A Reactive, Rigid GdIII Labeling Tag for In-Cell EPR Distance Measurements in Proteins

Article abstract of DOI:10.1002/anie.201611051

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5–10 μm.

Full text of DOI:10.1002/anie.201611051

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201611051
German Edition: DOI: 10.1002/ange.201611051
Structure Elucidation
A Reactive, Rigid GdIII Labeling Tag for In-Cell EPR Distance
Measurements in Proteins
Yin Yang+, Feng Yang+, Yan-Jun Gong, Jia-Liang Chen, Daniella Goldfarb,* and Xun-
Cheng Su*
Abstract: The cellular environment of proteins differs consid-
erably from in vitro conditions under which most studies of
protein structures are carried out. Therefore, there is a growing
interest in determining dynamics and structures of proteins in
the cell. A key factor for in-cell distance measurements by the
double electron–electron resonance (DEER) method in pro-
teins is the nature of the used spin label. Here we present
a newly designed GdIII spin label, a thiol-specific DOTA-
derivative (DO3MA-3BrPy), which features chemical stability
and kinetic inertness, high efficiency in protein labelling,
a short rigid tether, as well as favorable spectroscopic proper-
ties, all are particularly suitable for in-cell distance measure-
ments by the DEER method carried out at W-band frequencies.
The high performance of DO3MA-3BrPy-GdIII is demon-
strated on doubly labelled ubiquitin D39C/E64C, both in vitro
and in HeLa cells. High-quality DEER data could be obtained
in HeLa cells up to 12 h after protein delivery at in-cell protein
concentrations as low as 5–10 mm.
and it is generally applicable to a limited number of
proteins.[1a,2] DEER is a method that provides distance
distributions between pairs of, usually identical, spin labels
that are attached to a bio-macromolecule at well-defined
sites.[3] Its distance accessibility is usually in the 1.6–8 nm
range[3] and can increase to 16 nm if the protein is deuter-
ated.[4] Owing to its inherently high absolute sensitivity as
compared with NMR spectroscopy, and its insensitivity to
protein size and background signals, DEER can become an
efficient method for in-cell structural studies of proteins.[5]
The first in-cell DEER experiments using standard nitro-
xide spin labels were reported in oocytes, where the low
stability of the nitroxide radicals in the reducing cellular
environment was noted.[5a] This is not a problem when the
nitroxide spin label is exposed to the outside of the cell
environment,[6] but otherwise it pauses a significant limitation
on this methodology. Thus, for the in-cell DEER method to
become a significant and viable technique, stable spin labels
are required to explore the protein interactions and dynamics
in the cell. New nitroxide spin labels are currently under
development but so far they have not been demonstrated in
in-cell DEER experiments.[7] Recently, RIDME distance
measurements between a trityl spin label and intrinsic FeIII
in CP450, requiring 80 h accumulation time, was reported in
oocytes.[8] An attractive alternative approach is to use GdIII-
based spin labels, which have already been reported for many
in vitro GdIII–GdIII DEER applications.[9]
C
haracterization of the dynamics, interactions and struc-
tures of proteins is an important way of delineating their
functions. Current biophysical methods used to explore
protein dynamics and structures are generally applied in vitro,
under conditions that differ considerably from the cellular
milieu. In the cell, molecular crowding, sub-organelle local-
ization, post-translational modifications, and specific and
non-specific associations with cellular components may
inevitably affect the structure and conformational equilibria
of proteins.[1] Therefore, effective methods for exploring the
atomic resolution structure and dynamics of proteins in their
native cellular environment are highly desirable. Currently,
NMR spectroscopy is probably the most effective method for
determining structure and dynamics in the cell at atomic
resolution. However, its low sensitivity invariably requires
expensive isotope labeling and high protein concentrations,
We have recently reported on in-cell W-band GdIII–GdIII
DEER distance measurements of maleimide-DOTA-GdIII-
labeled proteins in human HeLa cells.[10] The drawback of the
maleimide-DOTA tag is its rather long and flexible tether. In
addition, there were reports that thiomaleimide conjugates do
not exhibit long-term stability in cells due to the thiol
exchange with glutathione and the hydrolysis of succinimide
ring.[11] Q-band DEER experiments on a 4-vinyl PyMTA-
GdIII conjugated peptide injected into oocytes was reported as
well.[12] However, the low reactivity of this tag towards protein
thiols results in unfavorable labeling conditions for a pro-
tein.[13] More recently, a lanthanide binding peptide was fused
into a helical bundle peptide both at the N- and C-termini,
GdIII was supplemented with high concentration (100 to
500 mm) through the growing media and W-band in-cell
spectra were recorded.[14] The main problem with this
approach is the low binding constant of GdIII and the long
measurement time (72 h) owing to the background of large
amounts of free GdIII, which affects the signal-to-noise ratio.
In general, efficient in-cell DEER measurements require
high stability of the spin label, a stable linker between the
protein of interest and the spin label and a minimal size of the
[*] Dr. Y. Yang,[+] Prof. D. Goldfarb
Department of Chemical Physics
Weizmann Institute of Science
Rehovot 76100 (Israel)
E-mail: daniella.goldfarb@weizmann.ac.il
F. Yang,[+] Y. J. Gong, J. L. Chen, Prof. X.-C. Su
State Key Laboratory of Elemento-organic Chemistry
Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Nankai University
Tianjin 300071 (China)
E-mail: xunchengsu@nankai.edu.cn
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201611051.
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spin label that will not affect the protein structure. Finally, the
linker should be short and rigid for obtaining a narrow
distance distribution that does not mask protein conforma-
tional changes.
The formation of a thioether bond between a protein and
a GdIII spin label is a promising way to introduce a para-
magnetic center in a protein for in-cell studies.[10,12] The stable
that the labeling does not affect the protein structure and to
assess the mobility of the spin label in the protein conjugate
(Figures S3–6 and Table S1). High-resolution spectra were
recorded and analyzed with 15N-labeled protein samples.
Large pseudo-contact shifts (PCSs) were generated for the
D39C-DO3MA-3BrPy-YbIII and E64C-DO3MA-3BrPy-YbIII
adducts, and similar paramagnetic tensors were determined
for the two protein adducts (using the Numbat program[22]).
The large PCS and the paramagnetic susceptibility tensor
parameters (Table S1) suggest that the protein-DO3MA-
3BrPy-LnIII conjugate is rigid. The excellent agreement
between the experimental and back-calculated PCSs indicates
reliable paramagnetic tensors in the two protein conjugates
(Figure S6), and that no significant structural perturbations
are observed when the paramagnetic tag is introduced.
Similar measurements were carried out on the doubly
DO3MA-3BrPy-YbIII labeled 15N-ubiquitin D39C/E64C
sample. Comparison of experimental PCSs determined in
the doubly labeled protein with the sum of PCSs from singly
labeled protein samples gave a very good agreement.
Compared with the singly labeled proteins, the high-quality
correlation of the experimental data and the calculated PCSs
from the individual paramagnetic tensors indicate that there
are no obvious structural variations in the doubly labeled
D39C/E64C adduct (Figure S6).
Following the encouraging results from the in vitro NMR
characterization, we proceeded to in vitro and in-cell DEER
measurements. The doubly DO3MA-3BrPy-GdIII-labeled
protein was delivered by hypotonic swelling[23] and electro-
poration[10a] into HeLa cells. Echo-detected EPR (ED-EPR)
and DEER measurements were carried out as a function of
time after the delivery into the cells.
Figure 2 presents the 10 K W-band ED-EPR spectrum of
ubiquitin D39C/E64C labeled with DO3MA-3BrPy-GdIII
recorded in vitro (frozen solution) and in HeLa cells. The
width of the central transition (full width at half height),
À
C S bond can be generated by either a nucleophilic sub-
stitution reaction between the solvent-exposed protein thiols
and haloacetateamide derivatives,[15] phenylsulfonyl[16] or
nitro-substituted pyridines,[17] methylsulfonylbenzothiazole
derivatives,[18] or a Michael addition-like thiol-ene reaction.[19]
The single-armed (4-phenylsulfonyl) pyridine-substituted
DOTA-like tags (DO3MA-Py, Py represents the 4-phenyl-
sulfonyl pyridine) were demonstrated as stable and rigid
paramagnetic tags in protein analysis by paramagnetic NMR
spectroscopy both in vitro and in living cells.[20] The coordi-
nation of the pyridine nitrogen to the metal ion restricts the
flexibility of the spin label and hence, it should report
narrower distance distributions in DEER measurements
compared with the maleimid-DOTA tag. However, the low
reactivity of these DO3MA-Py tags requires a high pH (ca.
9)[20a] or a high temperature and a long incubation time (408C
and 20 hours, respectively)[20b] for protein modifications.
These conditions are not suitable for efficient and general
protein modifications. Here we present a new reactive thiol-
specific
DOTA-derivative,
(2R,2’R,2’’R)-2,2’,2’’-(10-(5-
bromo-4-(phenylsulfonyl)pyridin-2-yl)methyl)-1,4,7,10-tet-
raazacyclododecane-1,4,7-triyl)tripropanoic acid (DO3MA-
3BrPy) (Figure 1), for efficient in-cell DEER measurements,
as demonstrated on ubiquitin in human HeLa cells.
The synthesis of the DO3MA-3BrPy tag and the forma-
tion of the GdIII complex are given in the Supporting
Information. A double cysteine mutant of ubiquitin, D39C/
E64C, was expressed and purified. The ligation of DO3MA-
3BrPy-GdIII with D39C/E64C ubiquitin at pH 8 was com-
pleted within six hours at room temperature with a ligation
yield of about 80% (see the Supporting Information). This is
significantly faster than the previously reported 4PhSO2-
PyMTA, DO3MA-Py, and DO8M-Py tags.[16,20] The increased
reactivity of DO3MA-3BrPy-GdIII towards solvent exposed
cysteines, imparted by the Br substitution is consistent with
earlier findings.[21]
In vitro paramagnetic NMR measurements were carried
out on the DO3MA-3BrPy-YbIII-tagged single and double
cysteine mutants, D39C, E64C, and D39C/E64C to ensure
Figure 2. The central transition region of the 10 K W-band ED-EPR
spectrum of ubiquitin D39C/E64C-DO3MA-3BrPy-GdIII in vitro (dashed
line) and in cells, frozen 2 hours after delivery by hypotonic swelling
(red) and 5 hours after delivery by electroporation (black), respectively.
The positions of the pump (n1) and observe (n2) frequencies are
denoted by arrows.
Figure 1. An efficient way of tagging proteins with a stable and rigid
GdIII tag for in-cell DEER measurements.
2
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180 MHz, is larger than that of the maleimide-DOTA-GdIII
distance of 4.3 nm[25] (see Figure S8). Considering that the
label (50 MHz).[10b] This difference is attributed to the larger
zero-field splitting (ZFS) arising from the lower symmetry
around the GdIII induced by the coordination of the pyridyl
nitrogen. Notably, strong cellular MnII EPR signals are
present in the in-cell samples.
DEER measurements on ubiquitin D39C/E64C-
DO3MA-3BrPy-GdIII, both in frozen solution and in frozen
HeLa cells, are presented in Figure 3. The position of the
observe and pump pulses are indicated in Figure 2 and the
experimental details are given in the Supporting Information.
labeling sites in the protein are not on a rigid secondary
element, this difference is expected and is likely to originate
from the protein flexibility. The reported distance distribution
widths measured for the ubiquitin S20C/G35C mutant with
MTSSL[5a] and maleimide-DOTA,[10b] were both 1.5 nm. This
is significantly larger than for the D39C/E64C conjugated
DO3MA-3BrPy-GdIII sample, with the reservation that the
labeling sites are different.
To account for the distance distributions, we used the
ubiquitin crystal structure[26] and anchored the DO3MA-
3BrPy-GdIII complex to the sidechains of cysteines incorpo-
rated at positions 39 and 64 (see the Supporting Information
and Figure S9 for details). The GdIII–GdIII distance was
obtained using MtsslWizzard[27] for random variations of the
torsion angles of the cysteine sidechains at the ligation sites
with the paramagnetic tag, whereas all other structural
segments were treated as a rigid body. The obtained distance
distribution, shown in Figures 3B and D as red traces, has
a maximum at 4.1 nm, which is in excellent agreement with
the experimental results.
The in-cell DEER data of D39C/E64C DO3MA-3BrPy-
GdIII delivered into Hela cells are shown in Figure 3 as well.
To test the stability of DO3MA-3BrPy-GdIII-conjugated
ubiquitin in living HeLa cells after hypotonic swelling and
electroporation delivery, the cells were incubated for different
times in the cell media (see the Supporting Information) and
then frozen for ED-EPR and DEER measurements. For
hypotonic swelling two-hour incubation was found to be
sufficient for full cell recovery,[10b,23] whereas for electro-
poration delivery five hours are needed.[10a] The similar
in vitro and in-cell GdIII–GdIII distance distributions indicate
that the structure of ubiquitin reported by the D39C/E64C
spin pair is essentially unchanged in cells. There are some
differences in the distance distribution width for the proteins
delivered by the different methods, of which electroporation
produces a wider distribution for most samples. The data
quality however is not sufficient for drawing unambiguous
conclusions from this observation. In terms of the DEER
modulation depth we observe a general reduction for the in-
cell protein samples compared to the in vitro ones. Moreover,
for hypotonic swelling, the modulation depth changed from
1.1% after 2 h to 0.9% after 7 h and to 0.8% after 12 h. For
the electroporation delivery, the 1 h sample resulted in
a modulation depth of 1.2%, which decreased to 0.7% for
5 h and to 0.5% after 12 h. The presence of cellular MnII
contributes to the background decay and reduces the
modulation depth, but it does not affect the modulation
frequency, that is, the distance distribution. This will be
discussed further later.
Figure 3. W-band DEER data after background correction of D39C/
E64C-DO3MA-3BrPy-GdIII in vitro (bottom trace in each panel) and
inside HeLa cells at different times after delivery by hypotonic swelling
A) and electroporation C) along with the fits obtained using DeerAnal-
ysis.[24] B) and D) are the distance distributions derived from the DEER
traces in (A) and (C), respectively. All traces were shifted for clarity.
The primary data are given in the Figure S7. The red traces in (B) and
(D) correspond to the calculated distance distribution using MtsslWiz-
zard,[27] and are depicted in both panels for better comparison with the
in-cell measurements.
The four-pulse DEER trace of ubiquitin D39C/E64C-
DO3MA-3BrPy-GdIII in vitro (75 mm) is shown in Figure 3A
(bottom trace); clear modulations with a modulation depth, l,
of 1.5% are observed. This l value is lower than that observed
for ubiquitin labeled with maleimide-DOTA-GdIII (5%)[10b]
owing primarily to the broader central transition of DO3MA-
3BrPy-GdIII. The distance distribution derived from the
DEER trace using DeerAnalysis,[24] shown in Figure 3B at
the bottom, has a maximum at 4.2 nm and a width at half
height of 0.7 nm (a standard deviation of 0.37 nm). For this
distance and the EPR central transition linewidth of 180 MHz
we do not expect significant broadening owing to the failure
of the weak coupling approximation used in the data
analysis.[25] This compares favorably to a width of 0.5 nm
(0.34 standard deviation) obtained for a rigid ruler molecule
bearing two GdIII–PyMTA complexes and a Gd(III)–Gd(III)
The ED-EPR spectra of in-cell samples (Figure S10)
reveal a decrease of the intensity of the GdIII signal with
incubation time relative to the MnII signal. We used these
EPR spectra to estimate the in-cell GdIII concentration. The
in-cell ED-EPR spectra were simulated as a superposition of
two spectra: one corresponding to the in-cell MnII back-
ground, which was obtained from the spectrum of cells
without the delivered protein, and the other corresponding to
the delivered protein, obtained from the in vitro sample with
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the known concentration. The bulk Gd(III) concentration,
namely, the concentration in the total volume of the sample,
was then estimated from the relative weight of the above two
spectra. (see Figure S10). We then estimated the in-cell GdIII
concentration, which is higher than the bulk concentration
because the cells do not comprise 100% of the sample
volume, taking into account the number of cells in the sample
and the cell volume (see the Supporting Information for
details). The dependence of the GdIII bulk and in-cell
concentrations on the incubation time for the two delivery
methods is shown in Figure 4. A continuous decrease from 2
to 12 h is observed for hypotonic swelling and the in-cell
concentration after 12 h was estimated to be about 5 mm
(corresponding to two GdIII per ubiquitin, which is equal to
[GdIII] = 10 mm). For electroporation, more protein was deliv-
ered into the cell and the decrease in GdIII concentration in
the cell with time was milder, and obvious changes were
observed after 5 h after the delivery.
that can be efficiently attached to a protein. The new GdIII
spin label demonstrated its excellent performance in W-band
in-cell DEER measurements down to 5–10 mm of in-cell
protein concentrations with accumulation times within 20 h.
Ubiquitin does not produce well resolved 15N-HSQC NMR
spectra in cells because the non-specific interactions with
cellular constituents broaden NMR signals.[28] Our in vitro
and in-cell DEER results indicate that these non-specific
interactions do not affect the protein conformation as
reported herein by the spin labeled D39C/E64C mutant.
The high stability of this new and rigid GdIII spin label up to
12 h in living HeLa cells, along with its relatively small size
and the high efficiency of its conjugation to protein cysteine
residues is likely to find wide applications in delineating the
structure and interactions of proteins in cells using DEER and
NMR methods.
Acknowledgements
This work was supported by Major National Scientific
Research Projects (2013CB910201 and 2016YFA0501202),
the National Natural Science Foundation of China (21473095
and 21273121) to X.-C.S and the Israel Science Foundation
(ISF grant number. 334/14) to D.G. This research was made
possible in part by the historic generosity of the Harold
Perlman Family (D.G.). D. G. holds the Erich Klieger
Professorial Chair in Chemical Physics. We thank Aleksei
Litvinov for carrying out the modeling with MtsslWizzard.
D.G. thanks Phil Selenko for his help with the electroporation
protocol and for many helpful discussions. The help of Yoav
Barak regarding the biochemical aspects of the work is highly
appreciated and so are discussions with Ami Navon regarding
the biochemical aspects of the work.
Figure 4. In-cell (black) and bulk (red) concentrations of ubiquitin
D39C/E64C-DO3MA-3BrPy-GdIII determined by EPR spectroscopy and
Western blots (blue) as a function of incubation time after delivery.
A) Hypotonic swelling delivery. B) Electroporation delivery. Note that
the GdIII concentration determined by EPR corresponds to 0.5 [GdIII],
which matches the protein concentration.
We also determined the in-cell ubiquitin concentration
using Western blots (see Figure S11), and the results pre-
sented in Figure 4 show that there is a reduction in the
concentration of monomeric ubiquitin in the cell with
incubation time. The agreement between the in-cell concen-
trations determined from the EPR spectra and the Western
blots is rather good, considering the error bars.
Conflict of interest
The authors declare no conflict of interest.
Keywords: EPR spectroscopy · protein dynamics ·
protein structures · spin labels · structure elucidation
Using the spectral deconvolution shown in Figure S10, we
estimated the relative contributions of the GdIII and MnII
signals at the observer frequency and found that they
correlate closely with the change in the modulation depth
for both delivery methods (see Figure S12). This indicates
that there is no significant leakage of GdIII from the tag nor is
there a significant detachment of the tag from the protein.
The reduction in the modulation depth is mostly due to the
reduction in the in-cell protein concentration and the
presence of overlapping signals of MnII. The former might
be due to extrusion of the delivered spin-labeled proteins
from the cells during incubation and maybe other degradation
processes.
[1] a) D. I. Freedberg, P. Selenko, Annu. Rev. Biophys. 2014, 43,
171 – 192; b) F.-X. Theillet, A. Binolfi, T. Frembgen-Kesner, K.
Hingorani, M. Sarkar, C. Kyne, C. Li, P. B. Crowley, L. Gierasch,
G. J. Pielak, A. H. Elcock, A. Gershenson, P. Selenko, Chem.
Rev. 2014, 114, 6661 – 6714.
[2] E. Luchinat, L. Banci, J. Biol. Chem. 2016, 291, 3776 – 3784.
[3] a) G. Jeschke, Y. Polyhach, Phys. Chem. Chem. Phys. 2007, 9,
1895 – 1910; b) G. Jeschke, Annu. Rev. Phys. Chem. 2012, 63,
419 – 446.
[4] T. Schmidt, M. A. Wꢀlti, J. L. Baber, E. J. Hustedt, G. M. Clore,
Angew. Chem. Int. Ed. 2016, 55, 15905 – 15909; Angew. Chem.
2016, 128, 16137 – 16141.
[5] a) R. Igarashi, T. Sakai, H. Hara, T. Tenno, T. Tanaka, H. Tochio,
M. Shirakawa, J. Am. Chem. Soc. 2010, 132, 8228 – 8229; b) M.
Azarkh, O. Okle, P. Eyring, D. R. Dietrich, M. Drescher, J.
In summary, we presented a new GdIII spin label
(DO3MA-3BrPy-GdIII) with a rigid tether, a high GdIII
binding constant and high reactivity towards protein thiols
4
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!

Angewandte
Communications
Chemie
´
Magn. Reson. 2011, 212, 450 – 454; c) I. Krstic, R. Haensel, O.
Romainczyk, J. W. Engels, V. Dçtsch, T. F. Prisner, Angew.
Chem. Int. Ed. 2011, 50, 5070 – 5074; Angew. Chem. 2011, 123,
5176 – 5180.
[15] G. T. Hermanson, Bioconjugate techniques, Elsevier, Academic
Press, Amsterdam, 2008.
[16] Y. Yang, J.-T. Wang, Y.-Y. Pei, X.-C. Su, Chem. Commun. 2015,
51, 2824 – 2827.
[6] a) B. Joseph, A. Sikora, E. Bordignon, G. Jeschke, D. S. Cafiso,
T. F. Prisner, Angew. Chem. Int. Ed. 2015, 54, 6196 – 6199;
Angew. Chem. 2015, 127, 6294 – 6297; b) B. Joseph, A. Sikora,
D. S. Cafiso, J. Am. Chem. Soc. 2016, 138, 1844 – 1847.
[7] a) M. J. Schmidt, A. Fedoseev, D. Summerer, M. Drescher,
[17] K. L. Gempf, S. J. Butler, A. M. Funk, D. Parker, Chem.
Commun. 2013, 49, 9104 – 9106.
[18] a) N. Toda, S. Asano, C. F. Barbas, Angew. Chem. Int. Ed. 2013,
52, 12592 – 12596; Angew. Chem. 2013, 125, 12824 – 12828; b) D.
Zhang, N. O. Devarie-Baez, Q. Li, J. R. Lancaster, M. Xian, Org.
Lett. 2012, 14, 3396 – 3399.
´
Methods Enzymol. 2015, 563, 483 – 502; b) A. P. Jagtap, I. Krstic,
N. C. Kunjir, R. Hansel, T. F. Prisner, S. T. Sigurdsson, Free
Radical Res. 2015, 49, 78 – 85; c) M. J. Schmidt, J. Borbas, M.
Drescher, D. Summerer, J. Am. Chem. Soc. 2014, 136, 1238 –
1241.
[19] A. Dondoni, A. Marra, Chem. Soc. Rev. 2012, 41, 573 – 586.
[20] a) F. Yang, X. Wang, B.-B. Pan, X.-C. Su, Chem. Commun. 2016,
52, 11535 – 11538; b) T. Mꢁntener, D. Hꢀussinger, P. Selenko, F.-
X. Theillet, J. Phys. Chem. Lett. 2016, 7, 2821 – 2825.
[21] J. L. Chen, X. Wang, F. Yang, C. Cao, G. Otting, X.-C. Su, Angew.
Chem. Int. Ed. 2016, 55, 13744 – 13748; Angew. Chem. 2016, 128,
13948 – 13952.
[8] J. J. Jassoy, A. Berndhauser, F. Duthie, S. P. Kuhn, G. Hage-
lueken, O. Schiemann, Angew. Chem. Int. Ed. 2017, 56, 177 – 181;
Angew. Chem. 2017, 129, 183 – 187.
[9] A. Feintuch, G. Otting, D. Goldfarb, Methods Enzymol. 2015,
563, 415 – 457.
[22] C. Schmitz, M. J. Stanton-Cook, X.-C. Su, G. Otting, T. Huber, J.
Biomol. NMR 2008, 41, 179.
[10] a) F. X. Theillet, A. Binolfi, B. Bekei, A. Martorana, H. M. Rose,
M. Stuiver, S. Verzini, D. Lorenz, M. van Rossum, D. Goldfarb,
P. Selenko, Nature 2016, 530, 45 – 50; b) A. Martorana, G.
Bellapadrona, A. Feintuch, E. Di Gregorio, S. Aime, D. Gold-
farb, J. Am. Chem. Soc. 2014, 136, 13458 – 13465.
[23] E. Di Gregorio, G. Ferrauto, E. Gianolio, S. Aime, Contrast
Media Mol. Imaging 2013, 8, 475 – 486.
[24] G. Jeschke, V. Chechik, P. Ionita, A. Godt, H. Zimmermann, J.
Banham, C. R. Timmel, D. Hilger, H. Jung, Appl. Magn. Reson.
2006, 30, 473 – 498.
[11] B. Q. Shen, K. Xu, L. Liu, H. Raab, S. Bhakta, M. Kenrick, K. L.
Parsons-Reponte, J. Tien, S. F. Yu, E. Mai, D. Li, J. Tibbitts, J.
Baudys, O. M. Saad, S. J. Scales, P. J. McDonald, P. E. Hass, C.
Eigenbrot, T. Nguyen, W. A. Solis, R. N. Fuji, K. M. Flagella, D.
Patel, S. D. Spencer, L. A. Khawli, A. Ebens, W. L. Wong, R.
Vandlen, S. Kaur, M. X. Sliwkowski, R. H. Scheller, P. Polakis,
J. R. Junutula, Nat. Biotechnol. 2012, 30, 184 – 189.
[25] A. Dalaloyan, M. Qi, S. Ruthstein, S. Vega, A. Godt, A.
Feintuch, D. Goldfarb, Phys. Chem. Chem. Phys. 2015, 17,
18464 – 18476.
[26] R. Ramage, J. Green, T. W. Muir, O. M. Ogunjobi, S. Love, K.
Shaw, Biochem. J. 1994, 299, 151.
[27] G. Hagelueken, R. Ward, J. H. Naismith, O. Schiemann, Appl.
Magn. Reson. 2012, 42, 377 – 391.
[12] M. Qi, A. Gross, G. Jeschke, A. Godt, M. Drescher, J. Am.
Chem. Soc. 2014, 136, 15366 – 15378.
[28] T. Sakai, H. Tochio, T. Tenno, Y. Ito, T. Kokubo, H. Hiroaki, M.
Shirakawa, J. Biomol. NMR 2006, 36, 179 – 188.
[13] a) Y. Yang, Q. F. Li, C. Cao, F. Huang, X. C. Su, Chem. Eur. J.
2013, 19, 1097 – 1103; b) F. H. Ma, J. L. Chen, Q. F. Li, H. H.
Zuo, F. Huang, X. C. Su, Chem. Asian J. 2014, 9, 1808 – 1816.
[14] F. C. Mascali, H. Y. V. Ching, R. M. Rasia, S. Un, L. C. Tabares,
Angew. Chem. Int. Ed. 2016, 55, 11041 – 11043; Angew. Chem.
2016, 128, 11207 – 11209.
Manuscript received: November 11, 2016
Revised: January 2, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Structure Elucidation
Y. Yang, F. Yang, Y. J. Gong, J. L. Chen,
D. Goldfarb,* X.-C. Su*
&&&& — &&&&
A Reactive, Rigid GdIII Labeling Tag for In-
Cell EPR Distance Measurements in
Proteins
Distance measurements: A reactive and
rigid GdIII spin label showed excellent
performances in distance measurements
using the double electron–electron reso-
nance (DEER) method in vitro and in
cells. High-quality DEER data could be
obtained in HeLa cells at protein con-
centrations as low as 5–10 mm up to 12 h
after the delivery of proteins.
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!