Protein Spin Labeling with a Photocaged Nitroxide Using Diels–Alder Chemistry

Article abstract of DOI:10.1002/cbic.201900318

EPR spectroscopy of diamagnetic bio-macromolecules is based on site-directed spin labeling (SDSL). Herein, a novel labeling strategy for proteins is presented. A nitroxide-based spin label has been developed and synthesized that can be ligated to proteins

Full text of DOI:10.1002/cbic.201900318

Accepted Article
Title: Protein Spin Labeling with a Photocaged Nitroxide Using Diels-
Alder Chemistry
Authors: Anandi Kugele, Bjarne Silkenath, Jakob Langer, Valentin
Wittmann, and Malte Drescher
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Protein Spin Labeling with a Photocaged Nitroxide Using Diels-
Alder Chemistry
Anandi Kugele,+[a] Bjarne Silkenath,+[a] Jakob Langer, Valentin Wittmann,* and Malte Drescher*
[a]
[a]
[a]
Abstract: EPR spectroscopy of diamagnetic bio-macromolecules is
based on site-directed spin labeling (SDSL). Here, we present a
novel labeling strategy for proteins. We developed and synthesized
a nitroxide-based spin label that can be ligated to proteins by an
inverse-electron-demand Diels-Alder (DAinv) cycloaddition to
genetically encoded non-canonical amino acids (ncAA). The
nitroxide moiety is shielded by a photoremovable protecting group
potentially cytotoxic catalyst further simplifies the reaction, while
water-solubility is a prerequisite for in vivo use. Gadolinium(III)-
and trityl-based spin labels are stable in cells. They have been
used for distance measurements but suffer either from a very
broad spectrum and resulting low modulation depths or from a
very narrow spectrum not suitable for double frequency
experiments.[
10b, 11]
Nitroxides provide ideal spectral width and
(
PPG) with an attached tetraethylene glycol unit to achieve water
additionally access to dynamic information. However, traditional
nitroxide-based labels feature limited redox stability in the
cellular environment and their EPR signal vanishes within
minutes.[ Thus, for routine in vivo use, it is crucial to increase
the nitroxide stability. Recently, a tetraethyl-modified maleimido-
proxyl-based spin label with enhanced stability was
introduced.[13]
solubility. We demonstrate SDSL of two model proteins with the
PaNDA (Photoactivatable Nitroxide for DAinv reaction) label. Our
strategy features high reaction rates combined with high selectivity,
and the possibility to deprotect the nitroxide in E. coli lysate.
12]
Electron paramagnetic resonance (EPR) spectroscopy together
with site-directed spin labeling (SDSL) is a valuable and
established tool to elucidate structure, function and dynamics of
proteins and protein complexes.[1] Nitroxide-based spin labels
are the best established and convenient ones,[2] as they are
small, non-disturbing and exhibit excellent spectroscopic
properties.[ In particular, they are perfect to display rotational
dynamics via line shape analysis[3a, 4] and can be used for
distance determination.[5] The most commonly used spin label
We here present a novel approach addressing both ncAA-
mediated spin labeling and nitroxide stability (Figure 1).
Introduction of the novel label is achieved by inverse-electron-
demand Diels-Alder (DAinv) reaction[14] of an 1,2,4,5-tetrazine
strained alkyne (cyclooctyne)[15] or alkene (trans-
with
a
[16]
cyclooctene, TCO).
This reaction forms the corresponding
3]
pyridazine and dihydropyridazine, respectively. Due to its
excellent water-compatibility, the DAinv reaction has proven
suitable for a broad range of biochemical applications both in
vitro and in vivo.[14, 17] Strain-promoted DAinv cycloadditions
for EPR spectroscopy is the methanethiosulfonate spin label
1b]
(
MTSSL).[
It reacts specifically with accessible sulfhydryl
have not been used for site-directed spin labeling of proteins so
moieties in proteins, i.e. cysteines. MTSSL application requires
elimination of native cysteines by genetic engineering and
introduction of strategically positioned ones. Alternative native
amino acids such as tyrosine were also utilized for spin
labeling.[6] However, the choice of native amino acids as tag for
spin labels limits bioorthogonality. When selecting genetically
encoded non-canonical amino acids (ncAA) as targets for SDSL
instead, selectivity can be achieved even in cells and removing
of functionally-relevant cysteines is not required.[7] Utilizing an
orthogonal aminoacyl-tRNA-synthetase (aaRS)-tRNA pair
far (notably, recently an in vitro transcribed RNA segment was
[18]
site-specifically labeled with
a tetrazine-nitroxyl moiety ).
Moreover, we aimed for addressing nitroxide stability by using a
protection strategy. So far, alkylation,[19] silylation,[20] acylation,
as well as photoremovable protecting groups (PPGs)[22] have
been demonstrated to protect nitroxides and to release them
when needed. Especially photoirradiation for deprotection is
interesting, as it enables spatial and temporal control over the
release of functional groups. In particular, o-nitrobenzyl
[21]
derivatives for diverse functionalities, even for native amino acid
side chains,[23] were pioneered since 1966 and are since the
capable of selectively charging a nonsense suppressor tRNA
8]
(
e.g. an amber codon) with an ncAA is an established method.[
best established PPGs.[24] Their application for the protection of
nitroxide spin labels during oligonucleotide synthesis has been
The expansion of the genetic code by incorporation of ncAA has
brought a plethora of potential conjugation techniques, as a
broad range of ncAA with specific reactivities is available.[9]
However, in combination with spin labeling only few ncAA and
corresponding chemical reaction schemes were employed to
date.[10] An ideal spin labeling procedure should exhibit high
reaction rates, but still be selective. The independence from any
[22]
introduced by Seven et al. in 2014 and continued by Weinrich
et al.[25]
Figure 1 A depicts the structure of the new spin label, named
PaNDA, referring to “Photoactivatable Nitroxide for DAinv
reaction”. It is composed of a 3,6-diarylsubstituted 1,2,4,5-
tetrazine as diene component of a DAinv reaction attached to an
o-nitrobenzyl-protected TEMPO derivative that delivers an EPR-
active nitroxyl radical upon photoirradiation and subsequent air
[a]
A. Kugele, B. Silkenath, J. Langer, Prof. Dr. V. Wittmann, Prof. Dr.
M. Drescher
Department of Chemistry and Konstanz Research School Chemical
Biology (KoRS-CB), University of Konstanz
Universitätsstraße 10, 78457 Konstanz (Germany)
E-mail: mail@valentin-wittmann.de
oxidation. Oligoethylene glycol chains have been used before to
enhance water solubility of tetrazine derivatives.[26] Accordingly,
we attached a tetraethylene glycol monomethyl ether to ensure
water solubility of the PaNDA label. This approach is elegant
insofar, as the rather bulky PPG along with the tetraethylene
glycol chain is cleaved off by irradiation.
malte.drescher@uni-konstanz.de
[+
]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. Protein labeling with the PaNDA label. A) Structure of the PaNDA spin label 1 with its particular functional features colored in orange (tetrazine moiety),
red (photocaged TEMPO), green (PPG with tetraethylene glycol chain in blue). B) Structures of the ncAA SCO-L-lysine 2a and TCO-L-lysine 3a used in this study.
The respective products upon reaction with 1 are shown as 2b and 3b for the ncAA 2a and 3a, respectively. C) Incorporation of ncAA into proteins (exemplarily
[
27]
shown for TRX-R74→2a (pdb entry 2TRX )), and in vitro labeling and deprotection conditions.
For the synthesis of PaNDA label 1, carboxy TEMPO 5 was
quantitatively transformed into hydroxylammonium salt 6 making
use of the acid-dependent redox triad of nitroxyl radical,
oxoammonium cation, and hydroxylamine (Scheme 1 A).[
Conveniently, the carboxylic acid is simultaneously protected
under these conditions. Nitrobenzaldehyde 7 was reduced to the
benzyl alcohol and the phenol group selectively alkylated with
tetraethylene derivative 8 to yield nitrobenzyl alcohol 9 (Scheme
are previous EPR studies available for data comparison.[10e, 30]
Both model proteins exhibit native cysteines, thus providing the
possibility to test for orthogonal labeling without affecting
cysteines. The first ncAA that can undergo a DAinv reaction, a
lysine-derived cyclooctyne, was introduced for copper-free click
28]
[
31]
before also a tetrazine,[32] norbornenes,[15a,
reaction in 2011,
3
3]
trans-cyclooctenes,[15a] and a spirohexene[34] were genetically
encoded. The rationally designed Methanosarcina mazei mutant
tRNAPyl/PylRSAF (Y306A, Y384F) possesses an enlarged
1
B). Appel reaction gave benzyl bromide 10 that was used to
alkylate 6 followed by ester hydrolysis yielding carboxylic acid 11. binding pocket suitable to incorporate these bulky ncAA in
Finally, amide bond formation with tetrazine derivative 12[
29]
response to the amber stop codon.
[15a, 31, 35]
Therefore, we co-
delivered PaNDA label 1 in an overall yield of 42 % starting from
nitrobenzaldehyde 7.
expressed amber stop codon mutants of C–terminally His6-
tagged model proteins together with the previously reported
tRNAPyl/PylRS synthetase in E. coli. We tested two different
AF
To initially test and optimize the deprotection we evaluated the
UV/Vis absorption spectrum of the PaNDA spin label (Figure 2
A). It exhibits a prominent feature for the photocage group at
approx. 300 nm. To this end, we tested the time-dependent
deprotection efficiency at 302 nm. LC-MS of the PaNDA spin
label proved almost complete conversion to the desired nitroxide
after irradiation for 10 min (Figure 2 B). Accordingly, an EPR
signal appeared only after irradiation (Figure 2 C).
To test and establish our labeling and deprotection method with
proteins, we chose the green fluorescent protein (GFP) and the
E. coli oxidoreductase thioredoxin (TRX). TRX was the first
protein to be modified by a DAinv reaction in 2008,[16a] and there
6
ncAA, i.e. N -((cyclooct-2-yn-1-yloxy)carbonyl)-L-lysine (SCO-L-
6
lysine 2a, Figure
1
B) and N -((((E)-cyclooct-2-en-1-
yl)oxy)carbonyl)-L-lysine (TCO-L-lysine 3a), which are well
known for high integration rates and which exhibit excellent
reaction kinetics.[
15a,
36]
The expression in presence of 1 mM
ncAA yielded between 4 and 7 mg/L of purified full-length GFP-
Y39→2a, GFP-Y39→3a, TRX-R74→2a or TRX-R74→3a,
respectively (Figure S11-S12 and S16-S17). We even
succeeded in the incorporation of SCO in response to two
amber stop codons in a protein, which was, to our knowledge,
not reported before (TRX-G34/R74→2a; Figure S13).
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Figure 2. Deprotection study of the PaNDA spin label 1. A) UV absorption
spectrum of 75 μM of 1 in methanol. B) LC-MS traces showing the absorption
at 254 nm of 1 before (purple) and after irradiation (black) for 10 min at 302
nm. After irradiation, 1 is virtually completely consumed and nitroxide 13 the
main product. Small amounts of intermediate 14 and side product 15 are
visible. C) EPR spectrum of 100 μM of 1 in water, before (purple) and after
irradiation (black).
By circular dichroism spectroscopy we further proved that the
labeled proteins kept their secondary structure throughout the
process (Figure S20). By analyzing the double integral of the
EPR spectra, we found that the deprotection of the PaNDA spin
label was maximal already after 2 min of irradiation at 302 nm
(
Figure S21). To substantiate our data, full-length ESI-MS
spectra were recorded (Figure S18-S19). Peaks can be
assigned to successfully labeled and deprotected protein, but no
mass peaks were found referring to still protected protein-label-
complex (occurrence of peaks with Δ = -150 Da for 2a or 2b, or
Δ = -152 Da for 3a or 3b indicated partial degradation of the
ncAA to lysine). Thus, the design of our spin label allows for fast
and selective labeling as well as convenient and quantitative
deprotection.
Scheme 1. Synthesis of PaNDA label 1. NMM = N-methylmorpholine, PyBOP
=
benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate.
We assessed the performance of our protection strategy in
biological environments. We reasoned, that reducing conditions
could potentially intervene with the spontaneous oxidation step
Next, we wanted to assess the spin labeling potential of the
ncAA-containing proteins with the PaNDA spin label in vitro (for
the general procedure see Figure 1 C). For this purpose, we
mixed the purified proteins with the PaNDA spin label and
established mild and universally applicable reaction conditions
converting the irradiation-derived hydroxylamine into
a
nitroxide.[22] To this end, we chose E. coli lysate, because it
contains relevant cell components and provides reducing
conditions. We first labeled TRX-R74→2a with the PaNDA spin
label and deprotected the obtained TRX-R74→2b as described
above. Then we incubated the lysate with this protein to confirm
nitroxide degradation. After 80 min, the EPR signal was < 1 µM
(
30 min, 20 °C). We separated the labeled proteins from excess
label via Ni-NTA resin, and measured EPR spectra before and
after irradiation (Figure 3). The data recorded before irradiation
indicate that the PPG is stable enough to survive the labeling
and washing procedure. EPR spectra after irradiation show, that
both model proteins comprising 2a or 3a were successfully
labeled and deprotection works. Spectra of the wildtype (wt)
proteins incubated with the PaNDA spin label exhibit no EPR
signal, indicating a high chemoselectivity of this reaction (Figure
(
Figure 4). Then, we incubated labeled and still protected TRX-
R74→2b with the same amount of fresh E. coli lysate for 80 min.
No EPR signal was detected in this period of time, meaning that
the protecting group remained stable on the protein. After
irradiation, the EPR signal increased, resulting in approx. 50 %
spin concentration compared to deprotection in buffered solution.
3, upper line).
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This demonstrates that a spontaneous oxidation step forming
the nitroxide is actually possible in reducing environment, and
that, in principle, the deprotection can be performed in E. coli
lysate to recover the EPR signal.
We envisage the application of the PaNDA spin label for in-cell
EPR measurements. Points that need to be addressed before
the system can be used in cells involve the delivery of the
PaNDA spin label into cells and removal of unbound label.
Moreover, labeling and deprotection efficiencies need to be
confirmed in cells. However, the DAinv reaction in general is
known to be well-suited for biorthogonal intracellular
reactions.[
37]
Moreover, o-nitrobenzyl and other PPGs were
shown to be cleaved upon irradiation even inside cells[38].
Addressing nitroxide lability after irradiation, samples for EPR
distance measurements will be frozen directly after being
irradiated, thus minimizing nitroxide degradation.
In conclusion, we have developed an innovative SDSL approach
based on genetically encoded ncAA amenable to DAinv
chemistry and on a newly designed spin label. The PaNDA spin
label provides an appealing combination of a tetrazine moiety
and a PPG. The use of DAinv chemistry for spin labeling
illustrates the assets and versatility of this reaction. For in-cell
labeling or for binding studies in cellular environments the PPG
potentially enables prolonged incubation times before data
acquisition. Thus, our method opens up new vistas for the study
of proteins by EPR spectroscopy.
Figure 4. Recovery of nitroxide radicals is possible in E. coli lysate. E. coli
cells were lysed and mixed with preliminarily irradiated TRX-R74→2b (black
squares) and the spin concentration was quantified at different time points until
the signal had vanished (< 1 μM) after 80 min. In comparison, a non-irradiated
sample of TRX-R74→2b (blue circles) was incubated with lysate for 80 min,
showing no EPR signal. After irradiation for
2 min at 302 nm, a spin
concentration of approx. 15 μM was found (see Figure S22 for EPR spectra at
each time point).
Acknowledgements
This project has received funding from the European Research
Council (ERC) under the European Union’s Horizon 2020
research and innovation programme (Grant Agreement Number:
772027 – SPICE – ERC-2017-COG) and from the Deutsche
Forschungsgemeinschaft (SFB 969, Projects B5 and C3). We
thank Edward Lemke and Daniel Summerer for plasmid
pEVOL_PylRS_AF.
Keywords: EPR spectroscopy • Cycloaddition • Bioorthogonal
chemistry • Non-canonical amino acids • Site-directed spin
labeling
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ChemBioChem
10.1002/cbic.201900318
COMMUNICATION
Entry for the Table of Contents
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COMMUNICATION
PaNDA (Photoactivatable Nitroxide for
DAinv reaction) is a novel spin label
for proteins. Kugele et al. developed
and synthesized this nitroxide-based
spin label that can be ligated to
proteins by an inverse-electron
Anandi Kugele, Bjarne Silkenath, Jakob
Langer, Valentin Wittmann,* and Malte
Drescher*
Page No. – Page No.
demand
Diels-Alder
(DAinv)
cycloaddition to genetically encoded
non-canonical amino acids.
Protein Spin Labeling with a
Photocaged Nitroxide Using Diels-
Alder Chemistry
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.