Biochem. J. (2011) 434, 353–363 (Printed in Great Britain)
353
doi:10.1042/BJ20101871
REVIEW ARTICLE
Pulsed electron–electron double resonance: beyond nanometre distance
measurements on biomacromolecules
Gunnar W. REGINSSON and Olav SCHIEMANN1
Biomedical Sciences Research Complex, Centre of Magnetic Resonance, University of St Andrews, St Andrews KY16 9ST, Scotland, U.K.
PELDOR (or DEER; pulsed electron–electron double resonance)
is an EPR (electron paramagnetic resonance) method that
measures via the dipolar electron–electron coupling distances in
the nanometre range, currently 1.5–8 nm, with high precision and
reliability. Depending on the quality of the data, the error can be as
small as 0.1 nm. Beyond mere mean distances, PELDOR yields
distance distributions, which provide access to conformational
distributions and dynamics. It can also be used to count the
number of monomers in a complex and allows determination
of the orientations of spin centres with respect to each other. If,
in addition to the dipolar through-space coupling, a through-bond
exchange coupling mechanism contributes to the overall coupling
both mechanisms can be separated and quantified. Over the last
10 years PELDOR has emerged as a powerful new biophysical
method without size restriction to the biomolecule to be studied,
and has been applied to a large variety of nucleic acids as well as
proteins and protein complexes in solution or within membranes.
Small nitroxide spin labels, paramagnetic metal ions, amino acid
radicals or intrinsic clusters and cofactor radicals have been used
as spin centres.
INTRODUCTION
The information that can be collected from PELDOR experiments
will be demonstrated by summarizing applications of PELDOR
to model systems, proteins and nucleic acids.
Molecular and structural biology are engaging ever larger
biomacromolecular complexes either isolated or within
membranes or whole cells. Thus biophysical methods are
needed which can provide structural and dynamic information
for these molecular architectures on the relevant nanometre
scale. Fluorescence-based methods, such as FRET (fluorescence
resonance energy transfer) are very powerful in this respect as
they can be applied at the single-molecule level and provide realtime dynamics over several time scales [1]. However, FRET lacks
precision when it comes to quantifying distances or distance
changes. In contrast, a pulsed EPR (electron paramagnetic
resonance) [2] method called PELDOR (or DEER; pulsed
electron–electron double resonance) yields reliably very precise
distances in the range ∼ 1.5–8 nm. Like FRET, PELDOR has
no molecular mass restriction, but it usually requires micromolar
concentrations and the sample has to be frozen. Thus both methods
are complementary. Milov et al. [3] were the first to introduce the
three-pulse version of PELDOR [3] (Figure 1A), and the groups
of Spiess [4] and Jeschke [5] extended it to the dead-time-free
four-pulse version (Figure 1B).
A more specialized method is the DQC (double quantum
coherence)-EPR filter experiment from the laboratory of Freed
[6]. Over the last few years several other review articles have
been published [7–12]. More experimentally focused articles with
detailed protocols for PELDOR measurements have also been
published [13–17].
The present review will briefly describe different ways of
spin-labelling biomacromolecules and then introduce the dipolar
coupling between unpaired electrons. This is followed by a
disussion about the signal the PELDOR pulse sequence generates.
Key words: biomacromolecule measurement, dynamics, nucleic
acid, pulsed electron–electron double resonance (PELDOR or
DEER), spin labelling.
SPIN LABELLING
In order to be able to apply EPR spectroscopic methods such
as PELDOR, the biomacromolecule needs to have at least one
unpaired electron or, in other words, it needs to have one or
more paramagnetic centre. These can, for example, be metal
ions (e.g. Cu2+ or Mn2+ ), metal clusters (e.g. FeS), organic
cofactors (e.g. semiquinones or flavin radicals) or amino acid
radicals (e.g. tyrosyl radicals) formed during catalytic cycles. In
cases where the biomolecule is diamagnetic and has no such
centre, paramagnetic labels carrying unpaired electrons can be
introduced. Commonly, these labels are nitroxides [18,19]. For
proteins, MTSSL (methanethiosulfonate spin label) [20] is mostly
used, which reacts with the SH-group of cysteine residues and
forms a covalent disulfide bridge (Figure 2). The site specificity
for this reaction is introduced by site-directed mutagenesis by
which unwanted cysteine residues are replaced by, e.g., serine
residues, and new cysteine residues are introduced at positions
where they are wanted [21]. In cases where cysteine residues
cannot be removed without loss of structure or function, ligation
techniques [22] or the incorporation of non-natural amino acids
via newly coded tRNAs [23] can be used.
Over the last decade, several strategies have been established
to also site-specifically spin label oligonucleotides [24]. For
example, nitroxides can be site-specifically attached to the
bases [25–29], the phosphate [30] or sugar group [31]. The
site specificity is introduced by incorporating a modified
Abbreviations used: ABC transporter, ATP-binding-cassette transporter; cw, continuous wave; EPR, electron paramagnetic resonance; FRET,
fluorescence resonance energy transfer; LHCII, light-harvesting chlorophyll a /b complex; MD, Molecular Dynamics; MTSSL, methanethiosulfonate spin
label; PELDOR (or DEER), pulsed electron–electron double resonance; PSII, photosystem II; RE, resonance echo; RNR, ribonucleotide reductase.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
354
G. W. Reginsson and O. Schiemann
If the distance between both electrons is small and thus the
dipolar interaction strong, the line splitting is large and might
be observed in a cw (continuous wave) EPR spectrum [40–
42]. For nitroxides measured at X-band frequencies, the upper
distance limit for a cw EPR observation of a dipolar splitting is
approx. 1.5–2 nm. Above this distance the dipolar splitting
is covered under the inhomogeneous linewidth. The advantage
of the PELDOR experiment is that it suppresses hyperfinecoupling contributions and selects the electron–electron coupling
which enables the measurement of much longer distances. The
longest experimentally verified distance obtained with PELDOR
is currently 8 nm [43], the shortest 1.5 nm [44]. Banham et al.
[45] investigated the distance regime in which both methods can
be applied.
Figure 1
Microwave pulse sequence for three- and four-pulse PELDOR
PELDOR PULSE SEQUENCE AND SIGNAL
(A) Three-pulse PELDOR pulse sequence. (B) Dead-time-free four-pulse PELDOR sequence.
Reproduced with permission, from Reginsson, G. W. and Schiemann, O. (2011) Biochem. Soc.
c the Biochemical Society.
Trans., 39, 128–139. Figure 2
MTSSL and its reaction with cysteine residues
phosphoramidite into the oligonucleotide at the desired place in
the sequence during automated oligonucleotide synthesis. The
modification can be a nitroxide label or a chemical group, which
reacts specifically with a corresponding group on a nitroxide.
These strategies are all limited by the need to synthesize the
oligonucleotide, which currently puts the length limit at approx.
80 bases. Obeid et al. [32] use enzymatic labelling to overcome
this problem. In 2010, a method was published in which a
spin-labelled cytosine analogue was non-covalently, but sitespecifically, attached to oligonucleotides [33]. The site specificity
is achieved with the help of an abasic site opposite a guanine. With
the possibility to buy oligonucleotide strands with abasic sites
from various companies, this labelling strategy no longer requires
an in-laboratory chemical step for labelling of nucleic acids. If the
biomolecule contains diamagnetic metal ions, e.g. Mg2+ , these can
be exchanged for paramagnetic ones, e.g. Mn2+ [34–37]. In any
case, labelling, mutagenesis and exchange of metal ions do lead
to alteration of the biomolecule, thus control experiments with
respect to the integrity of its structure and function are required.
DIPOLAR COUPLING
V(t) = V(t)intra V(t)inter
If a biomolecule contains two unpaired electrons their magnetic
dipole–dipole interaction gives rise to a splitting of their EPR
lines. Within the point-dipole approximation, which is fulfilled
for large distances and spin-localized systems [38], the dipolar
coupling ν dd is described by eqn (1) [39]:
vdd =
μ0 γA γB 1 − 3 cos2 θ
3
8π 2 rAB
(1)
where μ0 is the vacuum permeability, is the Planck constant
divided by 2π, γ A and γ B are the magnetogyric ratios of the two
spins A and B, rAB is the distance between them and θ is the angle
between the distance vector rAB and the external magnetic field
B0 .
c The Authors Journal compilation c 2011 Biochemical Society
Nowadays it is the dead-time-free four-pulse PELDOR
experiment that is routinely used (Figure 1B) [4,5]. The detection
sequence π/2-τ 1 -π-τ 1 -echo1 -τ 2 -π is applied at a microwave
frequency ν A . This pulse sequence creates after the time τ 2 ,
after the last π pulse, an RE (refocused echo) from spins (spins
A) in resonance with the microwave frequency ν A . The time
position of the RE does not change during the experiment, since
the time intervals of the detection sequence are not changed.
Introduction of the inversion pulse at frequency ν B during the
time interval T flips spins (spins B), which are in resonance with
this second frequency. Using two different and phase-independent
microwaves and keeping the RE at a fixed position in time strongly
suppresses hyperfine contributions. Incrementing the pump pulse
from t = 0 to t = T leads to an oscillation of the amplitude V
of the RE dependent on the time position t of the inversion pulse
if spins A and B are coupled. The frequency of this oscillation
is the dipolar coupling ν dd between both spins. The actual zero
time of this oscillation is when the inversion pulse crosses the
Hahn echo. Since the time axis t for the inversion pulse can
be chosen to start before the Hahn echo, one actually records
negative times for the dipolar evolution, but more importantly
one has no dead time for the dipolar evolution. In the three-pulse
PELDOR version (Figure 1A), a dead-time occurs because the inversion pulse cannot usually be applied on the π-pulse of the
detection sequence [46] without introducing too strong artefacts
at the beginning of the time trace.
Since PELDOR is not a single-molecule experiment (it usually
requires spin concentrations of 100 μM), the PELDOR signal V(t)
(Figure 3a) is the product of the contribution from the interaction
between the two spins in one molecule V(t)intra and the interaction
between spins on different molecules V(t)inter (eqn 2) [47]:
(2)
If the spins are randomly distributed in the sample, V(t)inter can
be written in the form of a monoexponential decay function
which depends on the radical concentration. Dividing V(t) by this
decay function leaves the wanted intramolecular signal V(t)intra
(Figure 3b), which can be described by eqn (3) [10]:
n n
1 {1 − λB [1 − cos (vdd t)]}
V(t)intra =
(3)
n A=1 B=1
B=A
ν dd is given by eqn (1), n is the number of radicals per biomolecule,
λB is the fraction of B spins inverted by the pump pulse, t is the
time delay of the pump pulse, and <· · ·> is the integration over
all values of rAB and θ .
PELDOR beyond distances
Figure 3
355
The PELDOR signal
(a) The green line is the convolute of intra- and inter-molecular interaction as obtained in a PELDOR experiment. The red line is the fitted intermolecular contribution. (b) The wanted intramolecular
signal obtained after dividing the original time trace [green line in (a)] by the intermolecular contribution [red line in (a)]. (c) Pake pattern obtained after Fourier transformation of the signal in (b).
Reprinted by permission from Macmillan Publishers Ltd: Nature Protocols [13], copyright (2007).
Figure 4 Schematic representation of a spin-labelled DNA–RNA duplex and the correlation between distances obtained from PELDOR and MD simulations
on a series of such duplexes with different numbers of base pairs between the labelled sides
Distances for RNAs are shown as 䊏 and from DNAs as 䊊. Adapted with permission from [54], Copyright 2004 American Chemical Society; and Piton, N., Mu, Y., Stock, G., Prisner, T. F., Schiemann,
O. and Engels, J. W. Base specific spin-labeling of RNA for structure determination. Nucleic Acids Res. 2007, 35, 3128–3143, by permission of Oxford University Press.
Each molecule in a frozen sample has a fixed and specific
orientation of rAB with respect to B0 and thus a specific value
for θ . However, since V(t)intra integrates over all molecules and
thus all values of θ (if the molecules are not aligned), Fourier
transformation of V(t)intra does not give a single line, but the socalled dipolar Pake pattern (Figure 3c). The peaks and edges of
the Pake pattern correspond to molecules with θ = 90 ◦ = θ ⊥ and
θ = 0 ◦ = θ ll respectively. Reading off the frequency at θ ⊥ = 90 ◦
and substituting it into eqn (4), the rearranged version of eqn (1)
for nitroxides, yields the distance rAB between the two spins:
r AB [nm] =
3
52.16
v (θ⊥ ) [MHz]
(4)
Instead of Fourier transforming the time traces, they are commonly analysed with the program DeerAnalysis from G. Jeschke
[48], which is based on Tikhonov regularization and the assumption that the full Pake pattern (all orientations of θ ) is excited.
The upper distance limit for PELDOR is determined by the
need to resolve one full oscillation period for a precise distance
determination. This requires that the time window t of the
experiment is long enough to include a full oscillation, which
in turn requires that the relaxation time T M of the system is long
enough. For example, to measure the so-far longest distance of
80 Å (1 Å = 0.1 nm), it was necessary to dissolve the bisnitroxide
model system in a special matrix to make T M long enough [43].
Interestingly, a recent paper by Ward et al. [49] demonstrated
that such long distances can even be accessed in aqueous buffer
solutions and for as large protein complexes as histones, if the
proteins and the buffer are fully deuterated.
The first PELDOR distance measurements on proteins have
been achieved in the laboratory of Kawamori using intrinsic
radicals in PSII (photosystem II) [50,51]. Persson et al. [52] were
the first to apply PELDOR to spin-labelled proteins, specifically
the human carbonic anhydrase II. Schiemann et al. [53,54]
established a PELDOR-based nanometer distance ruler for spinlabelled biomolecules in aqueous buffer solution using nucleic
acids (Figure 4) [26,54]. Also on nucleic acids, the laboratory of
Norman showed that PELDOR can resolve distances from up to
five different doubly labelled DNAs in one sample [55].
Today a wide range of biomolecules have been studied with
PELDOR. Below, a few examples are collected where PELDOR
has been used to resolve the arrangement of protein domains or
of proteins in complexes.
Park et al. [56] applied PELDOR to the chemotaxis
receptor kinase assembly of Thermotoga maritima to unravel
the arrangement of the CheA–CheW complex. The predicted
structure, constructed from 40 distances, matches nicely with
the crystal structure of this complex published in the same
paper. Based on the structure and biochemical data, suggestions
with respect to the functional mechanism of this complex have
been made. The laboratory of Fajer has published several papers
on troponin showing that the inhibitory region of the ternary
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356
Figure 5
G. W. Reginsson and O. Schiemann
Distance measurements on the KcsA ion channel using PELDOR
(a) Orientation-selective X-band PELDOR time traces from spin-labelled KcsA overlaid with the simulated time traces. (b) Structure of the spin-labelled tetrameric KcsA. Reproduced by permission
from [74]. Copyright 2009 American Chemical Society.
complex is α-helical [57] and that Ca2+ induces structural changes
[58]. Meyer et al. [59] determined the structures of the dimeric
full-length G-protein MnmE in the GTP and GDP states. The
observed distance changes of up to 1.8 nm clearly indicate large
conformational changes associated with the hydrolysis event. The
laboratory of Jeschke used a combined approach of modelling
and PELDOR to determine the structural arrangement of the
physiologically relevant homodimer of the Na+ /H+ antiporter
NhaA of Escherichia coli [60]. Grote et al. [61] used PELDOR to
study the conformational changes of an ABC transporter (ATPbinding-cassette transporter). The doubly spin-labelled series of
ABC maltose transporter variants (MalFGK2 -E) showed that
the substrate-binding protein MalE is bound to the transporter
throughout the transport cycle and that not only does the ATPbinding side undergo three conformational changes during function, but also so does the periplasmic MalF-P2 loop. This is suggested to be an important mechanistic feature of receptor-coupled
ABC transporters. Kay et al. [62] used the flavin cofactor bound to
each of the two subunits in the homodimer of the human
augmenter of liver regeneration. They reduced the two flavins
to the neutral radical form and obtained well-resolved PELDOR
time traces. The distances correlate well with those from the
X-ray structure. Bowman et al. [63] managed to assess the large
assembly of the tetrameric histone complex.
In the field of nucleic acids, PELDOR has been used to follow
the conversion of two complementary hairpin RNAs into a duplex
RNA [25], ligand binding to the neomycin riboswitch [64] and
magnesium(II)-ion-induced folding of the tertiary stabilized
hammerhead ribozyme [65]. Structures of damaged nucleic acids
yielding bended DNA double helices have also been studied
[46,66].
With respect to generating structures from PELDOR-derived
distances, Jeschke recently published a program called MMM
which is freely available on the Web [67], and Ward et al. [68]
reported a strategy to refine NMR structures using PELDORderived distances as long-range constrains.
MEMBRANES
In contrast with the PELDOR data discussed above, studies on
membrane-bound proteins show, in a lot of cases, only barely
visible dipolar oscillations or just multi-exponential decays. In
such cases, conclusions are mainly qualitative and should be
c The Authors Journal compilation c 2011 Biochemical Society
drawn carefully. This might also be considered when debating
whether α-synuclein adopts an extended [69] or horseshoe
[70] conformation. The problem with spin-labelled proteins
reconstituted into membranes is molecular crowding due to the
confinement into two dimensions and experimentally imposed low
lipid-to-protein ratios [71]. This leads to very close intermolecular
contacts and thus very short T M relaxation times, which shortens
the usable PELDOR time windows to a length of sometimes below
1 μs. In turn, this strongly limits the maximum distance that can be
extracted and causes problems in determining the intermolecular
background because the decay is no longer monoexponential.
The fast echo decay also strongly diminishes the intensity of the
PELDOR signal, which leads to PELDOR time traces with low
signal-to-noise. Thus, although PELDOR is, in principle, ideally
suited to study large membrane protein complexes in membranes
because it is not size-restricted and can assess the necessary
distance range, ways have to be found and sample preparation
protocols established to overcome these challenges. The current
state of knowledge is that molecular crowding can be reduced
by using nanodiscs [72]. In these nanodiscs, the membrane is
confined by proteins allowing the discs to host sometimes only
one membrane protein per disc, but this has to be shown more
widely. An additional possibility is to dilute the intermolecular
interaction by adding non-labelled protein. Below, a few examples
are given where the PELDOR data show modulations and are of
high quality. Zou et al. [73] used PELDOR to define the amplitude
and nature of the ATP-hydrolysis-induced conformational change
in the ABC transporter Msb. They obtained high-quality PELDOR
data for several doubly labelled mutants and distinguished them
from those cases where they don’t by taking the error into
account. Based on their analysis they found that residues on the
cytoplasmic side undergo a 2–3 nm closing motion, whereas a
0.7–1 nm opening motion is observed on the extracellular side.
The transmembrane helices undergo relative movement to create
the outward opening consistent with the X-ray model. Endeward
et al. [74] have been able to resolve the expected three distances
for the trimeric potassium ion channel KcsA both in detergent and
reconstituted into a lipid membrane (Figure 5).
DYNAMICS
The distance distribution is encoded in the damping of the
modulation. The faster the modulation is damped the broader
PELDOR beyond distances
Figure 6
357
Folding of membrane protein monitored by PELDOR
(a) Structure of monomeric LHCII with the spin-labelled residues indicated by numbers. (b) PELDOR time traces of double mutant 90/196 and the corresponding distance distribution. (c) Time course
of the folding event monitored on the two double mutants 106/160 and 90/196. Reprinted from Proc. Natl. Acad. Sci. U.S.A., 106, Dockter, C., Volkov, A., Bauer, C., Polyhach, Y., Joly-Lopez, Z.,
Jeschke, G. and Paulsen, H., Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR, 18485–18490, Copyright (2009), with permission from Elsevier.
Figure 7
Spin-labelling nucleic acids
Spin labels 1 [25], 2 [26] and 3 [27,28] are connected to the base moiety. Label 4 [30] is attached to the phosphate backbone and label 5 [31] to the 2’-sugar side. Label 6 [29] is a rigid
label fused on to a cytosine base, and label 7 [33] is its non-covalent analogue. Reproduced with permission from Reginsson, G. W. and Schiemann, O. (2010) Biochem. Soc. Trans. 39, 128–139.
c the Biochemical Society.
is the distance distribution and vice versa. As mentioned in
the introduction, PELDOR experiments are performed in the
frozen state, which means that real-time measurements of distance
changes/dynamics are not possible. However, freezing a sample
considerably rapidly freezes conformations of a biomolecule
populated at the freezing point of the sample. This means that
PELDOR does not access conformational distributions at 50 K
(the usual measuring temperature), but actually the one populated
at approx. 243 K (− 30 ◦ C), the freezing point of aqueous buffers
with cryoprotectant added. This is still not room temperature, but
much closer to the biologically relevant temperature. It has also
been shown repeatedly that the distance distribution can be correlated with conformational states and dynamics at room temperature
as long as they lead to distance changes between the spin labels.
The laboratory of Jeschke and colleagues demonstrated this nicely
on end-labelled phenyl-acetylene model systems of various length
and in matrixes with different freezing temperatures [75,76].
The dynamics of similar systems have also been analysed by
the laboratory of Prisner using a simple dynamics model [77].
Applications to biological systems are still rare. A very convincing
example is the study of Dockter et al. [78] on the folding of
the major LHCII (light-harvesting chlorophyll a/b complex) of the
photosynthetic apparatus. They used a combination of PELDOR
and rapid freeze–quench to monitor the folding of this complex by
means of taking snapshots along the folding trajectory (Figure 6)
[78].
An early correlation between MD (Molecular Dynamics) simulations and PELDOR-derived distances from doubly spin-labelled
c The Authors Journal compilation c 2011 Biochemical Society
358
Figure 8
G. W. Reginsson and O. Schiemann
Orientation-selective PELDOR measurements on DNA
(a) Schematic representation of a DNA duplex with the Ç spin labels coloured red. Next to it is a schematic representation of the orientation of the two labels with respect to each other and to r . (b)
Field sweep spectrum of the duplex in (a) with the position of the inversion pulse (green profile) and of the detection pulses indicated by arrows. (c) The PELDOR time traces obtained by moving the
detection pulses over the field sweep spectrum. Adapted from [83], with permission.
DNA double helices has been performed by Schiemann et al. [54].
Marko et al. (A. Marko, V. Denysenkov, D. Margraf, P. Cekan, O.
Schiemann, S. T. Sigurdsson and T. F. Prisner, unpublished work)
recently studied the breathing dynamics of 12 DNA duplexes
using the rigid spin label Ç (see Figure 7) and simulated the
orientation-selective X- and G-band (180 GHz) PELDOR time
traces with a correlated stretch–twist breathing model in which
the helix pitch is not changed, but the radius is.
ORIENTATION SELECTION
At X-band frequencies, the spectrum of a frozen nitroxide sample
has a width of approx. 190 MHz, which is governed by the
anisotropy of the hyperfine coupling tensor of the nitrogen
in the NO-bond. The pulse lengths most commonly used for
X-band PELDOR experiments are 16 ns and 32 ns for the π/2
and π pulses of the detection sequence respectively, and 12 ns
for the inversion pulse. The pulses of the detection sequence
and the pump pulse have thus an excitation bandwidth of 16 and
67 MHz respectively and do therefore only excite a fraction of the
nitroxide molecules. Whether this fraction samples all values of
θ depends on the flexibility of the spin-labelled biomolecule and
the field position at which the pulses are applied. Usually, the inversion pulse is applied at the centre of the cavity and at the
maximum of the central line where it excites all orientations
although not equally weighted. For nitroxides, the detection pulses
are usually applied at a 40–90 MHz higher frequency than the
inversion pulse. At a frequency offset of 90 MHz, the detection
sequence excites predominantly the mI = −1 component of AZZ of
the 14 N hyperfine tensor, whereas smaller frequency offsets select
more of the off-diagonal elements and deselect AZZ . At small
frequency offsets between detection sequence and pump pulse,
their excitation profiles overlap in the frequency domain, which
means that some spins are excited by both the detection sequence
and the pump pulse. These spins do not contribute to V(t)intra which
diminishes the PELDOR effect. In addition, the frequency overlap
increases the contribution of the unwanted hyperfine artefacts
in the PELDOR time trace. Those molecules contribute to the
intramolecular PELDOR signal V(t)intra in which one nitroxide
spin has been excited by the detection sequence and the second
by the pump pulse.
If the doubly labelled biomolecule, or at least the nitroxides, are
highly flexible then there is no correlation between the hyperfine
and g-tensors of the individual nitroxides and also not between
c The Authors Journal compilation c 2011 Biochemical Society
the nitroxides’ hyperfine/g-tensors and the distance vector rAB
connecting both. Thus positioning the detection pulses at the low
field edge where they excite those molecules with AZZ parallel to
the outer magnetic field B0 , they still do excite all orientations
of θ because AZZ and rAB have no correlation. However, if the
labels and the biomolecule is rigid, then selecting a specific
hyperfine tensor component selects at the same time a specific
value of θ [80]. Acquiring PELDOR time traces with different
frequency offsets and subsequently simulating them enables one
to determine the orientation of the two nitroxides with respect to
rAB . A second method for a direct extraction of orientations from
the set of time traces is based on Tikhonov regularization and has
been published by Marko et al. [81]. At X-band alone, one would
mainly be able to resolve the orientation of AZZ with respect
to rAB ; however, including PELDOR measurements at higher
fields/frequencies may allow resolving of all angles.
For nitroxides, orientation selectivity has been shown first on
bisnitroxide model systems using X- and S-band frequencies [44]
and later also at W-band [82]. Although these studies were able
to obtain orientation-selective PELDOR time traces, they were
hampered by the rather large rotational degree of freedom of the
nitroxides used. The laboratory of Sigurdsson recently designed
the so-called rigid spin label Ç for nucleic acids (Figure 7, label 6
[29]). The nitroxide ring of this label is fused on to a cytosine base
which base-pairs via three hydrogen bonds to an opposing guanine
base, which strongly reduces its rotational freedom. Utilizing this
label incorporated pairwise into DNA duplexes, Schiemann et
al. [83] showed that strong orientation selectivity can already be
obtained at X-band frequencies (Figure 8).
The laboratories of Steinhoff [84] and Prisner [74]
demonstrated that orientational information can also be obtained
from X-band PELDOR measurements using the conventional
spin label MTSSL if this label’s rotational degree of freedom
is inhibited by interactions with the protein. Beyond nitroxides,
the power of high-field/high-frequency PELDOR in determining
orientations has been shown on RNR (ribonucleotide reductase)
[85,86]. This study was able to fully resolve the orientation
of two tyrosyl radicals within the RNR by stepping the pump
and detection pulses with a fixed frequency separation over
the tyrosyl radical spectrum. It should be kept in mind that
analysis of PELDOR time traces affected by orientation selectivity
cannot be performed with the current version of DeerAnalysis
(DeerAnalysis2010), since it assumes that all orientations of θ
are excited [48].
PELDOR beyond distances
359
EXCHANGE COUPLING
An indication for a non-zero J is that the dipolar frequencies
ν dd for the perpendicular and the parallel orientation of rAB with
respect to B0 do not behave like 1:2. If the isotropic exchange
coupling constant J has to be taken into account, the coupling
between both spin centres is the sum of the dipolar contribution
ν dd and J (eqn 5) [10]:
vAB = vdd + J
(5)
Up to now, no example has been published where an exchange
coupling needed to be taken into account for the interpretation
of PELDOR data from a biomolecule. The reason for this is that
the distances measured with PELDOR are usually above 2 nm.
Since the through-space or direct orbital overlap contribution to
the exchange coupling constant decays exponentially with the
distance, this contribution can be neglected at such large distances.
The through-bond contribution can be considerable even at such
distances, but only if the connecting bonds provide a conjugated
bridge [87–89]. For the biomolecules studied up to now this is
not the case, but one might consider this carefully for nucleic acid
structures [90]. If J is non-zero, a recent study on bisnitroxide
model systems revealed that the magnitude and sign of J can
be determined from the PELDOR data [91]. The effect of J on
the Pake pattern is that the two halves of this pattern are shifted
against each other. How much depends on the magnitude of J
and the direction of the shift on the sign as predicted in silico
previously [10] and as known from ENDOR (electron-nuclear
double resonance) spectra. With the possibility of reading off
two frequencies for ν AB at the maximum and the edge of the
Pake pattern one can build two equations from eqn (5) which
can than be rearranged into eqns (6) and (7), yielding ν dd and J
respectively:
v⊥ − v
3
(6)
2v⊥ + v
3
(7)
vdd [MHz] =
J [MHz] =
However, as outlined in the same paper [91], this always yields
two solutions which one might distinguish using simulations. Also
here, a word of warning, the DeerAnalysis program in its current
version does not account for J.
MULTISPIN SYSTEMS
Most of the studies described above have dealt with two coupled
spin centres/two nitroxides per biomolecule. However, PELDOR
is also suited to resolve distances between more than two
nitroxides and it can be used to actually count the number of
interacting spin centres. The latter can be very useful if one wants
to know how many proteins are coming together to form a complex. In order to count the number of interacting spins in one
complex, one is not interested in the frequency of the modulation,
but the depth V λ of the modulation. V λ is the signal intensity after
intermolecular background correction and at long times t where
all modulation in the time trace is damped (Figure 3b) [47]. From
the value of V λ , the number of spins coupled in one complex can
be calculated according to eqn (8) as verified experimentally by
Bode et al. [92] (Figure 9):
n=
InVλ
+1
In (1 − λB )
(8)
Figure 9
spins
Dependence of the modulation depth on the number of coupled
Monomer (green line), dimers (blue lines), trimers (red line) and tetramer (black line).
Reproduced with permission from [92]. Copyright 2007 American Chemical Society.
The only free parameter in the calculation of n is λB , the
fraction of spins inverted by the pump pulse. The value of λB
can be determined from an independent PELDOR experiment
of a standard biradical measured under the same buffer and
spectrometer conditions (pulse lengths, cavity coupling, etc.).
From its V λ value, λB is calculated as 1 − V λ . In general, care
has to be taken that: (i) all modulation is damped, this requires
long time traces, which can be a problem for large distances; (ii)
the intermolecular background has to be fitted carefully, as bad
fits can give wrong V λ values; (iii) in the case of mixtures of
oligomers, one has to work with the weighted ratios and account
for dipolar relaxation differences [92]; and (iv) higher-order spin
correlations are currently not included in DeerAnalysis2010,
so using this program for multi-spin systems may give wrong
distance distributions [93].
Milov et al. [94] were the first to use PELDOR to estimate
that four trichogin GAIV molecules come together to form a
membrane-spanning channel [94] and Banham et al. [95] used it
to conclude that three von Willebrand A factors form the active
complex [95]. A study on the octameric carbohydrate export
channel WzA found good agreement between the modulation
depth and the octameric state of the complex in the crystal
[96]. Interestingly, for long time windows T, the observed
modulation depth of ∼ 50 % rather corresponded to a dimer.
However, shortening the pulse sequence revealed an increase in
V λ and, extrapolating V λ back to a pulse sequence length of zero,
yielded the V λ of 0.98 expected for an octamer. The rationale
for this behaviour was incomplete spin labelling and different
relaxation times for octamers carrying different amounts of spin
labels.
METAL IONS
PELDOR can be used to measure distances not only between
organic radicals, but also between metal centres or metal centres
and organic radicals. Metal centres can be challenging for
PELDOR measurements because of (i) very short T M relaxation
times leading to short PELDOR time windows and low signalto-noise ratios, (ii) very broad spectral widths due to large ganisotropies, which decreases the signal-to-noise ratio further and
can prevent, due to the limited bandwidth of the resonator, a free
c The Authors Journal compilation c 2011 Biochemical Society
360
G. W. Reginsson and O. Schiemann
Figure 10 PELDOR time traces (light grey, original time trace; black background, corrected time trace) of protein p75ICD labelled 2-fold with the gadolinium
complex shown on the far right
Adapted with permission from [108]. Copyright 2010 American Chemical Society.
choice of pump and probe positions, (iii) high-spin states that can
interfere with the PELDOR experiment, and (iv) delocalization
of spin density into ligands, in which case the point dipole
approximation has to be extended.
Copper(II) ions have been used several times for PELDOR
measurements because their T M values and spectral width are
not too extreme and it is an electron spin S = 1/2 system.
The laboratory of Huber was the first to measure Cu–Cu
distances in the covalently linked dimer of the electron transfer
protein azurin, showing that such measurements are possible,
and gaining information about the arrangement of the two
monomers in the complex without spin labelling [97]. Kay
et al. [98] published PELDOR measurements between two
copper(II) ions in the dicupric human serum transferrin and
lactoferrin [98]. The high-quality data supported the arrangement
of the two subdomains. Jeschke’s laboratory published in 2002
a first study between two nitroxides and a copper(II) ion in
a model complex demonstrating that the nitroxide–nitroxide
distance can be individually measured and distinguished from
the copper(II)–nitroxide distances by choosing adequate pump
and probe positions [99]. Subsequent publications on copper–
porphyrin–nitroxide model systems showed how to deal with spin
delocalization into the ligand and how distributions of exchange
coupling constants can be mistaken for a distribution in rAB
[100,101]. This analysis also included dynamics of the model
system and orientation selection. Another thorough analysis also
including dynamics of copper(II) ion materials has been published
by Lovett et al. [102,103].
The laboratory of Bittl analysed PELDOR measurements
between an iron–sulfur cluster and a nickel–iron centre in the hydrogenase from Desulfovibrio vulgaris Miyazaki F taking the spin
projection factors for the individual iron centres in the cluster into
account [104]. Already in the last century, Kawamori’s laboratory
did PELDOR measurements involving the manganese cluster in
PSII in its S = 1/2 state [105,106]. The laboratory of Goldfarb is
the first who successfully performed PELDOR measurements on
a high-spin metal centre, a gadolinium(III) complex [107]. They
1
could select the mS = +
− /2 transition by performing the PELDOR
measurements at 95 GHz. An interesting property of this complex is that the relaxation times are rather long and the EPR
spectrum is dominated by a single line. This, together with the
worked out derivatization of this complex with a thiol group,
makes this gadolinium complex a promising candidate for a new
spin label (Figure 10) [108].
c The Authors Journal compilation c 2011 Biochemical Society
CONCLUSIONS
PELDOR has emerged as a powerful biophysical method and is
applied to soluble and membrane proteins, nucleic acids and
complexes thereof. The biomolecules can either be spin-labelled
with nitroxides or one makes use of intrinsic radical cofactors,
amino acid radicals or paramagnetic metal centres. PELDOR
not only measures mean distances with high resolution and
reliability, but also yields distance distributions, which enable
access to dynamics and conformational distributions. It can count
the number of singly spin-labelled biomolecules in a complex
and, if an exchange coupling mechanism is present, it can be
easily disentangled. Importantly it can also resolve the orientation
of labels, which makes a correlation of the nitroxide–nitroxide
distance with the biomolecular structure easier. The latter two
properties, together with the distance precision, small labels and
that not two different labels are needed makes this method highly
complementary to FRET. And indeed, the first papers are appearing where both methods are combined [109]. Also promising
are recent technological advances that enable high field/highfrequency PELDOR measurements with high power/short pulses
[110,111]. These new spectrometers have increased sensitivity,
allowing concentrations as low as 1 μM to be measured [111],
and enable highly orientation-selective measurements.
FUNDING
Work in our laboratory is supported by the Biotechnology and Biological Sciences Research
Council [grant numbers BB/H017917/1, F004583/1]. O.S. thanks the Research Councils
of the U.K. for a fellowship and G.W.R. thanks the School of Biology, University of St
Andrews for a SORS (Scottish Overseas Research Students) Scholarship.
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Received 15 November 2010/30 November 2010; accepted 1 December 2010
Published on the Internet 24 February 2011, doi:10.1042/BJ20101871
c The Authors Journal compilation c 2011 Biochemical Society