Orientation of spin labels in de novo peptides

Article abstract of DOI:10.1002/mrc.1692

A series of de novo synthesised peptides including the artificial rigid paramagnetic amino acid TOAC at two positions with different distances from two to seven in the primary structure have been investigated by 9- and 94-GHz EPR spectroscopy under solid-state conditions. From simulations of the spectra of such two-spin systems, the distance and relative orientation of the paramagnetic centres can be deduced. This yields structural information on the peptides. A quantitative analysis of the spectra of individual peptides in different solvents as well as a qualitative analysis of the spectra of the peptide series shows that the peptides do not assume conformations corresponding to any of the common helical structures in proteins. Copyright

Full text of DOI:10.1002/mrc.1692

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2005; 43: S26–S33
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1692
Orientation of spin labels in de novo peptides†
1
2
2
1∗
¨
Celine Elsaßer, Bernhard Monien, Wolfgang Haehnel and Robert Bittl
1
Freie Universita¨ t Berlin, Institut fu¨ r Experimentalphysik, Arnimallee 14, 14195 Berlin, Germany
Universita¨ t Freiburg, Institut fu¨ r Biologie II/Biochemie, Scha¨ nzlestrasse 1, 79104 Freiburg, Germany
2
Received 4 May 2005; Revised 17 June 2005; Accepted 17 June 2005
A series of de novo synthesised peptides including the artificial rigid paramagnetic amino acid TOAC at
two positions with different distances from two to seven in the primary structure have been investigated
by 9- and 94-GHz EPR spectroscopy under solid-state conditions. From simulations of the spectra of such
two-spin systems, the distance and relative orientation of the paramagnetic centres can be deduced. This
yields structural information on the peptides. A quantitative analysis of the spectra of individual peptides
in different solvents as well as a qualitative analysis of the spectra of the peptide series shows that the
peptides do not assume conformations corresponding to any of the common helical structures in proteins.
Copyright  2005 John Wiley & Sons, Ltd.
KEYWORDS: EPR; multi-frequency; de novo peptides; spin labels; dipolar coupling
unique distance and orientation with respect to each other if
all molecular motions have correlation times that are large
compared to the timescale of EPR. This requires a rigid spin
label with defined orientation to the protein backbone and
solid-state conditions.
INTRODUCTION
EPR in conjunction with spin labelling provides the oppor-
tunity to determine molecular structures.1–3 In particular,
distance measurements between two-spin labels4 have been
widely used for investigations of protein structure.5–7 Most
commonly used spin labels for such studies are those of the
MTS family, which can be covalently attached to cysteine
amino acid residues.8 The link between the piperidine-oxyl
moiety and the protein backbone renders the label flexible.
This usually allows native folding of the protein because
the label can evade the steric restrictions of the protein. On
the other hand, the labels have structural variability over
Chemical synthesis of peptides12 provides the opportu-
nity to incorporate rigid (non-natural) spin-labelled amino
acids into peptides.13 The spin-label TOAC14 is an artifi-
cial amino acid and has only one degree of freedom, the
conformation of the six-membered ring (Scheme 1). This
label has repeatedly been used for the investigations of sec-
ondary structures of small peptides13,15–17 taking advantage
of the fact that TOAC narrows the distance distribution
significantly. However, these experiments were primarily
performed in liquid solutions with the proteins rotating
freely and therefore, only the isotropic parts of the exchange
coupling could be measured, while anisotropic parts as well
as the purely anisotropic dipolar coupling are averaged out.
Consequently, the orientation of the labels could not be
determined.
In this work, we investigate the potential to extract
not only the distance but also the complete orientational
information from EPR spectra of such doubly spin-labelled
peptides. By using the rigid spin-label TOAC and performing
measurements under solid-state conditions, we tried to
experimentally realise the concepts successfully applied to
spin-polarised species for stationary two-spin systems. We
investigated chemically synthesised peptides into which two
TOACs are built in at different positions. These peptides were
de novo designed to form an ˛-helix. Charged amino acids
are arranged such that in ˛-helical conformation salt bridges
can be formed. The goal is to find the relative orientation of
the two nitroxides and to correlate this with the geometry
expected for ideal helical peptides.
˚
several Angstroms. Therefore, a distance distribution, rather
than defined distances, is commonly observed. Further, the
distance is not a unique parameter to describe a protein struc-
ture. Studies on spin-correlated radical pairs have shown that
it is possible to obtain the orientation of radicals from EPR
spectra. Measurements on short-lived spin-correlated two-
žC
spin systems (radical pairs), e.g. on P
Až1ꢀ yielded the
700
orientation of the dipolar axis within the g-tensor system
of the electron acceptor A1žꢀ and thereby, the orientation of
Až1ꢀ with respect to the axis connecting A1 and the primary
9
donor P700
.
This was later extended to a complete model
for the relative arrangement of the two species P and A1žꢀ
žC
700
(for a review see Ref. 10). The potential to extract this struc-
tural information has been shown for pairs of spin labels
also.11 Most structural information can be extracted from
nitroxides that are rigidly bound to the protein, adopting
†Presented as part of a special issue on High-field EPR in Biology,
Chemistry and Physics.
ŁCorrespondence to: Robert Bittl, Freie Universita¨t Berlin, Institut
fu¨r Experimentalphysik, Arnimallee 14, 14195 Berlin, Germany.
E-mail: Robert.Bittl@Physik.FU-Berlin.DE
Although the peptides were designed for an aqueous
environment, we have investigated them in different solvent
Contract/grant sponsor: Deutsche Forschungsgemeinschaft;
Contract/grant number: Bi464/7.
Copyright  2005 John Wiley & Sons, Ltd.

Orientation of spin labels in peptides S27
(from Novabiochem) except that Fmoc-TOAC was crys-
tallised from a 1 : 1 mixture of ethyl ether and N-hexane
(v/v). Peptide synthesis on 0.3 mmol PAL-PEG-PS-Fmoc
(Applied Biosystems) resin began with standard solid
phase peptide synthesis (SPPS) using the Fmoc strat-
egy as described.24 The resin was removed from the
column and, before Fmoc-TOAC, was coupled manually
using HATU (N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5b]-
pyridin-1-ylmethylene]-N-methylmethanaminium Hexaflu-
orophosphate N-oxide) (Perseptive) and HOBt (N-hydroxy-
benzotriazole) in a 4.5 times (mol/mol) excess over the resin.
The amino acid following TOAC was coupled with extended
cycle times before standard SPPS was continued.
O
N
HN
HN
z
O
y
N
O
x
Scheme 1. The rigid spin-label TOAC built into a peptides
backbone has only one degree of freedom left, which is the flip
of the six-membered ring. TOAC’s methyl groups are omitted
for clarity.
systems to explore the influence of the environment on the
protein structure. A mixture of water and glycerol, forming
a glass upon cooling and therefore is a more homogeneous
environment, which is also used often for biological systems.
Fluorinated solvents such as TFE (2,2,2-trifluorethanol) and
HFIP (1,1,1,3,3,3-hexafluorisopropanol) and their mixtures
with water are considered to be structure stabilising in most
cases.18–21 We have therefore chosen to investigate, as an
example, the peptide mr18 in a mixture of TFE/H2O.
The effect of organic solvents on protein structures has
gained interest since model environments for membrane
proteins are needed. CHCl3 constitutes a non-polar and
aprotic environment; DMSO also provides an aprotic but
polar environment. Aprotic solvents that form no or only
very weak hydrogen bonds do not compete with the
peptides’ internal hydrogen-bonding networks. The polar
character of a solvent still allows it to stabilise partially
charged structures. By mixing CHCl3 and DMSO it is possible
to adjust the polarity of the solvent to a certain degree by
adjusting the ratio of the solvents.
The
spin-label
2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPO) (Aldrich) was used for comparison, as it has been
shown to have similar g-values as TOAC.25
The following solvents were used: TFE (99.5%, NMR
grade), Aldrich; CHCl3 (uvasol), Merck; DMSO (99.9%, acs),
Sigma-Aldrich. For the MOPS buffer, a 0.1 molar solution
of 3-morpholinopropansulfonic acid in aq. bidest was set to
pH 7.0 with NaOH and sterilised. Solutions of the peptides
were filled into EPR tubes and frozen in a liquid-nitrogen-
cooled ethanol bath.
EPR
EPR spectra at 9 GHz were recorded partly with an FT-
EPR spectrometer ESP 380E (Bruker) equipped with a
dielectric ring resonator, and partly with a laboratory-built
spectrometer. All spectra were recorded at 80 K. EPR spectra
at 94 GHz were recorded with an Elexsys E680 spectrometer
(Bruker).26 For field calibration Li : LiF was used.27
Description of a helix
The shape of an EPR spectrum of two coupled spin labels
is sensitive to the distance between the labels and their
relative orientation. The relative geometry is described by
six independent parameters. We have chosen the following
set of parameters:
EXPERIMENTAL
Peptides and solvents
Design and synthesis of the de novo peptides are already
described elsewhere.22 The primary sequences of the six pep-
tides are listed in Scheme 2. As spin labelfor EPR, the artificial
amino acid TOAC (4-amino-2,2,6,6-tetramethylpiperidine-1-
oxyl-4-carboxylic acid)14 was used.
Fmoc-TOAC was synthesised following the proce-
dure of Marchetto et al.23 using TOAC (from Acros
Organics) and N-(9-fluorenylmethoxycarbonyl)succinimide
– three Euler angles, ˛, ˇ, ꢀ, transforming the g-tensor
system of one spin label into the g-tensor axis system
of the second spin label by the rotation operation
RzꢁˇꢂRxꢁ˛ꢂRzꢁꢀꢂ;
– two angles ϑ, ϕ describing the orientation of the dipolar
axis, where ϑ is the angle between the spin–spin
connecting vector and the z-axis of the first spin labels
g-matrix and ϕ is the angle between the projection of the
connecting vector into the xy plane in this g-matrix and
its x-axis (Fig. 1);
1
5
10
mr57
mr58
mr59
NH2 KKKLAEEAXKXLQEA CONH2
NH2 KKKLAEEAXKLXQEA CONH2
NH2 KKKLAEEAXKLQXEA CONH2
– the distance R between the two centres of gravity of either
spin density.
The spin Hamiltonian
mr510 NH2 KKKLAEEAXKLQEXA CONH2
The EPR spectra of two coupled nitroxides are determined
by the g-tensor of the nitroxides (electron Zeeman interaction
eZ), the hyperfine coupling (hf) of the nitrogen to the electron
spin and the electron–electron coupling (D) between the
spins. The spin Hamiltonian of the system can be written as
mr17
mr18
NH2 KKKLXEEAAKXLQEA CONH2
NH2 KKKLXEEAAKLXQEA CONH2
Scheme 2. Primary sequences of investigated de novo
peptides, where X denotes the spin-label TOAC and the scheme
of spin-label positions.
O
O
O
O
H D HeZ C Hhf C HD
ꢁ1ꢂ
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S26–S33

S28
C. Elsa¨ßer et al.
different frequency bands can therefore be used to separate
contributions of different origin.
z
y
x
Two-spin order
For single spins, lowering the temperature raises the signal
intensity because of the increased population difference.
Furthermore, dynamic effects like protein movement and
rotation of side chains are reduced.
In coupled systems, the depopulation of higher energy
levels leads to a shift in population differences between
different ms levels. This effect is referred to as ‘two-spin
order’. Intensity shifts due to this effect can already be
observed at temperatures which are an order of magnitude
above the Zeeman temperature hꢄ D kTz.29 At temperatures
below the Zeeman temperature, mainly the lowest level is
populated so that half of the transitions disappear.
z
R
ϑ
ϕ
y
x
Figure 1. The relative orientation of two-spin labels is
E
determined by their distance jRj, the orientation of the dipolar
axis is defined by an angle ϑ between gz and the connecting
E
vector R and the angle ϕ between the projection of connecting
vector into the xy-plane and the x-axis of the first spin system.
At X-band, the Zeeman temperature is at Tz D 0.4 K,
which cannot be reached with standard EPR instrumentation.
At 94 GHz, temperature T D 5 K, close to the Zeeman
temperature (Tz D 4.5 K), can well be obtained. In Fig. 2,
the simulated spectra of two dipolar-coupled nitroxides
at different temperatures are shown. The g-tensors of the
nitroxides are collinear and the dipolar axis has an angle
ꢀ
ꢀ
D ꢃB
BgiSi C
IjAijSi C S1DS2
ꢁ2ꢂ
i
ij
In the simulations of the spectra, we omitted an exchange
contribution to the coupling term HD since for most of
the peptides a separation of the spin labels of more than
8 A is expected in any helical conformation. Only the dipolar
O
°
of 30 with the z-axis towards the x-axis. Therefore, the gx-
˚
and gz-components show a distinct splitting, whereas the
gy-component that lies close to the magic angle of the dipolar
tensor is merely broadened. The simulations of spectra at
different temperatures show distinct shifts of intensity in the
dipolar-split lines. These intensity variations upon cooling
can be used as additional spectroscopic information in multi-
frequency studies. Along gx the signal at higher fields loses
intensity, whereas along gz the lines at lower fields lose
intensity. This can be accounted for by the sign of the dipolar
coupling, which changes between x and z. Therefore, the
relative orientations of the dipolar axis with respect to the
g-tensor system can be determined.
O
coupling is taken into account for HD, which can be expressed
as
1 ꢃ0
gigj ꢃ2Bꢁ3 cos2 ϑ ꢀ 1ꢂ
ꢁ3ꢂ
O
Hdip
D
3
2h
rij
The dipolar coupling shows a rꢀ3 distance dependence
and therefore can be used to determine the distance between
the spins.
The electron Zeeman interaction and the hyperfine
coupling are anisotropic and are therefore represented by
tensors. The electron Zeeman anisotropy is a field-dependent
effect which is expressed via the g-tensor, and measurements
at different fields/frequencies are used to separate lines of
different g-value. The spectral separation of two lines g1 and
g2 is given by:28
Simulation routines
The spectra with two dipolar-coupled spin labels were
simulated using the program barley30 which diagonalises the
full spin Hamiltonian. The X-band data were fitted with a
routine written by E. Kirilina (Novosibirsk), based on second
order perturbation theory.
ꢁ
ꢂ
hꢄ
ꢃ
1
g1
1
g2
B D
ꢀ
.
ꢁ4ꢂ
The hyperfine and the dipolar interaction, on the other
hand, are field-/frequency-independent. Experiments at
The g-principal values and hyperfine values were taken
from measurements of TEMPO in the respective solvent.
gy
gx
gz
(a)
(b)
1
2
1
2
,
,
,
80 K
5 K
+
+
+
−
1
2
1
2
−
1
2
1
2
+
1 K
1
2
1
2
,
−
−
3340
B/mT
3350
hν< kBT
hν kBT
Figure 2. Two-spin order. (a) Population of the four electron spin levels in the high- and low-temperature regime. (b) Simulation of
W-band EPR spectra of two coupled nitroxides at different temperatures.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S26–S33

Orientation of spin labels in peptides S29
mr18 at X-Band is quite similar to the spectrum of mr57,
the spectrum of mr18 at W-band shows sharper features in
the gz-region (Fig. 3(B)). Again, upon cooling to 5 K rather
global intensity changes are observed. Similar to mr57, the
broad line features cannot be explained by a specific dipolar
coupling in any of the helix types. A possible origin for the
significant line broadening is an aggregation of the peptides
upon cooling. However, the samples stayed homogeneous
even after repeated freeze–thaw cycles. Therefore, we
consider aggregation as unlikely. Another source for line
broadening is the conformational variability of the spin
labels. Because of the flip of the six-membered ring, four
conformers giving rise to different EPR spectra are possible.
But even the superpositions of calculated spectra for these
four conformers result in clearly resolved spectra (data not
shown) so that further mechanisms of line broadening have
to be assumed.
Frozen aqueous solutions form a micro-crystalline envi-
ronment, causing strain effects and, therefore, line broaden-
ing. By this, mainly gx would be affected.28 Another effect,
which leads to a broadening of all lines corresponding to the
respective g-components, would be a conformational hetero-
geneity of the peptide that leads to a wide distribution of
dipolar couplings.
To investigate the influence of the micro-crystalline
environment, we used a buffer mixture with glycerol and
measured, as an example, the peptide mr18. In Fig. 4(A) the
spectrum measured at W-band is shown. Compared to the
spectrum of mr18 in water, the linewidth is significantly
reduced, but no resolved components are visible. Therefore,
no unique information about distance and orientation
between the labels can be deduced. Since the absence of
line splitting at the observed small linewidth indicates very
small dipolar couplings, we assume that the peptide is mostly
unfolded in the water/glycerol environment. As the next
solvent mixture, we consider TFE/H2O.
RESULTS
The primary sequences of the investigated doubly spin-
labelled de novo peptides are shown in Scheme 2. First, we
concentrated on two peptides, one with a single amino acid
between the labels, called mr57, and the other with seven
amino acids between the labels, called mr18. As solvent we
have chosen aqueous buffer (pH 7), resembling the natural
environment of soluble proteins. The X-band spectra of both
peptides are quite similar (Fig. 3(A)). Both spectra display the
shape of a single nitroxide that is broadened, presumably due
to dipolar coupling. However, in the two peptides the labels
should have completely different distances and orientations.
This difference can, in this case, hardly be deduced from
the X-band EPR spectra since both show line broadening.
In W-band, where field-dependent components are better
separated, the spectra of the two peptides are at 80 K clearly
different (Fig 3(B)). At 94 GHz, the spectrum of mr57 shows
substantial broadening of the gx component, which cannot be
explained by convolution with only one gaussian line. The gy
component is broadened to approximately 3.0 mT. In a well-
defined peptide, a dipolar coupling of such size should lead
to a line splitting since the linewidth of the spin label itself
is much smaller (about 0.4–0.6 mT). Furthermore, cooling
close to the Zeeman temperature (Fig. 3(C)) only leads to a
general intensity shift towards the low-field region but not
to the disappearance of individual lines, as demonstrated in
Fig. 2.
Simulation of the experimental spectra is possible only
under the assumption of a high inhomogeneous linewidth.
Therefore, a large set of parameters lead to similar spectra.
Also, the simulated spectra for an ˛- and a -helix are very
similar and both fit the experimental spectra. In a 310-helical
conformation the distance between the spin labels can be
˚
estimated to be about 5 A. In this case, the dipolar and
exchange coupling between the spin labels would be so
large that despite the high linewidth, a splitting should
be observed. Since this is not observed, the 310-helical
conformation can be excluded. Although the spectrum of
Spectra of mr18 in TFE/H2O (1 : 1) were recorded again
at 9 GHz (80 K) and at 94 GHz (80 K, 5 K) (Fig. 5). The
spectra of mr18 in TFE/H2O are better resolved than the
(a)
A
(b)
330
340
350
360
330
340
350
360
B/mT
B/mT
B
C
3340
3350
B/mT
3360
3340
3350
B/mT
3360
Figure 3. EPR spectra of mr57 (a) and mr18 (b). A: 9 GHz, 80 K, B: 94 GHz, 80 K, C: 94 GHz, 5 K.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S26–S33

S30
C. Elsa¨ßer et al.
at W-band. Distinct changes in intensity are observable in
the W-band spectra at the different temperatures. These
intensity shifts mainly affect the split hyperfine lines on
gz, but prominent intensity changes also occur in the still
unresolved gx-component.
A
Despite the well-resolved structure of the X-band spec-
trum, satisfactory simulations are possible using different
parameter sets. Only a simultaneous simulation of all three
spectra leads to a unique simulation, which is shown in Fig. 5
(the simulation parameters are given in the figure caption).
In addition, spectra of doubly labelled peptides have
been recorded in organic solvents. The spectra of mr18
in CHCl3 and in DMSO at 94 GHz are shown in Fig. 4(B)
and (C). The spectral features are extremely broadened
and an interpretation beyond the assumption of significant
structural heterogeneity is impossible.
B
C
D
In a mixture of CHCl3/DMSO (7 : 3) the spectrum
of mr18 (Fig. 4D) differs distinctly from that in water
and from that in TFE/H2O. However, in contrast to the
case of pure solvents, the linewidth is strongly reduced
compared with the spectrum in water and similar to the
spectrum taken in TFE/H2O. Comparing the spectra in the
solvent mixtures TFE/H2O and CHCl3/DMSO, the most
significant differences are visible along gx. In TFE/H2O the
gx-component shows no clear dipolar coupling, whereas
in CHCl3/DMSO gx is made up of two lines separated by
1.6 mT. This shows that the peptide adopts a well-defined but
different conformation in CHCl3/DMSO than in TFE/H2O.
It is important to note here, that the centre of the split lines
around gx is shifted to lower g-values (upfield) compared
to the gx-value of TEMPO in the same solvent. To obtain
additional structural information, we have recorded spectra
of a series of peptides with increasing distance between the
labels in the primary structure in the two solvents H2O and
TFE/H2O.
3340
3350
B/mT
3360
Figure 4. W-band spectra of mr18 in different solvents at 80 K.
A: H2O/glycerol (4 : 6), B: CHCl3, C: DMSO, D: CHCl3/DMSO
(7 : 3) (thin line spectrum at 5 K). Marked is the hyperfine and
dipolar splitting of the gz component.
A
335
340
345
350
355
360
B/mT
X-band EPR spectra of the peptide series in water show
repeated increase and decrease of the dipolar coupling
(Fig. 6(a)). The deviation from the spectral shape of a single
spin label increases from mr57 to mr59. In mr59 the dipolar
coupling clearly dominates the spectrum. Analysis of the
spectrum shows that it consists of two species. The central
part of the spectrum (341–349 mT) presents the lineshape of
a weakly coupled species while the outer features around
338 and 351 mT belong to a strongly coupled spin system.
In the peptide mr510, the dipolar coupling is so weak that
it only leads to a broadening of lines. The spectrum of mr17
has a peculiar shape since the line at the lowest field starts
negative. A positive signal at a lower field should be present,
but probably because of the strong broadening it has such
little intensity that it was not observed. The spectral shape
can be explained, assuming that mr17 adopts two different
conformations. The well-structured signal between 340 and
350 mT belongs to a very weakly coupled spin label, whereas
the signals at 337 and 357 mT belong to a strongly coupled
conformation. In mr18 the spectral shape is again uniformly
broadened.
B
3335
3340
3345
3350
3355
3360
3365
3370
B/mT
C
3335
3340
3345
3350
3355
3360
3365
3370
B/mT
Figure 5. mr18 in TFE/H2O. EPR spectra and simulations at
9.7 GHz (A), 80 K, 94 GHz, 5 K (B), 94 GHz, 80 K (C).
Simulation parameters: gx D 2.0086, gy D 2.0060, gz D 2.0020,
°
Axx D 17.0 MHz, Ayy D 21.0 MHz, Azz D 110.5 MHz, ˛ D 15 ,
°
°
°
°
ˇ D 352 , ꢀ D 329 , ϑ D 20 , ϕ D 85 , D D 1.2 mT.
spectra of mr18 in H2O. Already at X-band, several lines
are clearly recognisable. Clear splittings of the hyperfine
components in the gz region are visible in the spectra taken
The spectra of the peptide series in TFE/H2O (Fig. 6(b))
at X-band have a linewidth of approximately 0.6 mT, which
is significantly reduced, compared with the spectra in water.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S26–S33

Orientation of spin labels in peptides S31
(a)
(b)
mr57
mr58
mr59
mr510
mr17
mr18
330
340
350
B/mT
Figure 6. EPR spectra (9 GHz) of the series of peptides. (a) in water, pH 7; (b) in TFE/H2O (1 : 1).
360
330
340
350
360
B/mT
The spectra of the peptides in TFE/H2O have not only a
reduced linewidth compared to those dissolved in water, but
some of the spectra even have completely different shapes.
Especially, for mr57, mr58, mr17 and mr18 the structure of
the spectra changes significantly between the solvents. As in
water, the series of spectra at 9 GHz shows a periodic increase
and decrease of the spectral width. However, in H2O/TFE,
the spectral width is the largest in mr58, mr59 and mr18.
Closer inspection of the individual spectra shows that mr57
reveals a partially resolved splitting of the gz component.
The spectrum of mr58 is an exception in the series since it
shows very narrow lines with a linewidth reduced to 0.4 mT.
The spectrum of mr59 is similar to the one in H2O but shows
a smaller linewidth. The spectrum again is composed of two
different species with a similar ratio between the species. In
the spectrum of mr17 the strongly broadened species have
disappeared. Therefore, this peptide has adopted a uniform
conformation under the addition of TFE. The spectrum of
mr18 has been discussed above.
Therefore, we conclude that the spin labels adopt a unique
conformation in mr18. The simulations fit the experimental
spectra and their temperature dependence, particularly well
in the gz region. The observed intensity changes of the split
hyperfine components along gz indicate that both gz-axes
form an angle with the dipolar axis that is much smaller
than the magic angle. For none of the considered ideal helix
types (taking all four possible TOAC conformations into
account) this situation is realised. Consequently, we assume
that the orientation of the TOACs describe a very specific
conformation of the peptide.
Apparently, the bulky and charged amino acid residues
can lead to a modification of the helical structure and
the relative geometry between the two-spin labels. Such
a deviation from an ideal helical structure deduced so far
from the analysis of the X- and W-band spectra of the
peptide mr18 is consistent with the findings from the X-band
spectra of the peptide series. Using a series of peptides, it
should be possible to deduce the helix type, based on the
periodicity of the dipolar broadening of the spectra. In a
310-helix, one turn is built from exactly three amino acids,
resulting in an expected periodicity of three for the dipolar
broadening. For a typical ˛-helix the dipolar broadening
should have a periodicity of 3.6. In a -helix the periodicity
should be 4.4. In the peptide series in H2O, the spectral
width is high for mr59 and mr17, at least for one of the
sub-species, and medium for mr58 and mr18. This pattern
for the peptides in water does not correlate to any of the
expected periodicities for the conventional helix types. It is
possible that the incorporation of the non-natural amino acid
TOAC disturbs the folding of the peptide to its preferential
geometry in the absence of TOAC. Amino acids that are tetra-
substituted at C˛ have been found to destabilise ˇ-sheets31
and to support helical structures. Increased ratios of ˛-helical
structures19,21 but also of 310 helical structures32 have been
found by CD spectroscopy. Furthermore, the observation
of different spectral sub-species, at least for some of the
peptides, leads to the conclusion of a structural heterogeneity
of the peptides.
DISCUSSION
In the following, we combine the results from the dual
frequency analysis of individual peptides in different
solvents with results derived from the measurements of the
peptide series in water and H2O/TFE. As an example, we first
analyse the spectra of mr18 in H2O and H2O/TFE. Similar
to the attempts to fit the significantly broadened spectra of
mr57, no unique parameter set describing the orientation
between the two-spin labels could be obtained for mr18 in
H2O. This is remarkable because the spectra of mr18 at W-
band show a smaller linewidth of the hyperfine components
along gz. For mr18 in H2O/TFE the situation is different. The
simultaneous simulation of the X-band spectrum and the W-
band spectra recorded at 80 and 5 K resulted in a satisfactory
simulation with the parameters given in the caption of
Fig. 5. In this simulation, only one specific relative geometry
between the spin labels was included, even though each
spin label could adopt two different conformers (Scheme 1).
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S26–S33

S32
C. Elsa¨ßer et al.
In TFE/water, the spectral width of the peptide series
resembles in its periodicity an ˛-helical shape. However,
the peptide mr59 still adopts two different conformations.
Analysis of the peptide mr58 shows that despite the highly
resolved pattern, the spectrum could not be simulated with
the given g-values and under the assumption of pure dipolar
coupling. The W-band spectra of mr58 (Fig. 7) at 80 K and at
5 K show no difference. Inspection of these spectra shows that
the gz-component consists of five lines with a splitting that
is half of the usual hyperfine splitting on gz. This is possible
only if the spin labels are so close that the two S D 1/2
systems couple to a spin S D 1. In that case, the spin would
couple to two 14N with equal hyperfine coupling, resulting
in the observed pattern of five lines with an intensity pattern
of 1 : 2 : 3 : 2 : 1.
mr18
TEMPO
310-helix
α-helix
π-helix
However, if the spin labels in mr58 are much closer than
in mr59, again a strong deviation from the ˛-helical shape
occurs.
3330
3340
3350
3360
B/mT
Finally, we analyse the spectra of the peptide mr18 in
the organic solvents. A comparison of the W-band spectrum
of mr18 with the spectrum of the single spin label (Fig. 8)
in CHCl3/DMSO shows that the gx-component of the single
spin label (3337.2 mT) is at a lower field than the lowest
lying resonance in the coupled spectrum (3338.0 mT). Such
a shift cannot be caused by spin–spin interactions but can
only be explained by a lowering of gx itself. The value of
gx strongly depends on the polarity of the environment
and the hydrogen-bonding pattern to the nitroxide.33–35
The environment-dependent parameters gi and Aii were
determined independently by measuring the similar spin-
label TEMPO in the same solvent mixture. Since TEMPO
and TOAC have similar g-values,25 the decrease of gx has to
originate from a change in the local environment of the label.
In a peptide, one possibility is internal hydrogen bonding
Figure 8. Experimental spectrum of mr18 at 94 GHz and 80 K,
in comparison with the experimental spectrum of TEMPO in
CHCl3/DMSO and calculated spectra of two-spin labels
orientated as in the three helix types given. gx of TEMPO is
marked by the vertical line.
small peptide. This finding, however, shows that for further
structural investigations, singly- as well as doubly labelled
peptides are required to obtain the g-values for the labels at
specific sites.
In Fig. 8 the spectrum of mr18 in CHCl3/DMSO is
compared with simulations based on spin-label orientations
in typical helix conformations. Neglecting the exact position
of the spectral lines and comparing only the overall shape
of the spectra, the spectrum of mr18 resembles most the
simulation of an 310 helix. The structure-stabilising abilities
of aprotic solvents arise from the fact that they do not
effectively solvate polar groups and therefore do not compete
with intra-peptide hydrogen bonds. However, polar groups
at the ends of a peptide cannot form intra-peptide hydrogen
bonds and destabilise the structure. In tighter conformations
(as a 310-helix), the number of end groups is smaller than in
wider conformations (as a -helix), rendering the latter less
stable. However, theoretical37 and experimental38 evidence
has been given for the occurrence of -helical conformations
under similar conditions. Pulsed ELDOR experiments on a
similar peptide in CHCl3/DMSO gave indications for a 27
conformation of the peptide.6 Such a structure, however, can
C
to side chains of other amino acids. Here, NH3 groups of
lysines are close, and a hydrogen bond to an ammonium
group could lower gx. A shift of g- and hyperfine values
due to the peptide environment increases the number of free
parameters from 6 to 18, since the values can again differ for
the individual labels. While environment-induced g-factor
changes are well established in the interior of proteins and
are used to map polarity profiles,36 we did not expect such
an influence for the solvent-exposed spin labels attached to a
˚
be excluded for mr18, since the labels would be 22 A apart
and no splitting would be observed in that case.
CONCLUSION
This work shows how determination of distance and
orientation of two-spin labels can contribute to the structural
investigation of peptides. A series of peptides doubly
labelled at different positions with the rigid spin label TOAC
has been investigated. By the choice of solvents, different
secondary structures could be induced. It was shown that the
peptide assumes a well-defined conformation in a mixture of
3340
3350
B/mT
3360
Figure 7. Spectrum of mr58 at 94 GHz and 80 K (top) and 5 K
(bottom).
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S26–S33

Orientation of spin labels in peptides S33
Labels. Kluwer Academic/Plenum Publishers: Dordrecht; 2000;
Chapt. 3.
12. Bruce Merrifield R. J. Am. Chem. Soc. 1963; 85: 2149.
13. Hanson P, Millhauser G, Formaggio F, Crisma M, Toniolo C. J.
Am. Chem. Soc. 1996; 118: 7618.
14. Rassat A, Rey P. Bull. Chem. Soc. Fr. 1967; 3: 815.
15. Hanson P, Martinez G, Millhauser G, Formaggio F, Crisma M,
Toniolo C, Vita C. J. Am. Chem. Soc. 1996; 118: 271.
16. Anderson DJ, Hanson P, McNulty J, Millhauser G, Monaco V,
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6919.
17. Bolin KA, Millhauser GL. Acc. Chem. Res. 1999; 32(12): 1027.
18. Cammers-Goodwin A, Allen TJ, Oslick SL, McClure KF, Lee JH,
Kemp DS. J. Am. Chem. Soc. 1996; 118: 3082.
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20. Nelson JW, Kallenbach NR. Biochemistry 1989; 28(12): 5256.
21. Hong D-P, Hoshino M, Kuboi R, Goto Y. J. Am. Chem. Soc. 1999;
121: 8427.
22. Rau HK, DeJonge N, Haehnel W. Angew. Chem., Int. Ed. Engl.
2000; 39: 250.
TFE/water as well as in CHCl3/DMSO. The structures in the
different solvents differ significantly. The peptide mr18 was
investigated in TFE/H2O at different frequency bands and
in different temperature regimes. From the simultaneous
simulation of three spectra, a set of parameters could be
obtained, which uniquely describes the relative orientation
of the two TOACs. However, the conformation does not
correlate with any of the typical helix types adopted by
peptides. The analysis of the peptide series showed that the
peptides do not adopt a uniform structure either in aqueous
buffer or in TFE/water.
The difficulties in analysing the relative geometry of two
rigid spin labels under solid-state conditions, which became
obvious during this study, provides an explanation for the
rather conflicting results that have been given for similar
systems at different times by other groups.6,13,16,17,39,40
23. Marchetto R, Schreier S, Nakaie CR. J. Am. Chem. Soc. 1993; 115:
11042.
Acknowledgements
We thank Evgenia Kirilina for the simulation routine, and her
as well as Alexander Schnegg and Gunnar Jeschke for helpful
discussions. We thank Claudio Toniolo for support and advice,
and Christa Reichenbach for technical assistance. The authors thank
the coordinators of the ‘Schwerpunktprogramm Hochfeld-EPR in
der Biologie, Chemie und Physik’, Klaus Mo¨bius and Klaus-Peter
Dinse, for their efforts in establishing and running this program.
This work was supported by Deutsche Forschungsgemeinschaft
(Bi464/7) within the priority program ‘High-field EPR in Biology,
Chemistry and Physics’ (SPP 1051) and by VolkswagenStiftung (to
W. H.).
24. Rau HK, Haehnel W. J. Am. Chem. Soc. 1998; 120: 468.
25. Milov AD, Tsvetkov YD, Gorbunova EY, Mustaeva LG,
Ovchinnikova TV, Raap J. Biopolymers 2002; 64(6): 328.
26. Schmalbein D, Maresch GG, Kamlowski A, Ho¨fer P. Appl. Magn.
Reson. 1999; 16: 185.
27. Stesmans A, van Gorp G. Rev. Sci. Instrum. 1989; 60(9): 2949.
28. Wegener C. Multifrequenz-(9 GHz und 95 GHz) ESR-Spektroskopie
zur Analyse der Dynamik und Polarita¨t von Ortsspezifisch
Spinmarkiertem Bakteriorhodopsin, Dissertation, Ruhr-Universita¨t
Bochum, 2002.
29. Calvo R, Abresch EC, Bittl R, Feher G, Hofbauer W, Isaac-
son RA, Lubitz W, Okamura MY, Paddock ML. J. Am Chem. Soc
2000; 122: 7327.
30. Scha¨fer KO. Exchange Coupled Manganese Complexes: Model
Systems for the Active Centres of Redoxproteins Investigated with
EPR Techniques, Dissertation, TU Berlin, 2002.
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Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S26–S33