Covalent Probes for Aggregated Protein Imaging via Michael Addition

Article abstract of DOI:10.1002/anie.202015988

Covalent chemical reactions to modify aggregated proteins are rare. Here, we reported covalent Michael addition can generally occur upon protein aggregation. Such reactivity was initially discovered by a bioinspired fluorescent color-switch probe mimickin

Full text of DOI:10.1002/anie.202015988

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 11335–11343
International Edition: doi.org/10.1002/anie.202015988
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Fluorescent Probes
Hot Paper
Covalent Probes for Aggregated Protein Imaging via Michael Addition
Wang Wan, Yanan Huang, Qiuxuan Xia, Yulong Bai, Yuwen Chen, Wenhan Jin, Mengdie Wang,
Di Shen, Haochen Lyu, Yuqi Tang, Xuepeng Dong, Zhenming Gao, Qun Zhao, Lihua Zhang,
and Yu Liu*
Abstract: Covalent chemical reactions to modify aggregated
proteins are rare. Here, we reported covalent Michael addition
can generally occur upon protein aggregation. Such reactivity
was initially discovered by a bioinspired fluorescent color-
switch probe mimicking the photo-conversion mechanism of
Kaede fluorescent protein. This probe was dark with folded
proteins but turned on red fluorescence (620 nm) when it non-
covalently bound to misfolded proteins. Supported by the
biochemical and mass spectrometry results, the probe chemo-
selectively reacted with the reactive cysteines of aggregated
proteins via covalent Michael addition and gradually switched
to green fluorescence (515 nm) upon protein aggregation.
Exploiting this Michael addition chemistry in the malachite
green dye derivatives demonstrated its general applicability
and chemical tunability, resulting in different fluorescence
color-switch responses. Our work may offer a new avenue to
explore other chemical reactions upon protein aggregation and
design covalent probes for imaging, chemical proteomics, and
therapeutic purposes.
Introduction
Proteins fold into their defined 3-dimentional structures
to acquire proper biological functions.[1] These structures
Figure 1. Chemical reactivity upon protein aggregation has not yet
been fully explored. Defined structure and chemical reactivity of folded
proteins facilitate the design of their covalent probes. Aggregated
proteins are difficult to target via covalent modification due to the lack
of reported chemical reactivity. This work reported 1,4-Michael addition
can occur upon protein aggregation, yielding fluorescence color-switch
probes.
revealed by X-ray crystallography and electron microscopy
guide the design of non-covalent probes to regulate protein
functions. Further exploration of the chemical reactivity of
proteins has resulted in countless reaction types at their
folded state (Figure 1).[2,3] The inherent nucleophilicity of
amino acids enables chemoselective bioconjugations,[4] such
as activated ester to react with lysine, maleimide to react with
cysteine, and various protein chemical ligation methods.[5–7]
The unique catalytic environment of enzymes or binding
proteins offers vastly diverse covalent reactions, including
acylation, 1,4-Michael addition, alkylation, carbamylation,
and boronation etc.[8–10] These covalent reactions allow us to
develop covalent probes targeting folded proteins, including
fluorescent probes for bio-imaging, covalent drugs for ther-
apeutic purposes,[11–16] and activity based probes for chemical
proteomics[17–24] etc. Besides these naturally existing chemical
reactivity, introduction of unnatural amino acids enormously
broadens the horizon of chemical reactivity and enriches the
bioorthogonal chemistry toolbox for selective bioconjugation
and functionalization of folded proteins.[25–28]
Unlike folded proteins, the misfolded and aggregated
proteins caused by genetic mutations, environmental stresses,
chemical modifications, and ageing usually lose their 3-
dimentioanl structures. Aberrant protein misfolding and
aggregation in the cell often lead to numerous protein
conformational diseases, such as neurodegeneration, meta-
bolic disorders, cardiovascular diseases, and certain type of
cancers.[29–32] Though conventionally considered to be struc-
turally disordered, protein amyloid aggregation has been
demonstrated to exhibit organized b-sheet stacked structures
given the recent advances in the state-of-the-art microscopic
[*] W. Wan, Y. Huang, Q. Xia, Y. Bai, Y. Chen, W. Jin, M. Wang, D. Shen,
H. Lyu, Y. Tang, Q. Zhao, L. Zhang, Y. Liu
CAS Key Laboratory of Separation Science for Analytical Chemistry,
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
457 Zhongshan Road, Dalian 116023 (China)
E-mail: liuyu@dicp.ac.cn
X. Dong, Z. Gao
The Second Hospital of Dalian Medical University
467 Zhongshan Road, Dalian 116044 (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202015988.
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methods.[33] The structural information provides blueprint to
design non-covalent dyes and sensors that selectively bind to
amyloid aggregated proteins.[34] Organic dyes, like Thiofla-
vin T, Congo Red, carbazoles, perylenes, and their derivatives
have been demonstrated to selectively bind to amyloid
precursors and fibrils.[35] In particular, Tang,[36] Zhu,[37] and
Hong[38] et al. pioneered the design of AIEgens to report on
amyloid and unfolded proteins during protein phase separa-
tion. Materials based probes, such as luminescent polymers,
quantum dots, and nanoparticles opened new avenues for
amyloid sensors.[39,40] Mirica[41] and Marti[42] et al. bridged the
gap of organic dyes and inorganic materials by using
organometallic complexes to probe amyloid proteins. These
probes target protein amyloids via non-covalent intercalation
into the intermolecular space of amyloid aggregates. Dyes
like SYPRO orange and PROTEOSTAT that bind to
amorphous protein aggregates of no defined structure were
less reported.
While much light has been shed on the non-covalent
probes for aggregated proteins, covalent probes targeting
aggregated proteins have not been generally reported. This is
probably due to the fact that the local chemical reactivity
within aggregated proteins has not been fully explored.
Recently, Zhang[43,44] and Hong[45] et al. embarked on the
survey of the physical environment inside aggregated pro-
teins, but their work primarily revealed the changes in
polarity and viscosity upon protein aggregation using envi-
ronment-sensitive probes. However, it still remains elusive
how chemically reactive proteins are at their aggregated state.
Therefore, current powerful chemical sensing and bioconju-
gation strategies[46–48] may provide hints to address this
question.
Figure 2. Bio-inspired fluorescent protein chromophore mimic probe
can switch fluorescence color upon Michael addition. a) Kaede fluores-
cent protein switches its green fluorescence to red upon photolysis.
b) Proposed working mechanism of AggSwitch P1 probe. P1 turns on
red fluorescence upon binding to misfolded proteins and switches to
green fluorescence upon protein aggrega-tion via Michael addition.
c) Synthetic P2 and P3 surrogates mimic the Michael addition prod-
ucts of P1 reacting with nucleophilic cysteine (P2) or lysine residues
(P3). d) Fluorescence emission spectra of P1, P2, and P3 in glycerol
(5.0 mM). e) Absorption spectra of P1, P2, and P3 in methanol
(10.0 mM). f) Photo-physical parameters of P1, P2, and P3 in glycerol.
In this work, we reported 1,4-Michael addition reaction
can occur upon protein aggregation, which is useful to
develop covalent probes for bioconjugation and imaging
purposes (Figure 1). Such chemical reactivity was discovered
by a bio-inspired fluorescent color-switch probe. This probe
exhibited no fluorescence with folded proteins, red fluores-
cence in misfolded proteins, and green fluorescence in
aggregated proteins. Mass spectrometry analyses revealed
the fluorescence color-switch property was induced by
chemoselective covalent Michael addition with cysteine
residues that were reactive upon protein aggregation. We
further exploited this chemistry to develop other fluorogenic
color-switch probes and demonstrate its chemical tunability.
The transmission electron microscopic images and protein
proteolytic resistance results suggested that the compactness
of aggregated proteins influenced their chemical reactivity.
Last, we utilized this chemistry to selectively visualize
aggregated proteome in stressed live cells.
(Figure 2). This probe was bio-inspired and reversely engi-
neered from the chromophore of Kaede photo-convertible
fluorescent protein based on its photo-conversion mechanism
(Figure 2a). The Kaede fluorescent protein shifts its fluores-
cent color from green to red upon photolysis. Inspired by this
mechanism, our probe P1 (Figure 2b) was designed to bear an
electrophilic vinyl ester that is ready for covalent 1, 4-Michael
addition by nucleophilic amino acid side chains (Figure 2c,
cysteine and lysine). Probe P1 turned on red fluorescence at
645 nm whereas its Michael addition surrogates P2 and P3
exhibited green fluorescence around 520 nm in the viscous
glycerol (Figure 2d,f). Meanwhile, the chemical transforma-
tion from P1 to P2 and P3 can be reflected by the absorbance
spectrum shift upon Michael addition (Figure 2e,f).
Interestingly probe P1, named AggSwitch probe here-
after, remained fluorescently dark when the model protein
destabilized E. coli dihydrofolate reductase mutant (mut-
DHFR, M42T:H114R)[49] was folded (Figure 3a, black curve).
Immediately upon acid induced DHFR misfolding and
aggregation, we initially observed the occurrence of red
fluorescence (Figure 3a, red curve, emmax 620 nm, pH 4.4). As
the protein aggregation proceeded, the probe gradually
changed its fluorescence from red to yellow after 30 min
(Figure 3a, yellow curve), and completely switched to green
fluorescence after 100 min (Figure 3a, green curve emmax
515 nm, F = 0.07). As shown in the absorption spectra
Results and Discussion
Fluorescent Color-Switch Probe upon Protein Aggregation
Our initial hypothesis of the presence of chemical
reactivity inside aggregated proteins was made on the basis
of a fluorescent color-switch probe upon protein aggregation
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AggSwitch probe (P1) exhibits fluorescence color-switch
property as protein misfolds and aggregates.
Michael Addition upon Protein Aggregation
We next attempted to confirm that AggSwitch P1
covalently modified aggregated proteins via Michael addi-
tion. Prior to characterizations, we denatured, refolded, and
desalted DHFR proteins that were aggregated in the presence
of AggSwitch probe (Figure 4a) to minimize the interference
from heterogeneous phase separated proteins. We explored
series of biochemical and mass spectrometry experiments to
examine the presence of covalent modification.
First, the UV/Vis spectrum of the P1-modified refolded
proteins (459 nm) shifted towards P2 in MeOH (440 nm)
Figure 3. AggSwitch P1 probe is a fluorescent color-switch probe upon
protein aggregation. a) Fluorescence spectra of P1 in folded, misfold-
ed, and aggregated mut-DHFR proteins. P1 was dark in folded mut-
DHFR (M42T:H114R, 10 mM, pH 7.4), turned on red fluorescence
upon acid induced misfolding (pH 4.4, 0 min), and switched to green
color after 100 min in acidic buffer (pH 4.4). b) Absorption spectra of
P1 in misfolded (red curve) and aggregated (green curve) mut-DHFR.
c) Fluorescence of P1 gradually switched from red to green as mut-
DHFR misfolded and aggregated induced by acid over time at pH 4.4.
d) Images of samples in (c). e) Fluorescence of P1 gradually switched
from red to green as heat induced mut-DHFR misfolds and aggregates
over time at pH 6.23 from 258C to 558C. f & g) Fluorescence color-
switch property retained in aggregated sortase and SOD1(V31A)
proteins.
(Figure 3b), P1 in aggregated proteins shared similar photo-
physical properties as its Michael addition product P2 and P3,
suggesting that such fluorescence color-switch property may
be caused by the Michael addition of P1. We further present
such fluorescence color-switch property in a time-dependent
(Figure 3c) and a temperature-dependent (Figure 3e) protein
aggregation experiment. Both experiments confirmed AggS-
witch probe first turned on red fluorescence and gradually
changed to green fluorescence as mut-DHFR protein aggre-
gated (Figure 3d) but remained non-fluorescent with folded
proteins (Figure S1). Finally, we examined the generality of
P1ꢀs fluorescence color-switch property in different aggregat-
ed proteins (sortase in Figure 3 f, SOD1(V31A) in 3 g, WT-
DHFR in Figure S2a). Collectively, we demonstrated that
Figure 4. Covalent and chemoselective Michael addition of aggregated
proteins by the AggSwitch P1 probe. a) Experimental Scheme to
denature and refold aggregated DHFR modified by P1 prior to
characterizations. b) Absorption spectra of refolded DHFR aggregates
indicated covalent adduct of P1 onto DHFR proteins. c) SDS-Page
electrophoresis showed stable covalent adduct of P1 onto DHFR.
d) MALDI mass spectrometry identified two covalent modifications by
P1 probe on one DHFR protein molecule. e) Tandem mass spectrom-
etry identified C85 and C152 residues that were modified by P1.
f) Distinct local environment of C85 and C152 revealed by X-ray
crystallography (PDB:5CCC).
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compared to P1 in MeOH (513 nm) (Figure 4b). This result
indicated that stable covalent Michael addition occurred
between the protein and P1 upon DHFR aggregation even
after denaturation, refolding, and size exclusion purification
to remove non-covalent binders. Second, SDS-PAGE electro-
phoresis showed that DHFR was covalently modified by
a fluorescent probe (Figure 4c for heat induced aggregation,
Figure S3 for acid induced aggregation). Third, MALDI mass
spectrometry further identified the exact covalent adduct
product of DHFR-P1 conjugate, whereas the probe did not
modify folded DHFR under the same conditions (Figure 4d,
Figure S4). Similar SDS-PAGE gel, MALDI, and color-switch
results can be obtained using other model proteins, showing
that such Michael addition universally occurs upon protein
aggregation (Figure S5, S6). These lines of evidence support
that stable covalency present between aggregated DHFR and
AggSwitch P1 probe.
(U), misfolded soluble oligomers (M), and insoluble aggre-
gates (I) (Figure 5a). We are interested in the origin of
individual fluorescent color of P1 and the role of Michael
addition in this color-switch behavior. We next attempted to
attribute the fluorescence colors of P1 to different protein
misfolded states (U, M, and I) and test the proposed
mechanisms (Figure 5a).
We next utilized tandem mass spectrometry to reveal
which amino acid residue is responsible for this Michael
addition (Figure 4e). Multiple proteases digested aggregated
WT-DHFR proteins were subjected to tandem mass spec-
trometry analysis and showed good peptide coverage (Fig-
ure S7). We observed chemoselective conjugation of P1
towards Cysteine-85 (C85) and Cysteine-152 residues
(C152) of WT-DHFR protein (Figure 4e, S8). DHFR
C85S:C152S double mutant abolished the covalent reaction
shown by the MALDI result and the fluorescence color-
switch property of P1 upon DHFR aggregation (Figure S9).
Furthermore, tandem mass results showed a preferred con-
jugation towards C85 over C152 (Figure S10, 4.3:1 in labeling
efficiency). However, C85 residue of higher labeling efficien-
cy was less exposed to protein surface and thus less accessible
to probes than C152 shown by the DHFR X-ray crystallo-
graphic structure (Figure 4 f, PDB code: 5CCC). We asked
why a buried residue was labeled by P1 more effectively than
an exposed residue. Scrutinizing the neighboring residues of
C85 and C152 revealed their different chemical environment.
C85 is buried in a highly hydrophobic environment, whereas
C152 is in an exposed charged polar environment (Figure 4 f).
Therefore, the interaction between hydrophobic C85 pocket
and hydrophobic AggSwitch P1 probe (ClogP = 4.1) may be
more energetically favorable compared to the interaction
with hydrophilic C152 pocket due to the hydrophobic effect
during protein misfolding and aggregation. This result also
depicted that the buried C85 residue first flips inside-out upon
protein aggregation, binds P1 probe, and then exposes itself to
the probe for Michael addition reaction. Interestingly, cys-
teines at different locations of DHFR bear different reactivity
and reaction kinetics revealed by mutagenesis (Figure S23).
Overall, these multiple lines of evidence showed that covalent
Michael addition occurs inside aggregated proteins with
chemoselectivity to cysteine residues.
Figure 5. Mechanism of the fluorescence color-switch property of P1.
a) Proposed model of stepwise fluorescence turn-on and color-switch
upon protein aggregation. P1 is dark in folded and unfolded mono-
meric proteins (F & U), turns on red fluorescence upon binding to
misfolded soluble oligomers (M), and reacts with reactive cysteines
upon the formation of insoluble aggregates (I). b) Relative fluores-
cence intensity of P1 (5 mM) at different folding states of DHFR
(50 mM). c) Quantitative thermal shift curves of red fluorescence (red
curve), green fluorescence (green curve), and turbidity (black curve).
d) MALDI and fluorescence analyses revealed covalent modification
triggered the color-switch property. e) Chemical crosslinking experi-
ments revealed red fluorescence is from misfolded soluble oligomers
using Coomassie blue stained SDS-PAGE gel. f) Fractionation experi-
ment revealed green fluorescence in the insoluble fraction.
Michael Addition Triggers the Fluorescence Color-Switch
Property
Protein aggregation is a multi-step phase separation
process that involves folded proteins (F), unfolded proteins
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First, urea mediated DHFR protein unfolding resulted in
mechanism: AggSwitch probe P1 first binds to misfolded
minimal fluorescence increase in neither the red nor the green
fluorescence (Figure 5b, black curve). Thus, unfolded pro-
teins are not responsible for the turn-on mechanism of red
and green fluorescence. Second, the thermal shift curve of red
fluorescence (Tm = 324.38 K, Figure 5c, red curve) exhibited
a slightly lower melting temperature than that of turbidity
assay measuring insoluble aggregates (Tm = 326.59 K, Fig-
ure 5c, black curve), indicating the red fluorescence might be
from misfolded soluble oligomers that formed earlier than the
insoluble aggregates of DHFR. Meanwhile, MALDI mass
spectrometry result and the corresponding fluorescence
profile when DHFR transited from misfolded state to
insoluble state (Figure 5d, middle panel, M to I transition
state, pH 6.23, 538C for 5 min) showed incomplete modifica-
tion of DHFR proteins by P1 and partial fluorescent
conversion from red to green color. Chemical crosslinking
of DHFR aggregation intermediates (M to I transition state)
confirmed the presence of misfolded soluble oligomeric
DHFR (Figure 5e, M to I transition state, Figure S11 for
fluorescent gel). The disappearance of DHFR soluble oligo-
mers as protein aggregation proceeded (Figure 5e, I state,
pH 6.23, 648C for 60 min) matched the decrease in red
fluorescence (Figure 5d), again confirming the red fluores-
cence was from the misfolded oligomeric proteins. Together,
these data supported that P1 turned on red fluorescence upon
binding to the misfolded soluble oligomers but remained
unreacted. Finally, we showed complete conversion of
fluorescence from red to green color as aggregated DHFR
was all covalently modified by P1 (Figure 5d, lower panel, I
state, pH 6.23, 648C for 5 min). This result demonstrated
color-switch property was triggered by the covalent modifi-
cation.
soluble oligomeric proteins via non-covalent physical inter-
actions and turns on its red fluorescence; as proteins form
insoluble aggregates, they further compact and activate
nucleophiles to react with the probe via Michael addition
and switched to the green fluorescence (Figure 5a). The
alternative mechanism does not hold true as we observed no
covalent modification when protein is folded shown by the
MALDI results (Figure 5d) and the presence of red fluores-
cence during the transition from misfolded oligomers to
insoluble aggregates (Figure 5b, c, and d).
Exploiting the Michael Addition Chemistry to Develop Other
Protein Aggregation Color-Switch Sensors
To demonstrate this chemistry reported here can be
utilized to develop other fluorescence color-switch probes, we
designed MG probe (P4) based on the scaffold of malachite
green (MG) dye (Figure 6a). MG derivatives have been
reported to be fluorogenic upon binding to evolved peptide
sequences.[50,51] Thus MG scaffold may inherently bind to
aggregated proteins. MG of positive charge possesses electro-
positivity in the center of the molecule and has been reported
as a reversible Michael acceptor with thiol group (Fig-
ure S16b).[52] Encouraged by these pieces of evidence, we
tested the fluorescence color-switch property of P4 upon the
aggregation of mut-DHFR. Surprisingly, we observed exclu-
sively red fluorescence in aggregated mut-DHFR (Figure 6b,
ex.max 600 nm, em.max 652 nm) but no color-switch property as
Fractionation experiment showed green fluorescence
located in the insoluble fraction of aggregated DHFR (Fig-
ure 5 f, S26), indicating the green fluorescence is from
insoluble aggregates of DHFR proteins. Interestingly, AggS-
witch P1 probe switched to green fluorescence slightly slower
than the formation of DHFR insoluble aggregates shown by
the similar melting temperatures in the thermal shift curves of
green fluorescence (Tm = 327.57 K) and turbidity assay (Tm =
326.59 K) (Figure 5c, Figure S2b). This data indicated that the
green fluorescence was caused by insoluble DHFR aggre-
gates. It also highlighted the fast labeling kinetics of P1
towards aggregated proteins because no significant delay of
green fluorescence from the turbidity data was observed at
the given concentrations.
We next explored the impact of covalent linkage on the
green fluorescence of P1. P1ꢀs Michael addition product
surrogate probe P2 without Michael acceptor was not capable
of fully turning on the green fluorescence to the same
intensity as P1 upon protein aggregation (Figure S12).
Furthermore, addition of P1 after the formation of insoluble
aggregate protein aggregation could not effectively turn on
the green fluorescence either and the reaction is aggregation-
stage-depedent (Figure S13). These results indicated that
covalency occurring during protein misfolding and aggrega-
tion is necessary to fully switch on green fluorescence of P1.
Together, these lines of evidence supported a plausible
Figure 6. Fluorescent color-switch probe P4 revealed different chem-
ical reactivity inside different protein aggregates. a) Malachite green
(MG) dye derivative (P4) can reversibly react with thiols via covalent
Michael addition. b) Mut-DHFR’s aggregation exclusively turned on
the red fluorescence of P4. c) Mutant SOD1 (V31A) switched on the
blue fluorescence of P4. d) TEM images of aggregated mut-DHFR and
SOD1(V31A) proteins revealed different compactness inside the in-
soluble aggregates. e) The highly compact SOD1(V31A) aggregates
were more resistant to proteolysis than mut-DHFR aggregates.
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shown in P1. No fluorescence color-switch responses of P4
towards aggregated DHFR may arise from its decreased
chemical reactivity compared to AggSwitch P1 probe because
the electropositivity of its Michael acceptor is compromised
by the nearby p electrons through electron delocalization.
Meanwhile, P4 is also less accessible to nucleophilic conjuga-
tion due to its larger steric hindrance than P1. However,
fluorescence color-switch property of P4 was resumed when
using the other pathogenic model protein mutant superoxide
dismutase 1 (SOD1-V31A). We observed exclusively blue
fluorescence in aggregated SOD1-V31A (Figure 6c, ex.max
380 nm, em.max 434 nm).
We then questioned ourselves why P4 exhibited different
fluorescence color-switch responses towards different aggre-
gated proteins. Namely why do different aggregated proteins
possess differential chemical reactivity? One plausible ex-
planation is that P4 of lower chemical reactivity and higher
steric hindrance may require much higher compactness in
aggregated proteins in order to bring the probe closer to the
reactive nucleophiles for Michael addition. To validate this
hypothesis, we first utilized transmission electron microscope
to examine the compactness of aggregated DHFR and SOD1
proteins (Figure 6d). We found that aggregated SOD1 is less
transparent compared to aggregated DHFR, indicating its
higher compactness and density. Second, we used a reported
non-covalent protein aggregation sensor AggStain that can
detect the local viscosity inside aggregated proteins to reflect
its compactness (Figure S14). Supporting the TEM observa-
tion, we found aggregated SOD1 is more compact than
aggregated DHFR as the sensor exhibited higher fluores-
cence in aggregated SOD1 (Figure S14). Third, proteolytic
resistance of experiment pinpointed that SOD1 aggregate is
more compact and less accessible to proteinase K degradation
(Figure 6e), which may explain why SOD1 aggregation is
pathogenic in vivo but mut-DHFR was reported to be fast
degraded. Together, we showed that the compactness of
aggregated proteins influences the chemical reactivity of the
nucleophiles inside aggregated proteins. The differences of
chemical reactivity in aggregated proteins can be reflected by
the different fluorescence color-switch response of our
probes.
Figure 7. Decreasing MG chemical reactivity leads to partial fluores-
cence color-switch response to aggregated proteins. a) Decreasing the
chemical reactivity of MG dye resulting in P5 probe. b) Emission
spectra of P5 in aggregated SOD1(V31A). P5 partially switched its red
fluorescence to blue upon SOD1(V31A) aggregated. c) Quantitative
analysis of (b).
dye in conjugation with thiol functional group is reversible in
both previous study and our experiment (Figure S16b).
Overall, these results demonstrated that the Michael addition
chemistry upon protein aggregation reported in this work can
be modulated by fine-tuning the chemical reactivity of the
designed probe. Modulation of chemical reactivity can result
in different fluorescence color-switch response towards ag-
gregated proteins.
Michael Addition Reactivity of Cellular Aggregated Proteome
Finally, we examined whether this chemistry can occur in
the aggregated proteome of stressed live cells. Supported by
the above in vitro results, our proposed Michael addition
reaction model is that the probe first selectively binds to
misfolded proteins, turns on the red fluorescence, and then
switches to green color upon aggregation induced covalent
reaction. To examine this model in live cells, proteome
aggregation was first induced by attenuating cellular protein
degradation using proteasome inhibitor MG132. We attempt-
ed to visualize aggregated proteome by confocal microscopy
using AggSwitch P1 due to its better spectral alignment with
commercial laser source. As expected, we observed punctate
and peri-nuclear aggresomes exclusively in the green channel
upon addition of proteasome inhibitor MG132 in U251
human glioblastoma cells for 18 h (Figure 8, lower panel),
whereas the control cells at basal condition were not
fluorescent (Figure 8, upper panel). Fractionation of cellular
lysate further confirmed the green fluorescent aggregates
formed in the stressed proteome (Figure S18). Interestingly,
we observed the intermediate red fluorescence of P1 upon
short-term 5 h stress (Figure 8 middle panel), supporting our
in vitro model (Figure 5a). In addition, P2 added prior to or
after proteome aggregation was not capable of staining
aggregated proteome (Figure S17), indicating covalency is
required for efficient switch-on of the green fluorescence.
This result echoed our in vitro experiment shown in Fig-
Modulation of the Chemical Reactivity of Michael Addition
The above results suggest that the reactivity of this
Michael addition upon protein aggregation is affected by (1)
the compactness of aggregated protein and (2) the chemical
reactivity of the Michael acceptor in the fluorescent color-
switch probe. To further demonstrate the tunability of this
chemistry, we then tuned down the chemical reactivity of P4
via enhancing the electron donating capacity, resulting in
probe P5 (Figure 7a). Probe P5 of decreased Michael
addition reactivity partially converted its red fluorescence to
blue fluorescence in SOD1 aggregates (Figure 7b,c), reflect-
ing the compromised reactivity. However, we were not able to
observe covalency after denaturing and refolding the aggre-
gated SOD1 in conjugation with P4 and P5 by SDS-PAGE
analysis (Figure S15). This is possibly due to the fact that MG
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fluorogenicity was also reversible (Figure S19). Second, P1
itself first turned on its red fluorescence upon protein
misfolding, indicating P1 tends to bind misfolded proteins
prior to covalent conjugation (Figure 5). We then asked
whether all Michael acceptors can label aggregated proteins.
To test this hypothesis, we installed the vinyl ester Michael
acceptor onto a common BODIPY fluorophore scaffold (M3,
Figure S20) that is not an inherent binder for aggregated
proteins, resulting in P6 probe. We observed neither covalent
labeling of aggregated protein by P6 nor color-switch
property (Figure S20). Therefore, even though they harbor
a Michael acceptor, probes of no binding affinity to aggre-
gated proteins are not capable of covalent modification. This
is probably why such reactivity was not reported previously
given the wide applications of Michael acceptor probes.
Third, cysteine-free protein, like our DHFR mutant
(C85S:C152S, Figure S9), cannot be modified by P1, showing
that the protein needs to harbor nucleophilic cysteine for
Michael addition to occur. However, reaction with other less
nucleophilic residues (lysine or serine) may occur if the
electrophile of a probe is more reactive. If a fluorogenic
covalent sensor for aggregated proteins was desired for
imaging purpose, the scaffold may exhibit polarity or viscosity
dependent fluorescence upon binding to aggregated proteins
as our P1, P2, P3, P4, and P5 probes (Figure S21, S22).
Thus, a probe bearing inherent binding affinity to
aggregated proteins and a Michael acceptor can undergo
the Michael addition reaction reported here. The inherent
binding capacity of the probe will guide the probe into
misfolded proteins first. Upon protein aggregation, protein
structurally collapses and compacts to bring the probe and
reactive nucleophiles close enough for covalent reaction.
However, further elaboration of the scaffold diversity of
binding moieties and the chemical reactivity of the warheads
is desired to understand how to selectively target the
aggregates of specific protein-of-interest.
Figure 8. Detecting aggregated proteome in stressed live cells via the
Michael addition reaction with AggSwitch P1. Confocal images of
U251 cells stressed by MG132 to induce proteome aggregation.
Experimental details see Supporting Information.
ure S12. These imaging results suggested that probe P1 is
a fluorescent color-switch probe upon protein aggregation
induced by Michael addition and can be utilized to detect
aggregated proteome in stressed cells.
Discussion on the Design Strategy for Covalent Probes Targeting Conclusion
Aggregated Proteins
In summary, we have reported in this work that Michael
What structural features does a probe need to harbor in
order to selectively target the reactive nucleophiles in
aggregated proteins? Our results highlight three require-
ments for a designed probe to covalently label aggregated
proteins. First, the probe needs to harbor a Michael acceptor
or other potential reactive electrophiles for covalent conju-
gation. Second, the core structure of the designed probe tends
to inherently bind misfolded and aggregated proteins in order
to bring the probe close enough to the reactive nucleophiles
and trigger this chemistry. Third, the protein is not cysteine
free if using Michael addition.
Several lines of evidence support these notions. First, the
non-covalent analogues of P1 have been reported to exhibit
inherent binding affinity towards aggregated proteins and be
fluorogenic (Figure S19).[53] Therefore, this core scaffold of P1
serves as a guiding moiety to selectively bind to aggregated
proteins. However, without the Michael acceptor functional
groups, covalency was not expected to occur and the
addition reaction can occur upon protein aggregation. Based
on this chemistry, we designed color-switch fluorogenic
probes of tunable fluorescence responses towards different
protein aggregates. We revealed that different protein aggre-
gates may exhibit different compactness, proteolytic resist-
ance, and consequently chemical reactivity. In the future,
exploration of other potential chemical reaction types and
precise control over the chemical reactivity upon protein
aggregation may lead to novel chemical strategies to target
proteins at this challenging folding state. New covalent
labeling chemistry of aggregated proteins may offer oppor-
tunities to develop functional probes, such as sensors for
imaging, activity-based probes for aggregated proteome
profiling, and covalent PROTAC54 or LyTAC55 drugs for
clearance of pathogenic aggregation.
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This work was supported, in part, by funds National Natural
Science Foundation of China (21907091), the LiaoNing
Revitalization Talents Program from the Liaoning province
of China (XLYC1907048), Dalian Innovation Fund
(2020JJ26GX027), China Postdoctoral Science Foundation
Grant (2019M661138), the National Key Research and
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Keywords: bioconjugation ·fluorescent probe ·
fluorescent proteins ·protein aggregation ·protein chemistry
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