TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes.
ABSTRACT Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.
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ABSTRACT: Alamethicin is a 19-amino-acid residue hydrophobic peptide of the peptaibol family that has been the object of numerous studies for its ability to produce voltage-dependent ion channels in membranes. In this work, for the first time electron paramagnetic resonance spectroscopy was applied to study the interaction of alamethicin with oriented bicelles. We highlighted the effects of increasing peptide concentrations on both the peptide and the membrane in identical conditions, by adopting a twofold spin labeling approach, placing a nitroxide moiety either on the peptide or on the phospholipids. The employment of bicelles affords additional spectral resolution, thanks to the formation of a macroscopically oriented phase that allows to gain information on alamethicin orientation and dynamics. Moreover, the high viscosity of the bicellar solution permits the investigation of the peptide aggregation properties at physiological temperature. We observed that, at 35°C, alamethicin adopts a transmembrane orientation with the peptide axis forming an average angle of 25° with respect to the bilayer normal. Moreover, alamethicin maintains its dynamics and helical tilt constant at all concentrations studied. On the other hand, by increasing the peptide concentration, the bilayer experiences an exponential decrease of the order parameter, but does not undergo micellization, even at the highest peptide to lipid ratio studied (1:20). Finally, the aggregation of the peptide at physiological temperature shows that the peptide is monomeric at peptide to lipid ratios lower than 1:50, then it aggregates with a rather broad distribution in the number of peptides (from 6 to 8) per oligomer.Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
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ABSTRACT: The structure and energetics of alamethicin Rf30 monomer to nonamer in cylindrical pores of 5 to 11Å radius are investigated using molecular dynamics simulations in an implicit membrane model that includes the free energy cost of acyl chain hydrophobic area exposure. Stable, low energy pores are obtained for certain combinations of radius and oligomeric number. The trimer and the tetramer formed 6Å pores that appear closed while the larger oligomers formed open pores at their optimal radius. The hexamer in an 8Å pore and the octamer in an 11Å pore give the lowest effective energy per monomer. However, all oligomers beyond the pentamer have comparable energies, consistent with the observation of multiple conductance levels. The results are consistent with the widely accepted "barrel-stave" model. The N terminal portion of the molecule exhibits smaller tilt with respect to the membrane normal than the C terminal portion, resulting in a pore shape that is a hybrid between a funnel and an hourglass. Transmembrane voltage has little effect on the structure of the oligomers but enhances or decreases their stability depending on its orientation. Antiparallel bundles are lower in energy than the commonly accepted parallel ones and could be present under certain experimental conditions. Dry aggregates (without an aqueous pore) have lower average effective energy than the corresponding aggregates in a pore, suggesting that alamethicin pores may be excited states that are stabilized in part by voltage and in part by the ion flow itself.Biochimica et Biophysica Acta 09/2013; · 4.66 Impact Factor
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ABSTRACT: Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT1R and BKRB1 or BKRB2 G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S–S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac1-AngII and Toac0-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac3-AngII and Toac3-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT1R and BKRB cyclic constructs based on the structure of the CXCR4, an α-chemokine GPCR-type receptor.Amino Acids 44(3). · 3.91 Impact Factor
TOAC Spin Labels in the Backbone of Alamethicin: EPR Studies
in Lipid Membranes
Derek Marsh,* Micha Jost,yCristina Peggion,yand Claudio Tonioloy
*Max-Planck-Institut fu ¨r biophysikalische Chemie, Abteilung Spektroskopie, Go ¨ttingen, Germany; andyDepartment of Chemistry,
University of Padova, Padova, Italy
membranes. Analogues of the Glu(OMe)7,18,19variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone
have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a
helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and
orientation of the TOACnalamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic14N-hyperfine
couplings indicate that TOAC8and TOAC16are situated in the hydrophobic core of the membrane, whereas the TOAC1label
resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid
correspondingto atransmembrane orientation. Combination ofdatafrom the threespin-labeledpositions yieldsboththe dynamic
order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with
x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to
,30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from
transient or voltage-induced associations.
Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in
Alamethicin is a 19-amino-acid residue peptide from Trich-
oderma viride (with an N-terminal acetyl and a C-terminal
phenylalaninol) that is able to induce voltage-dependent ion
conduction across lipid membranes (1,2). The channel prop-
that consecutive conductance levels are generated by incor-
aggregate (4). The relative populations of the different con-
intrinsic curvature of the constituent lipids (5,6).
TOAC is a helicogenic nitroxyl amino acid that can be
incorporated directly in the backbone of synthetic peptides
(7–10). The nitroxide ring is rigidly attached to the Ca-atom
of the amino acid and therefore can be used as a spin-label
reporter of the orientation and dynamics of the peptide
backbone (11). Previous studies have demonstrated the util-
ity of conventional electron paramagnetic resonance (EPR)
spectroscopy to determine the location and orientation of
TOAC-labeled trichogin GA IV, a membrane-active peptide,
in lipid bilayers (12). Such methods exploit both the polarity
sensitivity (13) and the angular dependence (14) of the
nitroxide EPR spectra.
In this work, we investigate the association of TOAC-
labeled alamethicin analogs with phospholipid bilayer mem-
branes by using both conventional and saturation-transfer
(ST-) EPR spectroscopy. The TOAC residue is substituted at
one of three positions (1, 8, or 16) throughout the sequence
of alamethicin. Macroscopically aligned membranes are used
to demonstrate that the TOAC-labeled alamethicin assumes a
transmembrane orientation, consistent with the relative envi-
ronmental polarities of the different TOAC positions. Orien-
tational order parameters for the three TOAC positions allow
determination of both the angular amplitude of long-axis
motion and the intramolecular tilts of the individual nitro-
xides. Finally, ST-EPR, which is sensitive to much slower
rotational diffusion than conventional EPR (15), and the lack
of spin-spin interactions between monomers in the conven-
tional EPR, are used to obtaininformation on the aggregation
state of the peptide in the membrane.
MATERIALS AND METHODS
Dimyristoyl phosphatidylcholine (DMPC) was from Avanti Polar Lipids
(Alabaster, AL). Spin-labeled derivatives of alamethicin F50/5 [TOACn,
Glu(OMe)7,18,19], with n ¼ 1, 8, and 16, were synthesized according to ref-
Submitted July 4, 2006, and accepted for publication September 14, 2006.
Address reprint requests to Dr. Derek Marsh, Tel.: 49-551-201-1285; E-mail:
Abbreviations used: Ac, acetyl; Aib, a-aminoisobutyric acid; DMPC,
1,2-dimyristoyl-sn-glycero-3-phosphocholine; EDTA, N,N,N9,N9-ethylene-
diaminetetraacetic acid; EPR, electron paramagnetic resonance; Hepes,
N-(2-hydroxyethyl)piperazine-N9-2-ethanesulphonic acid; NHtBu, tert-
butylamino; OMe, methoxy; Phol, phenylalaninol; ST-EPR, saturation
transfer EPR; TOAC, 2,2,4,4-tetramethylpiperidine-1-oxy-4-amino-4-car-
boxylic acid; V1, first-harmonic absorption EPR spectrum detected in phase
with respect to the static magnetic field modulation; V29, second-harmonic
absorption EPR spectrum detected 90? out-of-phase with respect to the
static magnetic field modulation; Z, benzyloxycarbonyl.
? 2007 by the Biophysical Society
Biophysical JournalVolume 92January 2007 473–481473
where Aib is a-aminoisobutyric acid and Phol is the b-amino alcohol
L-phenylalaninol. Functional measurements demonstrate that the Gln7,18,19
to Glu(OMe)7,18,19substitution in F50/5 alamethicin does not dramatically
reduce the voltage-dependent membrane conductance that is induced by this
channel-forming peptide (18).
DMPC (1 mg) and ;1 mol % of the desired TOAC spin-labeled alamethicin
(in MeOH) were codissolved in CH2Cl2, and the solution then evaporated
with dry nitrogen. After keeping under vacuum overnight, the dry mixture
was hydrated in 50 ml of 10 mM Hepes (N-(2-hydroxyethyl)piperazine-N9-2-
ethanesulphonic acid), 10 mM NaCl, 10 mM EDTA (N,N,N9,N9-ethyle
nediaminetetraacetic acid), pH 7.8 buffer, with vortex mixing at 37?C. The
lipid dispersion was then transferred to a 1 mm-diameter glass capillary and
pelleted in a benchtop centrifuge. Excess supernatant was removed and the
capillaries were flame-sealed.
Aligned planar phospholipid bilayers were formed by evaporating the
CH2Cl2solution of DMPC plus 1 mol % of TOAC-alamethicin onto the
internal faces of a quartz flat cell (Wilmad model No. WG-812, Wilmad-
LabGlass, Buena, NJ) by using a stream of dry nitrogen. Residual solvent
was removed under vacuum overnight. The oriented lipid film was hydrated
with excess buffer containing 150 mM NaCl, at room temperature. The cells
were drained and sealed immediately before measurement, with sufficient
buffer retained to ensure complete hydration throughout the experiment.
EPR spectra were recorded on a Varian Century-Line 9-GHz spectrometer
(Varian, Palo Alto, CA) with 100 kHz field modulation. Sample capillaries
were accommodated in standard quartz EPR tubes that contained light
silicone oil for thermal stability. Temperature was regulated by thermostated
thermocouple situated in the silicone oil at the top of the microwave cavity.
Samples of ;5-mm height were centered in the rectangular TE102resonator,
to minimize microwave- and modulation-field inhomogeneities (19). The
microwave H1-field at the sample was measured as described in the latter
reference. Conventional EPR spectra were recorded in the in-phase first-
harmonic absorption mode (V1-display), and saturation transfer (ST-) EPR
spectra in the out-of-phase second-harmonic absorption mode (V29-display)
(20). Oriented bilayer spectra were obtained with the quartz flat cells in a
TE102rectangular microwave cavity mounted with its H1-field axis horizontal.
The entire cavity assembly was thermostated with nitrogen gas-flow.
Conventional EPR spectra were analyzed in terms of the outer and inner
hyperfine splittings, 2Amaxand 2Amin, respectively. The outer hyperfine
splitting is a useful empirical measure of the chain dynamics and ordering
that is valid in both slow and fast motional regimes of nitroxide EPR spec-
troscopy (21,22). In the motional narrowing regime, at high temperature,
Amaxisequalto theparallelelement, A//,of thepartially motionallyaveraged,
axial hyperfine tensor. The perpendicular element, A^, is derived from the
separation, 2Amin, of the inner extrema (23):
A?ðGaussÞ ¼ Amin11:3211:863log10ð1 ? SappÞ
A?ðGaussÞ ¼ Amin10:85forSapp,0:45;
where Sapp¼ ðAmax? AminÞ=½Azz?1
tensor with Cartesian elements (Axx,Ayy,Azz). The environmental polarity was
then characterized by means of the isotropic14N-hyperfine coupling, ao(13),
which is given by
2ðAxx1AyyÞ? for a spin-label hyperfine
In addition, the order parameter of the spin-label z axis, in the fast motional
regime, is given by
where the final factor on the right is a polarity correction to the hyperfine
tensor elements. Values taken for the hyperfine tensor elements are
(Axx,Ayy,Azz) ¼ (6.0 G, 7.3 G, 34.5 G) from 2,2,4,4-tetramethylpiperidine-
1-oxy in a toluene glass (24).
Saturation transfer EPR spectra were analyzed in terms of the diagnostic
line-height ratios, L$/L, C9/C, and H$/H, defined in the low-field, central,
and high-field regions of the spectra, respectively (15), and by the nor-
malized integrated intensity, IST(25). Effective rotational correlation times,
R, were obtained from ST-EPR line-height ratios, R, by using calibrations
with spin-labeled hemoglobin in solutions of known viscosity from Horva ´th
and Marsh (25). The calibrations can be expressed as (26)
R¼ k=ðR ? RoÞ ? b;
where Rois the rigid-limit value of R. The calibration constants k, b, and Ro
are given in Marsh (27). For shorter correlation times, polynomial ST-EPR
calibrations were used from Horva ´th and Marsh (28).
The crystal structure of the6T2twist-boat conformer of TOAC was taken
from molecule B of Z-TOAC-(L-Ala)2-NHtBu (29), which was obtained
from the Cambridge Crystallographic Data Centre (CCDC code: 123753). In
the available crystal structures of a-helical TOAC peptides,6T2is by far the
most prevalent conformer of TOAC (30). The crystal structure of native
alamethicin (31) was obtained from the Research Collaboration for Struc-
tural Bioinformatics protein database (32) (PDB code: 1amt). The TOAC resi-
due was substituted for the Aib residue at position 1, 8, or 16 in alamethicin
by constraining the transformed coordinates of the TOAC N, Caand C9 atoms
to coincide with those in alamethicin, by using nonlinear least-squares
The orientation uzof the nitroxide z axis of TOAC to the alamethicin
molecular axis was determined as described in Marsh (30). The molecular
axis of alamethicin was taken as the axis of the longer (N-terminal) helical
section. The latter was defined as the line equidistant from the Caatoms of
residues 4–14, for which the mean radial distance is 2.38 A˚, by nonlinear
least-squares fitting. The vector connecting the Caatoms of residues 1 and
19 was also used as an alternative definition of the molecular axis. The un-
published structure of [TOAC16, Glu(OMe)7,18,19]-alamethicin (33), and
variants in which the TOAC residue from position 16 was substituted for the
Aib1or Aib8residue, were used in an analogous manner to obtain the
orientation of the TOAC nitroxyl axes. This alamethicin analog has the6T2
twist-boat conformer of TOAC that is found in the Z-TOAC-(L-Ala)2-
NHtBu reference peptide.
RESULTS AND DISCUSSION
Conventional spin-label EPR spectra
Fig. 1 shows the EPR spectra of the three different TOAC1,
474Marsh et al.
Biophysical Journal 92(2) 473–481
in DMPC bilayer membranes. Spectra are shown at various
temperatures, both above and below the lipid chain-melting
temperature of 23?C. In the gel phase, the spectra are in-
dicative of strong immobilization on the nanosecond time-
scale, but that of the TOAC1derivative also evidences strong
spin-spin broadening. The latter is seen most clearly at 10?C
as a strong distortion of the baseline by a very broad un-
derlying component (dotted-line spectrum). This spin-spin
interaction is caused by aggregation of the spin-labeled
alamethicin in the gel-phase membrane. The sharp features
in the low-temperature spectra most probably arise from a
population of noninteracting spin labels, and therefore in-
dicate some heterogeneity in the degree of aggregation. The
aggregation observed by spin-spin interaction in the gel-
phase correlates well with functional studies on the effects of
the lipid chain-melting transition. The transmembrane cur-
rent density mediated by alamethicin in unsupported bilayers
of 1-stearoyl-3-myristoylphosphatidylcholine was found to
increase dramatically on entering the gel phase of the mem-
brane from the fluid phase (34). The current density in the gel
phase at 24?C corresponded to a pore concentration of ;106
pores/cm2and decreased to a low level representing only
1 pore/cm2in the fluid phase. Increased pore density implies
an increased degree of peptide aggregation and an increased
local concentration of alamethicin in the gel phase that
manifests itself here as an increase in spin-spin interactions.
Spin-spin broadening is absent from the EPR spectra in
the fluid phase at 1 mol % spin label. This finding indicates
that the spin-labeled alamethicin is randomly dispersed in
the lipid membrane at temperatures above the lipid chain-
melting transition. Similar conclusions have been reached
from EPR studies on alamethicin with a flexible spin label
at the N- or C-terminus (35,36). The spectra from the three
TOAC derivatives are still highly anisotropic, indicating high
ordering or limited motion, in the fluid phase. The extent
of spectral anisotropy decreases with increasing temperature.
At higher temperatures, the spectra display axial motional
averaging, as indicated by the well-defined outer and inner
hyperfine splittings, 2Amaxand 2Amin, respectively (see, e.g.,
Fig. 2 shows the temperature dependence of the outer hyper-
fine splitting, 2Amax, for alamethicin with the three different
positions of TOAC labeling in DMPC membranes. For the
TOAC8and TOAC16derivatives, there is a small but abrupt
decrease in the value of Amaxat the DMPC chain-melting
transition. This decrease in Amaxcorresponds to an increase
in rotational dynamics of alamethicin on lipid chain fluid-
ization. The apparent increase in Amaxfor the TOAC1deriv-
ative at 23?C most probably is an artifact arising from the
spin-spin broadening of this particular label in the gel phase.
The values of Amaxat temperatures immediately above the
lipid transition are still indicative of a high degree of order, or
limited amplitude of angular motion, of the spin-labeled
alamethicin. There are, nonetheless, differences between the
values of Amaxand their rate of change with temperature for
the three different label positions. For each label, the values
of Amaxdecrease steadily in response to the increased extent
of lipid chain motion with increasing temperature.
Isotropic hyperfine couplings
Fig. 3 gives the temperature dependence of the effective
isotropic hyperfine couplings, ao, defined by Eq. 3, for the
different positions of TOAC labeling. The true value of ao
should depend only on the polarity of the environment in
which the spin label is situated (see, e.g., (38,39)), and not on
the molecular motion. Therefore, it is expected to be ap-
proximately independent of temperature. Artifactual in-
creases in effective values with decreasing temperature are
found at lower temperatures, due to the breakdown of
motional narrowing theory (on which Eqs. 3 and 4 are based)
caused by slow-motional contributions to the spectra at the
lower temperatures. This provides a useful experimental cri-
terion by which to define the temperature region over which
motional narrowing theory is valid. Fig. 3 shows that the
values of aoare practically constant for the TOAC1and
TOAC8analogs at temperatures of 55?C or higher. Only
for the TOAC16analog is the temperature dependence
tra (V1-display) of [Glu(OMe)7,18,19]
alamethicin analogs with TOAC substi-
tuted for (A) residue 1, TOAC1; (B)
residue 8, TOAC8; and (C) residue 16,
TOAC16, in DMPC bilayers at the tem-
peratures indicated. (Solid lines, total
scan width ¼ 100 Gauss; dotted lines,
total scan width: 160 Gauss.)
Conventional EPR spec-
TOAC-Alamethicin in Membranes 475
Biophysical Journal 92(2) 473–481
fine couplings are: ao¼ 15.41 6 0.01 G, 15.01 6 0.01 G,
and 15.03 6 0.07 G, for the TOAC1, TOAC8, and TOAC16
analogs, respectively. The lower values indicate that the
TOAC8and TOAC16analogs are situated considerably
deeper in the hydrophobic core of the membrane than is the
TOAC1analog. For the TOAC16analog, only the values at
higher temperature were taken and are associated with a
higheruncertainty,asindicated.Fora TOACpeptide inwater,
the isotropic hyperfine coupling is considerably higher: ao¼
16.2–16.5 G, depending on the pH/charge state of the peptide
(40). This implies that the TOAC1analog is not directly
exposed to water, but is probably located close to the polar-
apolar interface of the membrane, as suggested also for
surface-associated trichogin GA IV and melanocortin (41,42).
Note that a higher water exposure of TOAC1than of TOAC16
(at high temperature) does not exclude the possibility of a
yet greater water exposure of a spin label attached to the
C-terminal phenylalaninol, especially at lower temperatures
Orientational order parameters
Fig. 4 gives the temperature dependence of the order param-
eters, Szz, for the three TOAC analogs of alamethicin in
DMPC bilayer membranes in the fluid phase. According to
the criterion of constant ao, the data in Fig. 3 suggest that
motional narrowing theory should be applicable above 55?C
for all except the TOAC16analog. As already noted, the
spectra are axially symmetric in this temperature regime (see
Fig. 1). The values given in Fig. 4 therefore should be rea-
sonably representative of the time-average angular amplitude
of the spin-label z axis, relative to the director for the uniaxial
rotational motion. At temperatures below those for which data
is given in Fig. 4, motional narrowing theory can no longer be
relied upon for determining order parameters.
Fig. 5 shows the conventional EPR spectra of the three
TOAC-containing analogs of alamethicin in aligned multi-
bilayers of fully hydrated DMPC in the fluid phase. Spectra
are shown for the static magnetic field parallel (solid lines)
and perpendicular (dashedlines) tothe normalto the orienting
quartz substrate on which the multibilayers are deposited.
Although the degree of alignment of the sample is probably
not completely homogeneous, and at these temperatures (30–
32?C) the rotational motion is not yet in the fast regime
(especially for the TOAC16analog), there is clear anisotropy
between spectra recorded with the parallel and perpendicular
orientations of the magnetic field. For all three TOAC posi-
tions, the largest hyperfine splitting of the first derivative-like
stant, Amax, for [Glu(OMe)7,18,19] alamethicin TOAC1(squares), TOAC8
(circles), and TOAC16(triangles) analogs in DMPC bilayers.
Temperature dependence of the outer hyperfine splitting con-
couplings, ao, for [Glu(OMe)7,18,19] alamethicin TOAC1(squares), TOAC8
(circles), and TOAC16(triangles) analogs in DMPC bilayers.
Temperature dependence of the effective isotropic hyperfine
Szz, for [Glu(OMe)7,18,19] alamethicin TOAC1(squares), TOAC8(circles),
and TOAC16(triangles) analogs in DMPC bilayers. Solid lines are experi-
mental measurements; dotted lines are a nonlinear least-squares fit of Eq. 6
to the temperature dependence, with constant uzfor each TOAC position
(see text and Fig. 6).
Temperature dependence of the effective order parameters,
476Marsh et al.
Biophysical Journal 92(2) 473–481
single absorption lines is obtained in the parallel orientation
with the magnetic field lying along the substrate normal (see,
e.g., (14)). This finding confirms that the director, N, for the
uniaxial rotation lies along the membrane normal, consistent
with a transmembrane orientation of the alamethicin molecule
as indicated by the isotropic hyperfine couplings.
For uniaxial motional averaging, the EPR order parameter
of TOAC is given by the addition theorem for spherical
where g is the angle that the principal rotational diffusion
axis, R, of the TOAC-labeled alamethicin molecule makes
with the membrane normal, N, and uzis the inclination of the
spin-label z axis to R (see Fig. 6). P2(x) ¼ (3x2?1)/2 is a
second-order Legendre polynomial and the angular brackets
indicate a time average over the rotational motion. Because
the TOAC spin label is rigidly attached to the peptide back-
bone, the different values of Szzin Fig. 4 imply different
orientations, uz, of the three TOAC residues to R. The crystal
structure of native alamethicin reveals an a-helical conforma-
tion thatis bent (31). This bend, togetherwith localdistortions,
could account for the dependence of the TOAC order param-
eters on residue position.
The dotted lines in Fig. 4 represent a nonlinear least-
squares fit of Eq. 6 to the temperature dependence of Szz
for all three TOAC labels, under the assumption that the
spin-label inclination to the diffusion axis is temperature-
independent. The order parameter of the diffusion axis, rela-
tive to N, then varies from ÆP2(cosg)æ ¼ 0.87–0.70 over the
temperature range 60–85?C. The local orientation of the indi-
vidual spin labels is characterized by the fixed values uz¼
30?, 25?, and 20? for TOAC1, TOAC8, and TOAC16, re-
spectively. Judging from the goodness of the fits in Fig. 4,
only for TOAC1are there significant changes in uzwith
temperature, possibly corresponding to a local unwinding of
the helix or other conformational reorientation at the first
TOAC orientation in alamethicin
Fig. 7 shows one of the molecules (A) in the crystal structure
of native alamethicin (31) into which the crystal structure of
the TOAC moiety from molecule B of Z-TOAC-(L-Ala)2-
NHtBu (29) has been incorporated at residue position 1, 8, or
16. This was done by constraining the coordinates of the
alamethicin analogs in aligned DMPC bilayers. (Solid lines, magnetic field
parallel to the membrane normal; dashed lines, magnetic field perpendicular
to the membrane normal.) (A) TOAC1-alamethicin at 30?C; (B) TOAC8-
alamethicin at 32?C; and (C) TOAC16-alamethicin at 32?C. Total scan
width ¼ 100 Gauss.
Conventional V1-EPR spectra of [TOACn, Glu(OMe)7,18,19]
brane. The principal molecular diffusion axis, R, is inclined at instantaneous
angle g to the membrane normal N. The nitroxide z axis is oriented at con-
stant angle uzto R. The experimental order parameter, Szz, of the nitroxide
z axis is given by Eq. 6, where, for axial symmetry, ÆP2(cosg)æ is the order
parameter of the alamethicin diffusion axis.
Orientation of TOAC-labeled alamethicin in a lipid mem-
TOAC-Alamethicin in Membranes477
Biophysical Journal 92(2) 473–481
TOAC N, Ca, and C9 atoms to coincide with those of Aib1,
Aib8, or Aib16in alamethicin. If the axis of the longer helical
segment is defined as the line that is equidistant from the Ca
atoms of residues 4–14, the inclination of the nitroxide z axis
to this axis is ua¼ 7?, 15?, and 34?, for TOAC at residue
positions 1, 8, and 16, respectively. For the recently solved
structure of [TOAC16, Glu(OMe)7,18,19] alamethicin (33),
the orientation of the spin-label z axis to the longer helical
axis is ua¼ 10–12? and, for this TOAC structure grafted
at residue positions 1 and 8, is ua¼ 4–7? and 8–9?, re-
spectively. In terms of residue position, these values for the
TOAC orientation uaare in the opposite order to those of uz
that are derived from the EPR results. From this, one must
conclude that the diffusion axis does not coincide with the
helical axis between residues 4 and 14, as defined above.
Taking the more recent crystal structure, the rotation axis R
is tilted relative to the principal helix axis by ;30?. This
value may be somewhat of an upper estimate because of the
effects of local helix distortions that were referred to above.
Note that taking the mirror-image2T6twist-boat conformer
of TOAC would predict nitroxide z-axis orientations that are
incompatible with the EPR order-parameter measurements
Saturation transfer spin-label EPR spectra
Fig. 8 shows the saturation transfer EPR spectra of the
three different TOAC1, TOAC8, and TOAC16analogs of
[Glu(OMe)7,18,19] alamethicin in DMPC bilayer membranes.
Spectra are shown at various temperatures, both above and
below the lipid chain-melting temperature of 23?C. The
spectra are scaled to line height, rather than to the absolute
intensity, which decreases with increasing temperature. In
the gel phase, the ST-EPR spectra have appreciable intensity,
not only in the overall spectrum, but also in the diagnostic
regions at intermediate positions in the low-, high-, and
center-field manifolds of the14N-hyperfine structure. These
nonvanishing ST-EPR intensities reflect the response of the
peptide to the extremely slow rotational diffusion of the
gel-phase lipids, with effective correlation times beyond the
microsecond regime (45,46). Immediately above the lipid
chain-melting transition, the overall intensity of the ST-EPR
spectrum drops abruptly and the spectral lineshape changes
because of preferential reduction in the line heights at the
diagnostic L99, C9, and H99 positions relative to the stationary
turning points at positions L, C and H, respectively (see, e.g.,
(15)). These spectra are characteristic of very fast motion, no
longer in the microsecond regime (at least for the central C9
region of the spectrum), and reflect the response of the
peptide mobility to the rapid lipid chain motions in the fluid
membrane phase (45,47).
Fig. 9 gives the temperature dependence of the normalized
ST-intensity, IST, and the diagnostic line-height ratios, L99/L,
C9/C, and H99/H, for the TOAC8alamethicin analog in
DMPC membranes. This is the analog with highest ST-EPR
intensities and, therefore, that most likely to exhibit any
microsecond motions that are associated with the whole
peptide. All four ST-EPR parameters clearly reflect the
change in overall peptide dynamics at the gel-fluid phase
transition, which occurs at ;23?C. The values of the C9/C
line-height ratio and of the ST integral are very low in the
fluid phase, beyond those for which ST-EPR calibrations
were made. This corresponds to an effective rotational
correlation time of ,2.9 3 10?8s (28). In addition, the L99/L
ratio is seen to increase with increasing temperature, i.e.,
with decreasing correlation time. This is a feature of incipient
motional narrowing in ST-EPR spectra (15), that again is
consistent with correlation times of ,10?7s. Such rapid
rotational reorientation suggests rather strongly that the
peptide is not aggregated in fluid DMPC membranes, as
was concluded already from the lack of spin-spin broadening
of the conventional EPR spectra. Standard hydrodynamic
theory (see, e.g., (48)) predicts a rotational correlation time
of 1.4–2.8 3 10?7s for a single transmembrane a-helix in a
membrane of effective viscosity 2.5–5 P (49). For a dimer,
this value increases to 3.5–7.0 3 10?7s, and for a tetramer to
5.6–11.3 3 10?7s. It therefore seems most likely that the
TOAC alamethicin analogs are monomeric (at a concentration
ecule A of native alamethicin ((31);
PDB: 1amt) with the TOAC structure
from molecule B of Z-TOAC-(L-Ala)2-
NHtBu ((29) CCDC: 123753) substi-
tuted for Aib at residue position 1, 8, or
16. The alamethicin molecule is ori-
ented relative to the membrane surfaces
as predicted in the OPM database (53).
Crystal structure for mol-
478Marsh et al.
Biophysical Journal 92(2) 473–481
of 1 mol %) in fluid DMPC bilayers, as suggested previously
foralamethicin in vesicles ofunsaturatedphosphatidylcholines
(35,36). Note that substitution of Gln residues, particularly
the conserved Gln7, by Glu(OMe) might reduce somewhat the
propensity of alamethicin to form pores, as suggested by the
lower conductance (18).
ties of the environments of the different TOAC positions
demonstrate that Glu(OMe)7,18,19alamethicin adopts a trans-
membrane orientation in fluid bilayer membranes of DMPC.
This is in agreement with other spectroscopic studies on un-
modified and chemically labeled alamethicins (43,50). Cer-
tain models for the induction of ion channels propose a
switching of the alamethicin long-axis from a surface to a
transmembrane orientation (see, e.g., (2)). The present
results show that the bulk of the peptide has a transmembrane
orientation and therefore such channels are most likely
formed by self-assembly within the membrane.
The combined order parameter measurements from the
different TOAC positions indicate that the tilt of the long
axis of the peptide, relative to the membrane normal, is fairly
small with values of ÆP2(cosg)æ corresponding to effective
tilt angles of 17–27? over the temperature range 60–85?C. It
is expected that the tilt of alamethicin is restricted because
the length of the molecule (;29 A˚from Caof residue 1 to
Caof residue 19) is relatively short compared with the
thickness of a DMPC bilayer. For the latter, the hydrophobic
thickness is ;26 A˚and the total thickness is ;37 A˚at 30?C,
which extrapolate to 21 A˚and 30 A˚, respectively, at 85?C
using an expansion coefficient of ?0.004 per degree (51).
Orientation of alamethicin according to the distribution of
polarity/hydrophobicity in the molecule, as reported in the
OPM database (see Fig. 7), predicts a transmembrane align-
ment of alamethicin with a hydrophobic depth of 28 A˚and a
tilt of 16 6 8? (52,53). This theoretical predictionis therefore
essentially in accord with the present experimental measure-
An interesting feature of the angular motion of the TOAC
spin labels, relative to that of spin-labeled lipid chains (see,
e.g., (54)), is that the rotational diffusion is slow on the EPR
timescale (;ns) in fluid membranes, except at rather high
[Glu(OMe)7,18,19] alamethicin analogs with TOAC substituted for (A)
residue 1, TOAC1; (B) residue 8, TOAC8; and (C) residue 16, TOAC16, in
DMPC bilayers at the temperatures indicated. Total scan width ¼ 160
Saturation transferEPRspectra(V29-display) of
circles), and diagnostic line-height ratios, L99/L (squares), C9/C (solid
circles), and H99/H (triangles), from the ST-EPR spectra of [TOAC8,
Glu(OMe)7,18,19] alamethicin in DMPC bilayers.
Temperature dependence of the integrated intensity, IST(open
TOAC-Alamethicin in Membranes479
Biophysical Journal 92(2) 473–481
temperatures (.60?C). This reflects the rigidity of the helical
backbone of alamethicin and the anchoring of the TOAC
ring at the Ca-position of the helix.
On the longer (ms) timescale of ST-EPR, however, rotation
about the long axis of alamethicin is relatively rapid. This
means that alamethicin is not forming large pore aggregates,
which would have rotational correlation times in the micro-
second regime. Most likely, the bulk of the alamethicin is
monomeric in the fluid membrane, and pore formation (and
growth) occurs via transient association of the monomeric
species. This is in accordance with the electrophysiological
channel behavior and proposals from other spectroscopic
We thank Frau B. Angerstein and Frau B. Freyberg for skillful technical
assistance, Dr. Marco Crisma for providing the coordinates of [TOAC16,
Glu(OMe)7,18,19] alamethicin, and Frau I. Dreger for help with preparing
1. Nagaraj, R., and P. Balaram. 1981. Alamethicin, a transmembrane
channel. Acc. Chem. Res. 14:356–362.
2. Sansom, M. S. 1991. The biophysics of peptide models of ion chan-
nels. Prog. Biophys. Mol. Biol. 55:139–235.
3. Gordon, L. G. M., and D. A. Haydon. 1972. The unit conductance
channel of alamethicin. Biochim. Biophys. Acta. 255:1014–1018.
4. Boheim, G., and H.-A. Kolb. 1978. Analysis of the multi-pore system
of alamethicin in a lipid membrane. I. Voltage-jump current-relaxation
measurements. J. Membr. Biol. 38:99–150.
5. Opsahl, L. R., and W. W. Webb. 1994. Transduction of membrane
tension by the ion-channel alamethicin. Biophys. J. 66:71–74.
6. Keller, S. L., S. M. Bezrukov, S. M. Gruner, M. W. Tate, I. Vodyanoy,
and V. A. Parsegian. 1993. Probability of alamethicin conductance states
varies with nonlamellar tendency of bilayer phospholipids. Biophys. J.
7. Nakaie, C. R., G. Goissis, S. Schreier, and A. C. M. Paiva. 1981. pH-
dependence of electron paramagnetic resonance spectra of nitroxides
containing ionizable groups. Braz. J. Med. Biol. Res. 14:173–180.
8. Nakaie, C. R., S. Schreier, and A. C. M. Paiva. 1983. Synthesis and
properties of spin-labeled angiotensin derivatives. Biochim. Biophys.
9. Marchetto, R., S. Schreier, and C. R. Nakaie. 1993. A novel spin-labeled
amino acid derivative for use in peptide synthesis—(9-fluorenylmethy-
ylic acid. J. Am. Chem. Soc. 115:11042–11043.
10. Toniolo, C., M. Crisma, and F. Formaggio. 1998. TOAC, a nitroxide
spin-labeled, achiral Ca-tetrasubstituted a-amino acid, is an excellent
tool in materials science and biochemistry. Biopolymers. 47:153–158.
11. Karim, C. B., T. L. Kirby, Z. Zhang, Y. Nesmelov, and D. D. Thomas.
2004. Phospholamban structural dynamics in lipid bilayers probed by a
spin label rigidly coupled to the peptide backbone. Proc. Natl. Acad.
Sci. USA. 101:14437–14442.
12. Monaco, V., F. Formaggio, M. Crisma, C. Toniolo, P. Hanson, and
G. L. Millhauser. 1999. Orientation and immersion depth of a helical
lipopeptaibol in membranes using TOAC as an ESR probe. Biopolymers.
13. Marsh, D. 2001. Polarity and permeation profiles in lipid membranes.
Proc. Natl. Acad. Sci. USA. 98:7777–7782.
14. Schreier-Muccillo, S., D. Marsh, H. Dugas, H. Schneider, and I. C. P.
Smith. 1973. A spin probe study of the influence of cholesterol on
motion and orientation of phospholipids in oriented multibilayers and
vesicles. Chem. Phys. Lipids. 10:11–27.
15. Thomas, D. D., L. R. Dalton, and J. S. Hyde. 1976. Rotational diffusion
studied by passage saturation transfer electron paramagnetic resonance.
J. Chem. Phys. 65:3006–3024.
16. Jost, M., C. Peggion, F. Formaggio, and C. Toniolo. 2006. Total synthesis
in solution and preliminary conformational analysis of TOAC-labeled
alamethicin F50/5 analogues. In Understanding Biology Using Peptides.
S. E. Blondelle, editor. American Peptide Society, Secaucus, NJ. 263–264.
17. Peggion, C., I. Coin, and C. Toniolo. 2004. Total synthesis in solution
of alamethicin F50/5 by an easily tunable segment condensation approach.
18. Baldini, C., C. Peggion, C. Toniolo, N. Vedovato, and G. Rispoli.
2007. Biophysical properties of alamethicin F50/5 and selective ana-
logues inserted in rod outer segment membranes. In Peptides 2006. K.
Rolka, editor. Kenes International, Geneva. In press.
19. Fajer, P., and D. Marsh. 1982. Microwave and modulation field
inhomogeneities and the effect of cavity Q in saturation transfer ESR
spectra. Dependence on sample size. J. Magn. Reson. 49:212–224.
20. Hemminga, M. A., P. A. De Jager, D. Marsh, and P. Fajer. 1984.
Standard conditions for the measurement of saturation transfer ESR
spectra. J. Magn. Reson. 59:160–163.
21. Rama Krishna, Y. V. S., and D. Marsh. 1990. Spin label ESR and
31P-NMR studies of the cubic and inverted hexagonal phases of
dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixtures.
Biochim. Biophys. Acta. 1024:89–94.
22. Schorn, K., and D. Marsh. 1996. Lipid chain dynamics and molecular
location of diacylglycerol in hydrated binary mixtures with phospha-
tidylcholine: spin label ESR studies. Biochemistry. 35:3831–3836.
23. Schorn, K., and D. Marsh. 1997. Extracting order parameters from
powder EPR lineshapes for spin-labeled lipids in membranes. Spec-
trochim. Acta [A]. 53:2235–2240.
24. Ondar, M. A., O. Ya. Grinberg, A. A. Dubinskii, and Ya. S. Lebedev.
1985. Study of the effect of the medium on the magnetic-resonance
parameters of nitroxyl radicals by high-resolution EPR spectroscopy.
Sov. J. Chem. Phys. 3:781–792.
25. Horva ´th, L. I., and D. Marsh. 1983. Analysis of multicomponent
saturation transfer ESR spectra using the integral method: application
to membrane systems. J. Magn. Reson. 54:363–373.
26. Marsh, D., and L. I. Horva ´th. 1992. A simple analytical treatment of
the sensitivity of saturation transfer EPR spectra to slow rotational
diffusion. J. Magn. Reson. 99:323–331.
27. Marsh, D. 1999. Spin label ESR spectroscopy and FTIR spectroscopy
for structural/dynamic measurements on ion channels. Methods Enzymol.
28. Horva ´th, L. I., and D. Marsh. 1988. Improved numerical evaluation of
saturation transfer electron spin resonance spectra. J. Magn. Reson. 80:
29. Flippen-Anderson, J. L., C. George, G. Valle, E. Valente, A. Bianco,
F. Formaggio, M. Crisma, and C. Toniolo. 1996. Crystallographic
characterization of geometry and conformation of TOAC, a nitroxide
spin-labeled Ca,a-disubstituted glycine, in simple derivatives and model
peptides. Int. J. Pept. Protein Res. 47:231–239.
30. Marsh, D. 2006. Orientation of TOAC amino-acid spin labels in
a-helices and b-strands. J. Magn. Reson. 180:305–310.
31. Fox, R. O., Jr., and F. M. Richards. 1982. A voltage-gated ion channel
model inferred from the crystal structure of alamethicin at 1.5 A˚reso-
lution. Nature. 300:325–330.
32. Berman, H. M., J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat,
H. Weissig, I. N. Shindyalov, and P. E. Bourne. 2000. The protein data
bank. Nucleic Acids Res. 28:235–242.
33. Crisma, M., F. Formaggio, M. Jost, C. Peggion, and C. Toniolo. 2005.
Crystal structure of a spin-labeled alamethicin analogue. In 1st European
Conference on Chemistry for Life Sciences: Understanding the Chemical
Mechanisms of Life. Book of Abstracts, DCSB 234. Rimini, Italy.
34. Boheim, G., W. Hanke, and H. Eibl. 1980. Lipid phase transition in
planar bilayer membrane and its effect on carrier- and pore-mediated
ion transport. Proc. Natl. Acad. Sci. USA. 77:3403–3407.
480 Marsh et al.
Biophysical Journal 92(2) 473–481
35. Archer, S. J., J. F. Ellena, and D. S. Cafiso. 1991. Dynamics and
aggregation of the peptide ion channel alamethicin. Biophys. J. 60:
36. Barranger-Mathys, M., and D. S. Cafiso. 1994. Collisions between he-
lical peptides in membranes monitored using electron paramagnetic
resonance: evidence that alamethicin is monomeric in the absence of a
membrane potential. Biophys. J. 67:172–176.
37. Marsh, D. 1981. Electron spin resonance: spin labels. In Membrane
Spectroscopy. Molecular Biology, Biochemistry and Biophysics, Vol.
31. E. Grell, editor. Springer-Verlag, Berlin, Heidelberg, New York.
38. Marsh, D. 2002. Membrane water-penetration profiles from spin labels.
Eur. Biophys. J. 31:559–562.
39. Marsh, D. 2002. Polarity contributions to hyperfine splittings of
hydrogen-bonded nitroxides—the microenvironment of spin labels.
J. Magn. Reson. 157:114–118.
40. Schreier, S., S. R. Barbosa, F. Casallanovo, R. Vieira, E. M. Cilli,
A. C. M. Paiva, and C. R. Nakaie. 2004. Conformational basis for the
biological activity of TOAC-labeled angiotensin II and bradykinin:
electron paramagnetic resonance, circular dichroism, and fluorescence
studies. Biopolymers. 74:389–402.
41. Monaco, V., F. Formaggio, M. Crisma, C. Toniolo, P. Hanson, and
G. Millhauser. 1999. Orientation and immersion depth of a helical
lipopeptaibol in membranes using TOAC as an ESR probe. Biopolymers.
42. Fernandez, R. M., R. F. F. Vieira, C. R. Nakaie, M. T. Lamy, and A. S.
Ito. 2005. Acid-base titration of melanocortin peptides: evidence of Trp
rotational conformer interconversion. Biopolym. Pept. Sci. 80:643–650.
43. Barranger-Mathys, M., and D. S. Cafiso. 1996. Membrane structure of
voltage-gated channel forming peptides by site-directed spin-labeling.
44. Hanson, P., D. J. Anderson, G. Martinez, G. Millhauser, F. Formaggio,
M. Crisma, C. Toniolo, and C. Vita. 1998. Electron spin resonance and
structural analysis of water soluble, alanine-rich peptides incorporating
TOAC. Mol. Phys. 95:957–966.
45. Marsh, D. 1980. Molecular motion in phospholipid bilayers in the gel
phase: long axis rotation. Biochemistry. 19:1632–1637.
46. Fajer, P., A. Watts, and D. Marsh. 1992. Saturation transfer, continu-
ous wave saturation, and saturation recovery electron spin resonance
studies of chain-spin labeled phosphatidylcholines in the low temperature
phases of dipalmitoyl phosphatidylcholine bilayers. Effects of rotational
dynamics and spin-spin interactions. Biophys. J. 61:879–891.
47. Bartucci, R., T. Pa ´li, and D. Marsh. 1993. Lipid chain motion in an
interdigitated gel phase: conventional and saturation transfer ESR of
spin-labeled lipids in dipalmitoylphosphatidylcholine-glycerol disper-
sions. Biochemistry. 32:274–281.
48. Marsh, D., and L. I. Horva ´th. 1989. Spin-label studies of the structure
and dynamics of lipids and proteins in membranes. In Advanced EPR.
Applications in Biology and Biochemistry. A. J. Hoff, editor. Elsevier,
Amsterdam, The Netherlands. 707–752.
49. Cherry, R. J., and R. E. Godfrey. 1981. Anisotropic rotation of bacte-
riorhodopsin in lipid membranes. Biophys. J. 36:257–276.
50. Vogel, H. 1987. Comparison of the conformation and orientation
of alamethicin and melittin in lipid membranes. Biochemistry. 26:
51. Nagle, J. F., and S. Tristram-Nagle. 2000. Structure of lipid bilayers.
Biochim. Biophys. Acta. 1469:159–195.
52. Lomize, A. L., I. D. Pogozheva, M. A. Lomize, and H. I. Mosberg.
2006. Positioning of proteins in membranes: a computational approach.
Protein Sci. 15:1318–1333.
53. Lomize, M. A., A. L. Lomize, I. Pogozheva, and H. I. Mosberg. 2006.
OPM: orientations of proteins in membranes database. Bioinformatics.
54. Schorn, K., and D. Marsh. 1996. Lipid chain dynamics in diacylglycerol-
phosphatidylcholine mixtures studied by slow-motional simulations of
spin label ESR spectra. Chem. Phys. Lipids. 82:7–14.
TOAC-Alamethicin in Membranes481
Biophysical Journal 92(2) 473–481