Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells

Article abstract of DOI:10.1002/anie.201609085

Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl–iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement.

Full text of DOI:10.1002/anie.201609085

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201609085
German Edition: DOI: 10.1002/ange.201609085
EPR Spectroscopy
Versatile Trityl Spin Labels for Nanometer Distance Measurements on
Biomolecules In Vitro and within Cells
+
+
J. Jacques Jassoy , Andreas Berndhꢀuser , Fraser Duthie, Sebastian P. Kꢁhn,
Gregor Hagelueken, and Olav Schiemann*
[
10,11]
Abstract: Structure determination of biomacromolecules
under in-cell conditions is a relevant yet challenging task.
Electron paramagnetic resonance (EPR) distance measure-
ments in combination with site-directed spin labeling (SDSL)
are a valuable tool in this endeavor but the usually used
nitroxide spin labels are not well-suited for in-cell measure-
ments. In contrast, triarylmethyl (trityl) radicals are highly
persistent, exhibit a long relaxation time and a narrow spectral
width. Here, the synthesis of a versatile collection of trityl spin
labels and their application in in vitro and in-cell trityl–iron
distance measurements on a cytochrome P450 protein are
described. The trityl labels show similar labeling efficiencies
and better signal-to-noise ratios (SNR) as compared to the
popular methanethiosulfonate spin label (MTSSL) and ena-
bled a successful in-cell measurement.
enhancement (RIDME)
better suited methods than
pulsed electron–electron double resonance (PELDOR or
[
12,13]
DEER).
Recently, trityl derivatives for labeling materi-
als, oligonucleotides and proteins have been reported.
However, trityl labeling of biomolecules is still very limited.
Oligonucleotides can only be labeled at their ends and the
two protein labels show either low labeling yields or long
[
14]
[7]
[15]
[
16]
[
17]
[
18]
linker groups.
Here, a series of trityl spin labels 1–4 (Figure 1) with
different bioconjugation moieties is presented, designed for
the SDSL of proteins, DNA, and RNA by means of disulfide
bridging, thioether bonding, Sonogashira coupling or copper-
(I)-catalyzed alkyne–azide cycloaddition (CuAAC) reactions.
Their labeling efficiencies are tested on the Pseudomonas
putida cytochrome P450 CYP101 mutant C58 for labels 1–3,
and on the membrane protein FeoB mutant R152 from E. coli
BL21 for label 4. Labels 1 and 3 were then used to
I
n the last two decades, EPR based dipolar spectroscopy has
III
become an increasingly effective tool for the measurement of
demonstrate first applications of a trityl–Fe distance mea-
surement on cytochrome P450 in vitro and within cells.
Previous in-cell measurements focused on interlabel distances
[1]
nanometer distances in biomolecules. Up to date, the most
frequently applied kind of spin labels are nitroxides, which
have been successfully used for a large variety of different
distance measurements. However, it is well-known that
nitroxides are sensitive to reducing environments such as
[2]
[3]
encountered within cells and exhibit generally short relax-
[4]
ation times at room temperature in aqueous solutions. To
overcome these limitations, alternative spin labels are cur-
rently under development. Among them are derivatized
[
5]
III
[6]
nitroxides,
Gd -complexes
and triarylmethyl radical
[
7]
(
trityl) spin labels. The latter ones show microsecond
[8]
relaxation times in buffer at room temperature and are
[9]
stable under the reducing conditions of the cell. They are
thus promising candidates for in-cell measurements at
physiological temperatures. Furthermore, their narrow spec-
tral width allows for a better SNR than achievable for
nitroxides, helps avoiding orientation selectivity and can
make single frequency distance measurements techniques
like for example, relaxation-induced dipolar modulation
[
+]
[+]
[
*] Dipl.-Chem. J. J. Jassoy, Dipl.-Chem. A. Berndhꢀuser,
B. Sc. F. Duthie, B. Sc. S. P. Kꢁhn, Dr. G. Hagelueken,
Prof. Dr. O. Schiemann
Institute of Physical and Theoretical Chemistry
University of Bonn, Wegelerstr. 12, 53115 Bonn (Germany)
E-mail: schiemann@pc.uni-bonn.de
+
[
] These authors contributed equally to this work.
Supporting information, including synthesis protocols, cw EPR
simulations, and original RIDME time traces, and the ORCID
identification number(s) for the author(s) of this article can be found
under http://dx.doi.org/10.1002/anie.201609085.
Figure 1. The synthesized trityl spin labels 1–4 and the trityl precursor
5 (bioconjugating moieties set in bold).
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[
6]
[19,20]
involving gadolinium and nitroxide
spin pairs. Recently,
The labeling procedures for trityl labels 1–3 were estab-
lished on the C58 mutant of the cytochrome P450 CYP101
from Pseudomonas putida, and for trityl label 4, on the FeoB
mutant R152 carrying the unnatural amino acid 4-azido-l-
phenylalanine (Scheme 1). All trityl labeling reactions where
a trityl/nitroxide spin pair was investigated in native mem-
[
15]
brane proteins. The report here shows distance measure-
ments between a trityl spin label and a native metal cofactor
in vitro and within cells.
The precursor compound for all presented spin labels is
the Finland Trityl 5 (Figure 1), which was synthesized follow-
[
21–24]
ing a combination of known protocols.
Slight modifica-
tions led to a noticeable increase of the overall yield for 5
[
23]
from 40% to 56% compared to the latest benchmark.
The idea was to attach the bioconjugating moieties to 5
using a mild and viable esterification reaction. The use of
conventional esterification reagents such as DCC and HOBt/
BOP results in amidification products of 5 or no conversion at
all. However, using 2-chloro-1-methylpyridinium iodide
[
25]
(
CMPI) as activating reagent,
obtained in high-yielding statistical esterification reactions
details see the Supporting Information). The monofunction-
compounds 1–4 were
(
alization is essential, since it prevents label-mediated bridging
between investigated sample compounds. In addition, the two
carbon acid groups in 1, 3 and 4 support the water solubility of
the labels while the fully esterified and therefore more
hydrophobic label 2 can be advantageous for the labeling of
Scheme 1. Labeling reactions with trityl labels 1–4. DDM=n-Dodecyl-
b-d-maltoside; TBTA=Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.
[
15]
membrane proteins
thesis.
and of oligonucleotides during syn-
[
26]
performed under similar conditions as for the corresponding
nitroxides but using a pH of 8 in order to increase the water
solubility of the trityl labels (details see the Supporting
Information). Labels 1 and 2 represent trityl analogs to the
widely used nitroxide spin label MTSSL and connect to
cysteine residues by disulfide bridging. The linker formed
with 1 and 2 is three bonds longer than with MTSSL, but is
one bond shorter than the linker of the recently published
All four labels were characterized by high-resolution mass
spectrometry (see the Supporting Information) and continu-
1
ous-wave (cw) EPR measurements (see Figure 2). The H and
1
3
C hyperfine coupling constants, linewidths and g-values are
[18]
typical for trityl radicals (see the Supporting Information).
[
18]
trityl label TAM-MTS. However, in silico predictions show
that this has only minor effects on the label volume.
Compound 2 is not water soluble and the corresponding
labeling procedure requires the usage of detergents. Label 3
also targets cysteine residues but forms a more stable
thioether bond to the protein, thus making it suitable for in-
[
6,27]
cell experiments.
non-canonical amino acids carrying an azide moiety and is
Label 4 targets commercially available
[
28]
bioconjugated by a CuAAC click reaction. It can thus be
used for orthogonal labeling strategies and in cases where
cysteines are not suitable labeling sites. Additionally, com-
pound 4 may also be attached to oligonucleotides either by
CuAAC or by palladium-catalyzed coupling reactions.
The persistence of the trityl radical against reducing agents
sodium ascorbate) is essential for the CuAAC method and
[29]
[30]
(
the formed triazole linkage is again well suited for in-cell
experiments.
Figure 2. Room-temperature cw X-band EPR spectra of labels a) 1,
b) 2, c) 3 and d) 4 in dry, degassed DMSO.
The labeling efficiencies of labels 1–4 were determined by
cw X-band EPR (see the Supporting Information) and
compared to the same cytochrome P450 mutant labeled
with the nitroxide MTSSL. The labeling efficiency of 1 and 2
is with ꢀ 80% of the same order of magnitude as for MTSSL.
However, it should be noted that the low polarity of 2 causes
a visible amount of protein to precipitate during the labeling
procedure. The labeling efficiency for 4 is with 56% notice-
ably lower than that of MTSSL, but is comparable with the
labeling efficiencies reported for such a reaction involving
The stability of radical centers 1–4 was tested by following
their EPR signal intensity at room temperature over a time
span of 3 h in DMSO and in TRIS-buffer (pH 7.4) in the
presence and absence of oxygen (see the Supporting Infor-
mation). None of the labels showed a decay under these
conditions rendering the radical center stable under the
aqueous and atmospheric conditions of the labeling proce-
dures.
2
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[
28]
a modified Alexa fluorophore (50%) as well as with the
reaction of an inversely functionalized nitroxide radical
(
Information), as the double integral of the iron signal for
equally concentrated samples is 171 a.u. and 263 a.u. for
C58T1 and C58R1, respectively, yielding a ratio of 0.65, which
matches the ratio of the modulation depths. These differences
in iron contents in CYP101 preparations are a known
[
31]
64%). The reason for the lower labeling yield as compared
to MTSSL is that the in vivo protein expression leads to
partial reduction of the non-canonical azide group of the
[
32]
[31,35,36]
amino acid. The labeling efficiency for 3 is with 36% lower
phenomenon.
than what was reported for a similar reaction in the literature
The distance distribution for C58T1 (Figure 3b) reveals
two maxima at 2.0 nm and 3.5 nm with a standard deviation of
0.2 nm and 0.15 nm, respectively. To translate this spin–spin
distances into biologically relevant CAlpha distances, it is
necessary to match them with in silico predictions, for
[
33]
(
70%),
which is attributed to the comparatively mild
labeling conditions that were applied here.
Since CYP101 contains iron(III) as an intrinsic para-
magnetic cofactor, labeling the protein with one trityl only is
sufficient for enabling distance measurements between the
trityl label and the iron center. Such measurements are
desirable since metal cofactors play a critical role in many
biological processes and their localization within a biological
[37]
example, using mtsslWizard. At this point, however, the
mtsslWizard prediction differs from the experimental result in
terms of mean distance and width of distribution, although it
encompasses the experimental distribution and also predicts
the experimentally obtained bimodality, which is probably
due to label–protein interactions. In order to exclude that the
bimodality is an artifact of the background correction, the two
peaks were demonstrated to prevail for various backgrounds
(see the Supporting Information). Compared to C58R1,
which revealed a nitroxide–Fe distance of 3.06 nm with
a standard deviation of 0.11 nm (Figure 3d), the T1 side-
chain shifts the distribution to 0.4 nm longer distances
because of the three bonds longer linker and causes a broad-
ening of the distribution.
[34]
structure
is often crucial for the understanding of for
example, a function of protein. Previous studies indicated,
that for distance measurements involving the CYP101 low-
[
35]
spin iron(III), RIDME is better suited than PELDOR.
Therefore, trityl labels 1 and 3 were tested for such RIDME
measurements. Labels 1 and 3 were chosen because 1 is the
analog of the nitroxide standard MTSSL and 3 enables in-cell
measurements.
In Figure 3a, the in vitro Q-band RIDME time trace of
CYP101 labeled with 1 (C58T1) and the corresponding
distance distribution are shown. The time trace is well-
Also, in the case of CYP101 mutant C58 labeled with 3
(C58T3), a modulated time trace could be obtained with
À1/2
modulated and has a high SNR of 20.84 S/Nmin . For the
À1/2
sake of comparison, also the RIDME data for the MTSSL-
labeled analog is shown (C58R1). Here, the SNR was
a SNR calculated as 10.85 S/Nmin . The decrease in SNR
relative to CYP101 mutant C58T1 is due to the lower labeling
efficiency of 3. The modulation depth of this sample is only
7% and thus a factor of 3 lower than found for C58T1, but
matches again with an iron content reduced by the same
factor (see the Supporting Information). However, even with
this rather low labeling efficiency and low iron content a good
quality RIDME time trace could be obtained. The distance
À1/2
calculated as 13.08 S/Nmin . Thus, the trityl sample pro-
vides a 60% better SNR than the MTSSL sample, which
matches expectations, as only about 40% of the nitroxide
spectrum but all of the trityl spectrum is excited. The
observed modulation depth for C58T1 is 15% lower than
the 35% obtained for C58R1. However, this can be attributed
to a lower iron content in CYP101 C58T1 (see the Supporting
III
distribution for C58T3 reveals a trityl–Fe distance with
maxima at 2.4 nm and 3.5 nm and standard deviations of
0
.25 nm and 0.12 nm, respectively. Within error, the mtsslWi-
zard prediction matches the experimental inasmuch as it
predicts a bimodal distribution, but again differs in terms of
mean distance and width of distribution. The distance
distributions obtained for labels 1 and 3 are of a smaller
width than those found in the literature for other trityl labels
where standard deviations of 0.19 nm and 0.37 nm were
[
15,17]
reported.
This is partially due to a smaller label volume
and a smaller number of rotatable bonds (see the Supporting
Information), but it should be considered that the width of the
distribution is also dependent on the labeling site and
therefore not immediately comparable between different
proteins.
In order to demonstrate that in-cell measurements are
feasible, xenopus laevis oocytes were injected with CYP101
C58T3 (final in-cell concentration 250 mm). The injected cells
were placed in a quartz tube, shock frozen and subjected to
Figure 3. a) Q-band RIDME time traces of CYP101 mutant C58 labeled
with 1 (blue solid line), 3 (green solid line) and MTSSL (red solid line)
overlayed with the corresponding DeerAnalysis fits (black dashed
lines). The corresponding distance distributions are shown in b) for
C58T1, c) C58T3 and d) C58R1. The purple lines are the mtsslWizard
predictions.
III
trityl-Fe Q-band RIDME measurements (Figure 4).
The obtained RIDME time trace (see the Supporting
Information) was background corrected using a polynomial of
the same order as for the in vitro time trace revealing
À1/2
a modulation depth of 4% and a SNR of 1.97 S/Nmin . The
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and standard deviations of 0.16 nm and 0.2 nm, respectively.
The smaller peaks at 1.8 and 3.1 nm are attributed to the
noise. The in-cell distance distribution is thus slightly broader
than the distribution obtained in vitro, and shows a shift of the
mean distances by roughly 1 ꢀ. This can either be due to the
effect of noise on the DEER analysis fit, or may signify
a small change of conformation of the protein under in-cell
conditions. The current data does not allow a distinction
between the two possibilities, but future studies of different
labeling positions may allow estimating the extent of a pos-
sible structural difference in vitro and in cell.
Figure 4. a) The time trace of in-cell RIDME on 3 (dark blue) and its
DEER Analysis fit (cyan). b) The corresponding distance distribution
(
blue line) overlayed with the in vitro distribution (green line).
In summary, the synthesis of four new trityl spin labels, the
corresponding labeling procedures and a first application in
III
modulation depth is of a similar order of magnitude as
trityl–Fe distance measurements using RIDME in vitro and
III
compared to Q-band PELDOR results published for Gd
within cells was presented. To the best of our knowledge this
is the first in-cell distance measurement with trityl spin labels
and also the first measurement between a spin label and
a native metal cofactor in cells. The presented trityl labels
work very well for the RIDME experiment, where they have
a significantly better SNR than nitroxide labels and allow an
[
6]
spin labels, although it should be stressed that the low
modulation depth here is due to the difficulty in controlling
the iron content of the CYP101 protein. Also, it should be
II
noted that oocytes contain 40 mm Mn which may reduce the
modulation depth because of relaxation enhancement of the
Fe center compared to the in vitro measurement of the
identically labeled protein. The in-cell SNR is less than for the
in vitro measurement by a factor of 10, because of the factor
III
unambiguous distance determination as opposed to Gd
labels, where higher harmonics of the dipolar coupling
complicate the analysis of the distance distributions, although
it was demonstrated on model compounds that this problem
can be compensated.
used for room-temperature distance measurements because
1
0 smaller protein amount injected into the cells. While no
[
41,42]
III
exact numbers are given for the aforementioned Q-Band
In addition, Gd labels cannot be
III
III
Gd PELDOR measurements, the average SNR of the Gd
[43]
experiments is visibly better than what was achieved for the
measurement presented here. The most likely reason for this
is the 10 fold higher label concentration used for the protein
of their fast relaxation times,
so that trityl spin labels
represent an important addition to the spin label repertoire.
The presented study does thus hold promise for the future
investigation of metal ion binding sites under truly biological
III
sample in the Gd case. Very good SNR was also achieved for
III
[20]
a Gd model compound in cell where the injected amount of
conditions.
spins exceeds the amount of trityl used here by one order of
magnitude higher taking spin labeling efficiencies into
III
III
account. Other works on Gd –Gd distance measurements Acknowledgements
in E. coli cells show a moderately increased SNR but exhibit
only a weak modulation depth of 0.6% or less, at in-cell
concentrations that are again one order of magnitude higher
than what is shown here. Such high concentrations could
not be achieved for the CYP101 protein used here and are
Funding by the Deutsche Forschungsgemeinschaft through
SPP1601 and SFB813 is gratefully acknowledged.
[38]
beyond being biologically relevant in-cell concentrations. Conflict of interest
III
However, recent works on Gd at W-band could achieve
timetraces with better SNR than the timetraces presented
here at concentrations as low as 20 mm. While these results are
very encouraging, the shown timetraces so far did not show
any modulations beyond the initial decay and yielded very
broad distance distributions, although this was to be expected
The authors declare no conflict of interest.
Keywords: distance measurements · EPR spectroscopy ·
in-cell spectroscopy · protein structures · spin labeling
[
39,40]
because of the high flexibility of the investigated protein.
III
The SNR achieved in the trityl–Fe measurements presented
here and its comparison to the results achieved with other
spin labels shows that at this point, distances of about 3.5 nm
constitute an upper limit for what can be measured within
cells. This is mainly due to the drastic SNR decrease after
[
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Manuscript received: September 16, 2016
Revised: November 3, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
EPR Spectroscopy
J. J. Jassoy, A. Berndhꢁuser, F. Duthie,
S. P. Kꢂhn, G. Hagelueken,
O. Schiemann*
&&&& — &&&&
Versatile Trityl Spin Labels for Nanometer
Distance Measurements on
Biomolecules In Vitro and within Cells
Distance measurements: Four new triar-
ylmethyl (trityl) labels for the site-directed
spin labeling of proteins and oligonucle-
otides have been synthesized. Their ap-
plication in EPR distance measurements
is demonstrated on CYP101 protein
under in vivo conditions
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!