In the L/P range of 40:1–30:1, the difference in F values
between 12PCSL and the other PCSLs was much less than in
the absence of peptide, and no difference in F was observed
between 5- and 7PCSL. Again, as the L/P ratio was further
decreased to 25:1, the PCSL depth profile reverted to a
pattern similar to that observed in the absence of peptide.
TABLE 1 Motional parameters for 5PCSL and 7PCSL
54.2 6 0.2
54.9 6 0.3
54.7 6 0.1
55.4 6 0.7
55.0 6 0.9
55.2 6 0.5
55.5 6 0.8
56.1 6 1.2
52.0 6 0.1
53.0 6 0.3
52.4 6 0.2
52.3 6 0.1
53.2 6 0.6
53.0 6 0.9
52.8 6 0.7
52.8 6 0.6
Motional parameters for 5- and 7PCSL as a function of L/P molar ratio.
Mean 6 SD of 2T//are from at least three independent experiments. All
values are in gauss (G).
TABLE 2 Motional parameters for 12PCSL
3.72 6 0.5
3.71 6 0.5
3.74 6 0.7
3.66 6 0.4
3.72 6 0.5
3.88 6 0.8
3.44 6 0.5
3.49 6 0.5
3.63 6 0.04
3.52 6 0.04
3.55 6 0.06
3.50 6 0.03
3.53 6 0.04
3.60 6 0.07
3.63 6 0.04
3.63 6 0.04
Motional parameters for 12PCSL as a function of L/P molar ratio. Mean 6
SE of the peak-peak width of the center line (DH0) and rotational corre-
lation time (t) are from two independent experiments.
PCSLs to NiEDDA. Large unilamellar liposomes (PE/PG/CL, molar ratio
70:25:5) containing 1 mol % of 5PCSL (squares), 7PCSL (circles), or
12PCSL (triangles) were mixed with CM15 to give the desired L/P ratio,
incubated overnight at room temperature and the CW saturation parameter
P1/2determined under N2. Final NiEDDA concentration was 20 mM.
Effect of wild-type CM15 binding on the accessibility of
(squares) is plotted against the L/P molar ratio. The spin labels were at a
concentration of 1 mol % in PE/PG/CL LUVs. Error bars represent the stan-
dard deviation from at least three independent experiments.
Effect of wild-type CM15 binding on the EPR depth param-
Membrane Perturbation by CM151657
Biophysical Journal 93(5) 1651–1660
AMPs interact with membranes by a variety of mechanisms
that can result in disruption of bilayer structure and loss of
the differential permeability barrier. Several studies have
indicated that for most linear, a-helical AMPs, including
CM15, initial interactions (i.e., at low concentrations of
membrane-bound peptide) occur primarily near the mem-
brane surface, with the peptide aligned parallel to the plane
of the bilayer (14–19). Peptide insertion near the hydropho-
bic-hydrophilic interface of the membrane is postulated to
result in expansion of the outer leaflet of the bilayer, with
continued accumulation of bound peptide leading to mem-
brane thinning as a prelude to pore formation or detergent-
like disintegration of the bilayer (21,45–47). The goals of
this study were to examine peptide-induced changes in the
lipid phase of the membrane and to explore potential changes
in peptide localization and aggregation state at increasing
concentrations of membrane-bound CM15.
Using phosphatidylcholine-analog spin labels, we find
that CM15-induced changes in lipid motional dynamics are
minimal and occur primarily near the membrane surface,
even at relatively high concentrations of membrane-bound
peptide (i.e., up to 4 mol %). For 5PCSL, spin label mobility
gradually decreased across the entire range of P/L ratios ex-
amined, as might be expected if peptide insertion increased
the membrane lateral pressure near the interfacial region.
This result is consistent with a previous spin labeling study
of a 34-residue cecropin B analog in which decreased mo-
bility of PCSLs upon peptide binding to liposomes was also
observed (48). In contrast to CM15 however, the most active
cecropin B analog caused decreased lipid mobility (i.e.,
increased order) at all depths of the bilayer (48), which may
reflect a fundamental difference in membrane interactions
between the shortened 15-residue peptide studied here and
full-length cecropins. The observation that lipid perturbation
by CM15 is restricted to near the membrane surface is con-
sistent with a toroidal-pore model for permeabilization, as
proposed for other linear a-helical AMPs such as the
magainins (49), magainin derivatives (50),andLL-37 (15,51).
In this model, peptides remain associated with lipid head-
groups even as they transition from an orientation parallel to
the membrane surface to an alignment along the bilayer nor-
In contrast to the minimal effects on lipid motion, peptide
binding significantly increased accessibility to the polar re-
laxation agent NiEDDA at all depths of the membrane. For
example, PCSL accessibility to NiEDDA increased by 1.5–
2-fold at an L/P ratio of 100:1, with 3–4-fold increases ob-
changes in P1/2, is a reflection of the diffusion-concentration
product of the relaxation agent. Given the absence of large
changes in membrane fluidity, it seems unlikely that the dif-
fusion coefficient of NiEDDA has significantly increased.
Consequently the observed increases in accessibility most
likely indicate an increase in NiEDDA concentration within
the bilayer, reflecting a disruption of the permeability barrier
to polar solutes. The observed changes in accessibility to the
polarsolute NiEDDA clearlyindicate that interactionofCM15
with the membrane alters the physical properties of the lipid
phase of the bilayer. Such changes are not readily apparent
using methods (e.g.,31P- and2H-NMR and spin label motion
analysis) that examine lipid dynamics.
Differences in NiEDDA accessibility at different bilayer
depths (5 . 7 . 12PCSL) were maintained up to an L/P ratio
of 50:1. However, at concentrations of bound peptide with
L/P ratios in the range of 40:1–30:1 differences between
12PCSL and the other two PC spin labels were diminished,
and no difference was observed between 5- and 7PCSL. We
speculate that this may be a reflection of membrane thinning.
studies have provided strong evidence that peptide accumu-
lationleads tomembranethinningbeforepeptide reorientation
and insertion into the bilayer. Our data showing diminished
differences in the NiEDDA accessibilities of 5-, 7-, and
12PCSL may be an indication of membrane thinning by
CM15 (thus reducing differences in the apparent depths of
the PCSL isomers), although further studies under conditions
where thinning of the bilayer is known to occur are needed to
support this conclusion. Our data also indicate that CM15-
induced changes in bilayer structure undergo an abrupt
transition at L/P ratios of 30:1–25:1. This is consistent with a
two-state mechanism proposed by Huang and co-workers for
the interaction of several AMPs with lipid bilayers, based
primarily on oriented CD studies in which peptides associate
parallel to the membrane surface up to a given critical con-
centration, at which point they reorient along the bilayer nor-
mal and insert into the membrane (20,22). Observed threshold
concentrations for various AMPs depend on both the peptide
reversion of the NiEDDA accessibility profile for PCSL spin
labels at an L/P of 25:1 to one similar to that observed in the
absence of peptide suggests that as this critical concentration
of bound peptide is reached CM15 may become sequestered
(i.e., into localized pores), leaving the remaining bulk lipid
phase relatively unperturbed.
With regard to the effects of CM15 accumulation on
peptide localization, our data indicate that the spin label side
chain at position 4 of the CM15-C4 analogs becomes more
deeply immersed in the membrane as the concentration of
bound peptide is increased. Decreased interaction with
NiEDDA and a concomitant increased interaction with O2
occurred gradually across the full range of bound peptide
concentrations, again followed by an abrupt transition be-
tween L/P ratios of 30:1 and 25:1. The decrease in NiEDDA
accessibility is even more remarkable given that the overall
bulk lipid concentration of this polar relaxation agent in-
creases with peptide binding at all depths of the bilayer, as
indicated by the PCSL data. The decreased interaction of
the C4-spin label side chain with NiEDDA may reflect a
1658 Pistolesi et al.
Biophysical Journal 93(5) 1651–1660
‘‘sinking’’ of the entire peptide (25) or a reorientation of the
peptide so that the N-terminal domain is more deeply buried.
Alternatively, a localized thinning of the bilayer in the region
of the peptide might allow the C4 side chain, positioned on
the nonpolar face of the amphiphathic helix, to reach a more
hydrophobic region of the bilayer while maintaining surface
exposure of the hydrophilic face of the helix. Such localized
effects on bilayer structure have recently been postulated
(22). The formation of peptide aggregates, sequestering the
spin label away from NiEDDA, could also produce a similar
changes in spin label mobility, b) the absence of spin-spin
interaction, and c) the fact that interaction with O2increases
with the accumulation of bound peptide.
One of the goals of this study was to investigate associa-
tion between membrane-bound peptides in the formation of
transmembrane pores. Previous studies using osmoprotec-
tants have provided strong evidence for pore formation by
CM15 in intact Escherichia coli cells, with an estimated pore
diameter in the range 2.2–3.8 nm (31). However, no spin-
spin interactions were observed even at the highest concen-
trations of membrane-bound peptide. In addition, there were
no evident changes in line width as a function of P/L ratio
in the frozen state, again indicating a lack of dipole-dipole
interaction. These results suggest that no stable aggregates
are formed in which spin labels are within the ;20 A˚range
of dipolar coupling for CW EPR (43,52) and would seem to
rule out the existence of stable ‘‘barrel-stave’’-type channels,
which should place labels within this distance. This observed
absence of dipolar broadening is best explained by formation
of a toroidal pore-type structure in which peptides are sepa-
rated by intervening phospholipids. However, we cannot rule
out the formation of transient pores or channel formation
by only a small fraction of membrane-bound peptides. In
addition, it may be that the labeling site employed in this
study—positioned on the nonpolar face of the peptide—may
tion was chosen as a conservative (hydrophobic-hydrophobic)
replacement; however CM15 analogs spin labeled at sites on
the more polar face of the peptide (e.g., at Ala-10) do retain
biological activity (H. Sato and J. B. Feix, unpublished data)
and may bebettersuitedfor studies ofchannel formation.Fur-
ther studies using pulsed EPR methods, which extend sen-
sitivity to dipolar interactions up to 50 A˚or more (53,54),
and/or the use of other labeling sites (i.e., those on the hydro-
philic face of the peptide) may allow us to further characterize
the nature of the putative transmembrane pore.
In summary, these studies provide further insights into the
disruptive effects of AMPs on membrane bilayers. We have
shown that as the concentration of membrane-bound CM15
increases, the nonpolar face of the peptide samples an
increasingly hydrophobic environment. There was no evi-
dence of peptide-peptide association, suggesting that trans-
membrane pores formed by the peptide are either transient or
that individual peptide monomers are separated by .20 A˚.
Binding of CM15 to model membranes with a lipid compo-
sition mimicking the bacterial inner membrane caused only
slight perturbations in membrane lipid dynamics, and changes
in lipid dynamics were observed only for 5PCSL, suggesting
that CM15 remains in a region of the bilayer near the hydro-
philic interface even as the concentration of bound peptide is
increased. In contrast, CM15 binding significantly increased
the accessibility of lipid-analog spin labels to the polar solute
NiEDDA. Accessibility studies with bothspin-labeledpeptide
and lipid-analog spin labels indicated an abrupt structural
change at an L/P ratio of ;25:1. Overall, these results are
most consistent with the toroidal pore model as the mecha-
nism of bilayer disruption by CM15.
We thank Dr. Candice Klug and Dr. Hiromi Sato for critically reading the
This work was supported by National Institutes of Health grant GM068829.
The National Biomedical EPR Center is supported by National Institutes of
Health grant EB001980.
1. Boman, H. G. 1995. Peptide antibiotics and their role in innate immunity.
Annu. Rev. Immunol. 13:61–92.
2. Hancock, R. E. W., and D. S. Chapple. 1999. Peptide antibiotics.
Antimicrob. Agents Chemother. 43:1317–1323.
3. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms.
4. Brogden, K. A. 2005. Antimicrobial peptides: pore formers or meta-
bolic inhibitors in bacteria? Nat. Rev. Microbiol. 3:238–250.
5. Jenssen, H., P. Hamill, and R. E. Hancock. 2006. Peptide antimicrobial
agents. Clin. Microbiol. Rev. 19:491–511.
6. Tossi, A. http://www.bbcm.univ.trieste.it/;tossi/pag2.htm.
7. Giangaspero, A., L. Sandri, and A. Tossi. 2001. Amphipathic alpha-
helical antimicrobial peptides. Eur. J. Biochem. 268:5589–5600.
8. Zelezetsky, I., and A. Tossi. 2006. Alpha-helical antimicrobial peptides—
using a sequence template to guide structure-activity relationship studies.
Biochim. Biophys. Acta. 1758:1436–1449.
9. Zhang, L., J. Parente, S. M. Harris, D. E. Woods, R. E. Hancock, and
T. J. Falla. 2005. Antimicrobial peptide therapeutics for cystic fibrosis.
Antimicrob. Agents Chemother. 49:2921–2927.
10. Wade,D.,A.Boman,B.Wahlin,C. M.Drain,D.Andreu,H.G.Boman,
and R. B. Merrifield. 1990. All-D amino acid-containing channel-
forming antibiotic peptides. Proc. Natl. Acad. Sci. USA. 87:4761–4765.
11. Bessalle, R., A. Kapitkovsky, A. Gorea, I. Shalit, and M. Fridkin. 1990.
All-D-magainin: chirality, antimicrobial activity and proteolytic resis-
tance. FEBS Lett. 274:151–155.
12. Bland, J. M., A. J. De Lucca, T. J. Jacks, and C. B. Vigo. 2001. All-
D-cecropin B: synthesis, conformation, lipopolysaccharide binding,
and antibacterial activity. Mol. Cell. Biochem. 218:105–111.
13. Merrifield, R. B., P. Juvvadi, D. Andreu, J. Ubach, A. Boman, and
H. G. Boman. 1995. Retro and retroenantio analogs of cecropin-melittin
hybrids. Proc. Natl. Acad. Sci. USA. 92:3449–3453.
14. Silvestro, L., and P. H. Axelsen. 2000. Membrane-induced folding of
cecropin A. Biophys. J. 79:1465–1477.
15. Henzler-Wildman, K. A., D. K. Lee, and A. Ramamoorthy. 2003.
Mechanism of lipid bilayer disruption by the human antimicrobial
peptide, LL-37. Biochemistry. 42:6545–6558.
Membrane Perturbation by CM151659
Biophysical Journal 93(5) 1651–1660
16. Bhargava, K., and J. B. Feix. 2004. Membrane binding, structure, and Download full-text
localization of cecropin-mellitin hybrid peptides: a site-directed spin-
labeling study. Biophys. J. 86:329–336.
17. Bechinger, B. 1999. The structure, dynamics and orientation of antimicro-
bial peptides in membranes by multidimensional solid-state NMR
spectroscopy. Biochim. Biophys. Acta. 1462:157–183.
18. Ramamoorthy, A., S. Thennarasu, D. K. Lee, A. Tan, and L. Maloy.
2006. Solid-state NMR investigation of the membrane-disrupting
mechanism of antimicrobial peptides MSI-78 and MSI-594 derived
from magainin 2 and melittin. Biophys. J. 91:206–216.
19. Sato, H., and J. B. Feix. 2006. Peptide-membrane interactions and
mechanisms of membrane destruction by amphipathic alpha-helical
antimicrobial peptides. Biochim. Biophys. Acta. 1758:1245–1256.
20. Huang, H. W. 2000. Action of antimicrobial peptides: two-state model.
21. Lee, M. T., F. Y. Chen, and H. W. Huang. 2004. Energetics of
pore formation induced by membrane active peptides. Biochemistry.
22. Huang, H. W. 2006. Molecular mechanism of antimicrobial peptides:
the origin of cooperativity. Biochim. Biophys. Acta. 1758:1292–1302.
23. Matsuzaki, K., O. Murase, N. Fujii, and K. Miyajima. 1996. An
antimicrobial peptide, magainin 2, induced rapid flip-flop of phospho-
lipids coupled with pore formation and peptide translocation. Bio-
24. Papo, N., and Y. Shai. 2003. Can we predict biological activity of
antimicrobial peptides from their interactions with model phospholipid
membranes? Peptides. 24:1693–1703.
25. Yandek, L. E., A. Pokorny, A. Floren, K. Knoelke, U. Langel, and
P. F. F. Almeida. Mechanism of the cell-penetrating peptide transportan
10 permeation of lipid bilayers. Biophys. J. 92:2434–2444.
26. Blondelle, S. E., K. Lohner, and M. Aguilar. 1999. Lipid-induced con-
formation and lipid-binding properties of cytolytic and antimicrobial
peptides: determination and biological specificity. Biochim. Biophys.
27. Epand, R. F., M. A. Schmitt, S. H. Gellman, and R. M. Epand. 2006.
Role of membrane lipids in the mechanism of bacterial species selec-
tive toxicity by two alpha/beta-antimicrobial peptides. Biochim. Biophys.
28. Fink, J., R. B. Merrifield, A. Boman, and H. G. Boman. 1989. The
chemical synthesis of cecropin D and an analog with enhanced anti-
bacterial activity. J. Biol. Chem. 264:6260–6267.
29. Andreu, D., R. B. Merrifield, H. Steiner, and H. G. Boman. 1985.
N-terminal analogues of cecropin A: synthesis, antibacterial activity,
and conformational properties. Biochemistry. 24:1683–1688.
30. Andreu, D., J. Ubach, A. Boman, B. Wahlin, D. Wade, R. B. Merrifield,
size reduction retains potent antibiotic activity. FEBS Lett. 296:190–194.
31. Sato, H., and J. B. Feix. 2006. Osmoprotection of bacterial cells from
toxicity caused by antimicrobial hybrid peptide CM15. Biochemistry.
32. Pace, C. N., F. Vajdos, L. Fee, G. Grimsley, and T. Gray. 1995. How to
measure and predict the molar absorption coefficient of a protein.
Protein Sci. 4:2411–2423.
33. Gennis, R. B. 1989. Biomembranes Molecular Structure and Function.
Springer-Verlag, New York.
34. Stewart, J. C. M. 1980. Colorimetric determination of phospholipids
with ammonium ferrothiocyanate. Anal. Biochem. 104:10–14.
35. Lobley, A., L. Whitmore, and B. A. Wallace. 2002. DICHROWEB: an
interactive website for the analysis of protein secondary structure from
circular dichroism spectra. Bioinformatics. 18:211–212.
36. Bates, I. R., J. M. Boggs, J. B. Feix, and G. Harauz. 2003. Membrane-
anchoring and charge effects in the interaction of myelin basic protein
with lipid bilayers studied by site-directed spin labeling. J. Biol. Chem.
37. Altenbach, C., S. L. Flitsch, H. G. Khorana, and W. L. Hubbell.
1989. Structural studies on transmembrane proteins. 2. Spin labeling
of bacteriorhodopsin mutants at unique cysteines. Biochemistry. 28:
proteins and peptide-membrane interactions. In Biological Magnetic
editor. Plenum Press, New York. 252–281.
39. Altenbach, C., D. A. Greenhalgh, H. G. Khorana, and W. L.
Hubbell. 1994. A collision gradient method to determine the im-
mersion depth of nitroxides in lipid bilayers: application to spin-
labeled mutants of bacteriorhodopsin. Proc. Natl. Acad. Sci. USA.
40. Yu, Y. G., T. Thorgeirsson, and Y.-K. Shin. 1994. Topology of an amphi-
philic mitochondrial signal sequence in the membrane-inserted state:
a spin labeling study. Biochemistry. 33:14221–14226.
41. Langen, R., K. J. Oh, D. Cascio, and W. L. Hubbell. 2000. Crystal
structures of spin labeled T4 lysozyme mutants: implications for the
interpretation of EPR spectra in terms of structure. Biochemistry. 39:
42. Columbus, L., T. Kalai, J. Jeko, K. Hideg, and W. L. Hubbell. 2001.
Molecular motion of spin labeled side chains in alpha-helices: analysis
by variation of side chain structure. Biochemistry. 40:3828–3846.
43. Altenbach, C., K. J. Oh, R. J. Trabanino, K. Hideg, and W. L. Hubbell.
2001. Estimation of inter-residue distances in spin labeled proteins at
physiological temperatures: experimental strategies and practical limita-
tions. Biochemistry. 40:15471–15482.
44. Subczynski, W. K., and J. S. Hyde. 1981. The diffusion-concentration
product of oxygen in lipid bilayers using the spin-label T1 method.
Biochim. Biophys. Acta. 643:283–291.
45. Heller, W. T., A. J. Waring, R. I. Lehrer, T. A. Harroun, T. M. Weiss,
L. Yang, and H. W. Huang. 2000. Membrane thinning effect of the
beta-sheet antimicrobial protegrin. Biochemistry. 39:139–145.
46. Chen, F. Y., M. T. Lee, and H. W. Huang. 2003. Evidence for mem-
brane thinning effect as the mechanism for peptide-induced pore forma-
tion. Biophys. J. 84:3751–3758.
47. Mecke, A., D. K. Lee, A. Ramamoorthy, B. G. Orr, and M. M.
Banaszak Holl. 2005. Membrane thinning due to antimicrobial peptide
binding—an atomic force microscopy study of MSI-78 in lipid bilayers.
Biophys. J. 89:4043–4050.
48. Hung, S. C., W. Wang, S. I. Chan, and H. M. Chen. 1999. Membrane
lysis by the antibacterial peptides cecropins B1 and B3: a spin-label
electron spin resonance study on phospholipid bilayers. Biophys.
49. Ludtke, S. J., K. He, W. T. Heller, T. A. Harroun, L. Yang, and H. W.
Huang. 1996. Membrane pores induced by magainin. Biochemistry.
50. Hallock, K. J., D. K. Lee, and A. Ramamoorthy. 2003. MSI-78, an
analogue of the magainin antimicrobial peptides, disrupts lipid bilayer
structure via positive curvature strain. Biophys. J. 84:3052–3060.
51. Henzler-Wildman, K. A., G. V. Martinez, M. F. Brown, and A.
Ramamoorthy. 2004. Perturbation of the hydrophobic core of lipid
bilayers by the human antimicrobial peptide LL-37. Biochemistry. 43:
52. Rabenstein, M. D., and Y. K. Shin. 1995. Determination of the distance
between two spin labels attached to a macromolecule. Proc. Natl. Acad.
Sci. USA. 92:8239–8243.
53. Pannier, M., S. Veit, A. Godt, G. Jeschke, and H. W. Spiess. 2000.
Dead-time free measurement of dipole-dipole interactions between
electron spins. J. Magn. Reson. 142:331–340.
54. Zhou, Z., S. C. DeSensi, R. A. Stein, S. Brandon, M. Dixit, E. J.
McArdle, E. M. Warren, H. K. Kroh, L. Song, C. E. Cobb, E. J.
Hustedt, and A. H. Beth. 2005. Solution structure of the cytoplasmic
domain of erythrocyte membrane band 3 determined by site-directed
spin labeling. Biochemistry. 44:15115–15128.
1660Pistolesi et al.
Biophysical Journal 93(5) 1651–1660