Highly selective fluorescent probe for vicinal-dithiol-containing proteins and in situ imaging in living cells

Article abstract of DOI:10.1002/anie.201101317

It pays to be direct: In a rapid and specific approach to the detection of vicinal-dithiol-containing proteins (VDPs), the use of a fluorescent probe (see picture) enabled the direct readout of fluorescence. This approach based on fluorescence polarization, electrophoresis, and the direct imaging of VDPs permits the noninvasive study of VDPs both in vitro and in living cells and offers insight into their potential roles in cell function. Copyright

Full text of DOI:10.1002/anie.201101317

DOI: 10.1002/anie.201101317
Redox Proteomics
Highly Selective Fluorescent Probe for Vicinal-Dithiol-Containing
Proteins and In Situ Imaging in Living Cells**
Chusen Huang, Qin Yin, Weiping Zhu,* Yi Yang,* Xin Wang, Xuhong Qian, and Yufang Xu*
The protein thiol group is vulnerable to oxidation by reactive
oxygen species and reactive nitrogen species, and plays a
principal role in maintaining an appropriate oxidation–
reduction state of the protein,[1] regulating signal pathways,[2]
and responding to various diseases,[3] such as cancer,[4]
diabetes,[5] and neurodegeneration.[6] As the reductive end
of this redox buffer network, vicinal-dithiol-containing pro-
teins (VDPs) are attracting more and more attention. VDPs
are proteins that contain two thiol groups that are close to
each other in space. In fact, most contain the thiol groups as a
-CXnC- (n: normally 2–6, X: amino acid) motif instead of a
CC sequence.[7] In contrast to the situation when a single thiol
group is present, the proximity of a second thiol group could
promote immediate oxidation to form a disulfide following
the transient intermediacy of an oxidative species, such as a
sulfenic acid, a thiyl radical, or an S-nitrosothiol.[8] Therefore,
as one final end of the redox buffer network, protein vicinal
dithiols show a higher tendency to cope with redox changes
and are more sensitive and prone to free-radical oxidation.[9]
Because of the function of protein vicinal dithiols, it is of
considerable significance to measure the proteome of vicinal
dithiols directly in biological systems. Since the first report of
the selective binding of arsenic(III) with VDPs,[10] several
strategies for detection and functional studies have been
employed. Arsenite-affinity chromatography[11] could enrich
VDPs and made proteomic detection possible. However, the
highly reactive vicinal dithiols were exposed to an open and in
vitro oxidative environment during the lysis and affinity
purification procedures, and many underwent oxidation and
disulfide exchange, which made data interpretation difficult.
Biotinylated conjugates of phenylarsine oxide (PAO)[12]
bound to VDPs immediately, but the application of these
nonfluorescent conjugates with poor membrane permeability
in the detection of VDPs in living cells without cell lysis was
limited. VDPs were also traced by another redox proteomic
methodology, whereby PAO and a thiol-alkylation agent were
used to block vicinal dithiols and thiols. The vicinal dithiols
were then labeled after the removal of PAO.[13] This method
can be used to selectively detect VDPs in vitro through
indirect procedures, but it is restricted by the stability of PAO
itself, and cannot trace the VDPs directly in cells. Dimalei-
mide fluorogens were reported to react with a target peptide
containing a vicinal dithiol and have been applied in protein
labeling.[14] Biarsenical fluorescent analogues, such as FlAsH
(Scheme 1), have been successfully employed in the imaging
of proteins in living cells on the basis of the interaction of the
[*] C. Huang,[+] Q. Yin,[+] Prof. Dr. W. Zhu, Prof. Dr. Y. Yang, X. Wang,
Prof. Dr. X. Qian, Prof. Dr. Y. Xu
State Key Laboratory of Bioreactor Engineering
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy
East China University of Science and Technology
Meilong Road 130, Shanghai, 200237 (China)
E-mail: wpzhu@ecust.edu.cn
yiyang@ecust.edu.cn
yfxu@ecust.edu.cn
[+] These authors contributed equally.
Scheme 1. Chemical structures of FlAsH, NPE, and CTNPE.
[**] We are grateful for financial support from the National Natural
Science Foundation of China (grants 90713026, 21076077), the
National Basic Research Program of China (973 Program,
2010CB126100), the National High Technology Research and
Development Program of China (863 Program, 2011AA10A207), the
China 111 Project (grant B07023), the Fok Ying Tung Education
Foundation (grant No. 111022), the Shanghai Leading Academic
Discipline Project (B507), and the Fundamental Research Funds for
the Central Universities. We also thank Prof. Dr. Charles Yang for the
improvement of this article.
arsenic centers with two pairs of vicinal thiols in tetracysteine
motifs (CCXXCC) that were genetically fused to the target
protein.[15] Both of these approaches focused on the develop-
ment of strategies for the labeling of target proteins, and the
target peptides containing vicinal dithiols were exogenously
introduced onto the target proteins in cells. As endogenous
vicinal thiols play a key role in redox homeostasis, there is an
urgent need to develop a methodology for the selective and
direct detection of VDPs in living cells.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201101317.
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In this study, we selected naphthalimide as a fluorophore
diglycol amine (Figure 1), which enhances both the water
solubility and the cell compatibility of the probe. A six-
carbon-atom spacer (Figure 1) was used between the cyclic
dithiaarsane and the naphthalimide fluorophore. NPE was
synthesized in six steps; a control compound without a cyclic
dithiaarsane (CTNPE, Scheme 1) was also synthesized (see
the Supporting Information).
We next investigated the spectroscopic characteristics of
NPE. The UV absorption and fluorescence intensity of NPE
showed negligible change at pH 6–8 (see Figure S1a,b in the
Supporting Information). The UV absorption and fluores-
cence spectra of CTNPE were almost the same as those of
NPE in HEPES buffer (see Figure S1c; HEPES = 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid); the FF value
of NPE was lower than that of CTNPE (see Table S1 in the
Supporting Information). Thus, all in vitro tests were con-
ducted in HEPES buffer (10 mm HEPES, 5 mm ethylenedia-
minetetraacetic acid, pH 7.4, 0.5% dimethyl sulfoxide). As
VDPs are related to the redox environment of living cells, we
next investigated the effect of redox agents on the fluores-
cence intensity of NPE. Different concentra-
and cyclic dithiaarsanes as the binding group to design and
synthesize a highly selective and cell-permeable fluorescent
probe (NPE, Scheme 1) for the rapid detection and visual-
ization of VDPs both in vitro and in living cells. In comparison
with the presently available method of arsenite-affinity
chromatography[11] for the detection of VDPs, the fluores-
cence of NPE ensures the more rapid and effective detection
of VDPs through direct fluorescence readout without the
need for cell lysis or purification steps. This approach enables
protein vicinal dithiols to remain in their native state in both
in vitro and living-cell assays. Furthermore, the noninvasive
determination of VDPs by the use of NPE in living cells
enables us to collect more information on the global local-
ization, distribution, and dynamic changes of endogenous
VDPs.
Our investigations began with the design of NPE for the
selective detection of VDPs (Figure 1). Small-molecular
fluorescent probes are widely used in detecting, tracing, and
visualizing the function of proteins because of their insignif-
tions of dithiothreitol (DTT) and H2O2
induced little change in the fluorescence inten-
sity of NPE (see Figure S1d). Thus, NPE
exhibits stable fluorescence characteristics in
various redox environments. Further experi-
ments also demonstrated that NPE was stable
and biocompatible (see Figures S2 and S5).
We then deduced a plausible mechanism
for the selective detection of VDPs with NPE
(Figure 2a). The exchange of EDT in the cyclic
dithiaarsane of NPE with vicinal dithiols in
VDPs induces covalent-bond formation
between NPE and VDPs. To confirm this
mechanism, we used NPE for the specific
labeling of thioredoxin (Trx; see the amino
Figure 1. Design of NPE for the specific labeling of VDPs. Thioredoxin structure model:
reduced human thioredoxin 2, chain A (PDB ID: 1W89).[16]
icant steric bulk and fast labeling kinetics.[17] For the inves-
tigation of specific VDPs both in vitro and in living cells, the
probe must be selective, stable, water-soluble, and cell-
permeable.[18] PAO is a popular specific ligand for protein
vicinal dithiols, but it is readily oxidized,[19] which results in
the loss of specific affinity for vicinal dithiols. Furthermore,
the high polarity of the As(OH)2 group of PAO makes it cell-
impermeable. In this study we used a more stable cyclic
dithiaarsane as the ligand (red in Figure 1) for selective
binding to VDPs and also to improve the lipophilicity of NPE.
The crystal structure of the tolylarsenic 2,3-dimercaptopro-
panolate complex reveals that two sulfur atoms form a stable
five-membered ring with the arsenic atom.[20] As monothiols
in proteins have much lower affinity than vicinal dithiols for
trivalent arsenic centers,[21] the interchange of 1,2-ethanedi-
thiol (EDT) in cyclic dithiaarsanes of NPE with vicinal
dithiols in proteins could selectively discriminate vicinal
dithiols from other forms of thiols in proteins. Naphthalimide
(Figure 1) was introduced as a fluorophore with a suitable
excitation wavelength, stable fluorescence signal, and mod-
erate quantum yield (FF) under physiological conditions (see
Table S1 in the Supporting Information). To tune the ratio of
lipophilicity to hydrophilicity, we introduced a biocompatible
acid sequence of Trx and its mutation in the Supporting
Information). The reduced form of Trx (rTrx) is a typical VDP
which only contains one pair of vicinal thiols. Different forms
of Trx were incubated with NPE and CTNPE, and then
separated by electrophoresis.
A fluorescent band was
observed only in the lane loaded with rTrx and NPE, whereas
oxidized Trx (oTrx-1 and oTrx-2) or the monothiol mutant of
Trx (rTrx-M) and its oxidized form (oTrx-M) exhibited no
fluorescence signal (Figure 2b). Coomassie Brilliant Blue
(CBB) staining demonstrated that the fluorescent band
corresponded to the formation of an rTrx–NPE complex
(Figure 2b). There was no fluorescent band observed when
CTNPE, which lacks of the cyclic dithiaarsane moiety, was
used for labeling. These results indicated that the five-
membered dithiarsolane ring in NPE was cleaved by rTrx
through the exchange of EDT in the cyclic dithiaarsane for
the vicinal dithiol in rTrx to form the fluorescent rTrx–NPE
complex. The results also demonstrated the selectivity of NPE
for protein vicinal dithiols.
The selectivity, reversibility, reaction kinetics, and sensi-
tivity of NPE were then explored in a fluorescence polar-
ization (FP) assay. The “mix and measure” FP assay is applied
extensively in studying the interaction of proteins with small
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Figure 2. a) Plausible mechanism for the specific detection of VDPs with NPE. b) Selectivity of NPE as shown by SDS-PAGE. c) Selectivity of NPE
in the FP assay. Blue bars: without the addition of Cys, Hcy, or GSH; red bars: with Cys, Hcy, or GSH (1 mm); black bars: with Cys, Hcy, or GSH
(10 mm). FP data were acquired at 258C in HEPES buffer with an excitation filter (485 nm, 20 nm bandwidth) and an emission filter (528 nm,
20 nm bandwidth). The data shown are the average of quadruplicate measurements. Error bars: Æstandard error of the mean (SEM); “+”: the
molecule was present in the detection system; “À”: the molecule was not present in the detection system. rBSA=reduced BSA (with vicinal
dithiols), oBSA=oxidized BSA (without thiols), oTrx-1=thioredoxin (Trx) oxidized during expression and purification, oTrx-M=thioredoxin
mutation (Trx C69G) oxidized immediately by hydrogen peroxide (H2O2), oTrx-2=thioredoxin (Trx) oxidized immediately by hydrogen peroxide
(H2O2), rTrx-M: reduced thioredoxin mutation (Trx C69G), rTrx=reduced thioredoxin.
molecules without the separation of free and bound ligands.[22]
FP reflects the particle spinning and overall molecular weight
of the fluorophore in solution; therefore, it is a convenient
way to monitor the binding of NPE to VDPs (for a detailed
discussion of the FP assay, see the Supporting Information).
To enhance DFP (defined as the difference between the
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polarization value of free NPE or CTNPE and that of bound
related to the concentration of EDT (Figure 3b). PAO, which
competitively binds vicinal dithiols, decreased the FP
response of NPE to rBSA in a dose-dependent manner
(Figure 3c). A decrease in the DFP values was observed for
the solution of NPE and rBSA in the presence of PAO within
10 min, which indicates that PAO inhibited the association of
NPE with rBSA.
NPE or CTNPE) upon the binding of NPE to VDPs, we used
a VDP with a medium molecular weight, bovine serum
albumin (BSA), instead of thioredoxin as the model protein
for a vicinal dithiol and disulfide.
On the basis of the FP signal of NPE, no labeling was
observed with oxidized BSA (oBSA), in which the vicinal
dithiol groups were oxidized (Figure 2c); however, a remark-
able increase in the FP signal was observed after the addition
of the reduced BSA (rBSA), whose disulfide groups were
reduced to vicinal dithiols (Figure 2c). Negligible change in
the FP signal was observed for CTNPE in the presence of
rBSA (Figure 2c); thus, NPE could be used to selectively
detect VDPs in the FP assay. Moreover, binding still occurred
even in the presence of a high concentration of reduced
glutathione (GSH), cysteine (Cys), or homocysteine (Hcy;
Figure 2c). These results further confirmed the specificity of
NPE toward vicinal dithiols.
The FP assay was used for further studies on the kinetics
and reversibility of binding between NPE and VDPs. During
the association of rBSA with NPE, the DFP value increased
quickly within 2 min and then reached a plateau; this result
demonstrates that the interaction of NPE with rBSA was
nearly completed in 2 min (Figure 3a). Upon the addition of
EDT to the complex, an approximately 80% decrease in the
strength of the FP signal was observed within 10 min (Fig-
ure 3a). The ability to dissociate the rBSA–NPE complex was
We next evaluated the sensitivity of NPE for rBSA. When
NPE was treated with increasing amounts of rBSA, a
substantial increase in the FP signal was observed. In contrast,
for oBSA, negligible enhancement of the FP signal was
observed. With CTNPE, the FP value was not changed upon
the addition of increasing amounts of rBSA (see Figure S3a).
We also observed that the detection limit of NPE for the
rBSA is relatively low (1–150 nm), and that the FP signal
increased linearly with the concentration of rBSA (see the
inset in Figure S3b); therefore, the quantitative detection of
rBSA over this concentration range may be possible. We
determined the apparent dissociation constant (Kd) of the
rBSA–NPE interaction to be Kd = 0.14 Æ 0.02 mm (see Fig-
ure S3b and details about the determination of the Kd value
in the Supporting Information). Negligible change in the
fluorescence intensity of NPE was observed during the in
vitro assay (see Figures S1d and S4). Moreover, other
common biological reductants, such as ascorbic acid and
CuI, had negligible effect on the labeling of rBSA with NPE
(see Figure S6). These results further demonstrated the
substantial applicability of NPE for quantification of the
changes in VDPs in living cells on the basis of changes in the
fluorescence signal.
The FP assay was introduced for the selective detection of
VDPs with NPE in vitro. This approach ensures that VDPs
exist in their native state, as the FP assay requires no
separation of the sample. The fast kinetics of binding between
NPE and VDPs prevents the instability of vicinal dithiols in
the process. The FP assay provides a rapid approach to the
specific detection of VDPs in vitro, and the fast kinetics and
reversibility of binding between NPE and VDPs also provide
a suitable method for high-throughput screening for novel
inhibitors of VDPs on the basis of this assay.
NPE makes in situ imaging possible for endogenous
VDPs. By loading the cells with NPE, we ascertained the
intracellular distribution of VDPs for the first time. A strong
fluorescence signal was observed in cells treated with NPE
(Figure 4a), but not for cells treated with CTNPE (Fig-
ure 4b), for which only a background signal was observed.
These results indicated the specific binding of NPE to
endogenous VDPs in living cells; this binding occurred
through the reactive cyclic dithiaarsanes. To further explore
this selective binding, we coincubated DTTor PAO with NPE
in Chang liver cells. In agreement with the in vitro results
(Figure 3a,c), the labeling of cellular vicinal dithiols with
NPE was inhibited by PAO (Figure 4c) owing to the
competitive binding of PAO with protein vicinal dithiols.
Similarly, coincubation with DTT, which competitively binds
to NPE, greatly decreased the labeling of cellular vicinal
dithiols with NPE (Figure 4d). All these results demonstrated
that cell-permeable NPE can be used for the in situ imaging of
endogenous VDPs in living cells and to trace changes in
Figure 3. Association kinetics and reversibility of the association of
NPE. a) Kinetics of binding of NPE to rBSA. EDT was injected into the
detection system after NPE had been incubated with rBSA for 30 min,
whereas PAO was coincubated with samples from the start. b) Effect
of the dose-dependent dissociation of EDT on the association of NPE
and rBSA. c) Dose-dependent inhibitory effect of PAO on the associa-
tion of NPE and rBSA. DFP data were acquired at 258C in HEPES
buffer with an excitation filter (485 nm, 20 nm bandwidth) and an
emission filter (528 nm, 20 nm bandwidth). The data shown are the
average of quadruplicate measurements. Error bars: ÆSEM.
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cellular VDPs on the basis of variations in fluorescence
intensity.
The localization of intracellular VDPs was also inves-
tigated in Chang liver cells. When observed by confocal
microscopy, the fluorescence signal of the NPE-labeled VDPs
showed a punctuated pattern mainly concentrated in a small,
eccentric perinuclear zone; the intensity was low in the
cytoplasm and nucleus (Figure 4a). This pattern suggested
mitochondrial localization, which was further confirmed by
colocalization studies with subcellular organelle-specific
labels, that is, ER-Tracker Blue-White DPX for the endo-
plasmic reticulum (ER) and Mito-Tracker Deep Red for
mitochondria. The merged image of the fluorescence of NPE
and mitochondria stained with Mito-Tracker Deep Red
showed convincing yellow fluorescence in a punctuated
pattern (Figure 4 f), which implied the colocalization of the
VDPs with mitochondria. Some colocalization of the VDPs
with the ER was also observed (Figure 4e), but to a much
lesser extent than with the mitochondria.
Triple staining also supported the view that the VDPs
were mainly localized in the mitochondria (Figure 4g). The
individual fluorescence signal for the ER (blue) was largely
preserved, and the mitochondria (red) appeared yellow,
which convincingly indicated colocalization with the NPE
signal.
It is interesting that protein vicinal dithiols seem to be
abundant in mitochondria, as demonstrated by NPE labeling
of living cells. In a previously described image of a FlAsH-
labeled exogenous tetracysteine tracer,[15a] a weak back-
ground signal also came from mitochondria, maybe as a
result of weak binding of the probe with vicinal dithiols. The
proteomic identification of arsenic-binding proteins in cell
lysate also proved the existence of abundant mitochondria-
related proteins.[12b] As the key organelle for respiration,
mitochondria are the main site for many redox-related life
processes. We previously reported that S-nitrosoproteins exist
mainly in the mitochondria and perimitochondrial compart-
ment, which suggests that mitochondrial proteins are prone to
modification and functional regulation in the presence of
nitric oxide (NO).[23] An image of the protein disulfide
proteome in mammalian cells also showed a weak signal in
mitochondria that markedly increased upon oxidative chal-
lenge.[24] Thus, these disulfides remained in the reductive form
(vicinal dithiols) in their normal state. Furthermore, both S-
nitrosothiol and disulfide formation are determined by
mitochondrial respiration and the generation of reactive
oxygen species. Now we have further shown the mitochon-
drial localization of cellular VDPs by in situ labeling and
imaging. These results suggest that mitochondria are the
central participants in thiol redox regulation and may have
profound effects on protein function.
Figure 4. In situ imaging and cellular response of NPE-labeled Chang
liver cells. a–d) Fluorescence of cells labeled with NPE (5 mm, a),
CTNPE (5 mm, b), NPE (5 mm) in the presence of PAO (5 mm, c), and
NPE (5 mm) in the presence of DTT (2 mm, d). e) Colocalization of
NPE with ER-Tracker. f) Colocalization of NPE with Mito-Tracker Deep
Red. g) Triple staining of the ER, mitochondria, and VDPs. h) Response
of cellular vicinal dithiols in proteins to different redox environments
induced by treatment with DTT (2 mm) for 12 h or treatment with
Aldrithiol-2 (AT-2) or Diamide (0.1 or 1 mm) for 0.5 h. Green: NPE
fluorescence signal; red: signal after staining with Sypro Ruby. Scale
bar: 10 mm. *: molecular weight calculated by Quantity One software.
By using this live-cell-labeling technique, we further
showed that the level of intracellular protein vicinal dithiols
changed upon redox regulation. The fluorescence intensity of
NPE-treated cells increased gradually from that of DTT-
untreated cells as the concentration of DTTwas increased. In
contrast, the fluorescence intensity of NPE-treated cells
decreased as the concentration of Diamide was increased
(see Figure S7).
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different responses of different proteins. The signal observed
upon staining with Sypro Ruby (red) showed no clear
difference in protein abundance between the samples, but
significant variation was revealed by the signal of VDPs
labeled with NPE (green). Furthermore, different proteins
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Keywords: fluorescent probes · in situ imaging · protein labeling ·
redox proteomics · vicinal dithiols
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