A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells

Article abstract of DOI:10.1021/ja500170h

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY

Full text of DOI:10.1021/ja500170h

Communication
pubs.acs.org/JACS
A Rapid and Fluorogenic TMP-AcBOPDIPY Probe for Covalent
Labeling of Proteins in Live Cells
Wei Liu,†,‡,∥ Fu Li,†,‡,∥ Xi Chen,†,‡,∥ Jian Hou,§,∥ Long Yi,‡ and Yao-Wen Wu*,†,‡
†Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, 44227 Dortmund, Germany
§
‡Department of Physical Biochemistry, Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology,
Otto-Hahn-Str. 11, 44227 Dortmund, Germany
S
* Supporting Information
modification,4 ligand binding domains,5 and self-labeling
enzymatic domains.6 These methods have profoundly enriched
ABSTRACT: Protein labeling is enormously useful for
characterizing protein function in cells and organisms.
Chemical tagging methods have emerged as a new
generation protein labeling strategy in live cells. Here we
have developed a novel and versatile TMP-AcBOPDIPY
probe for selective and turn-on labeling of proteins in live
cells. A small monomeric tag, E. coli dihydrofolate
reductase (eDHFR), was rationally designed to introduce
a cysteine in the vicinity of the ligand binding site.
Trimethoprim (TMP) that specifically binds to eDHFR
was linked to the BOPDIPY fluorophore containing a
mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY
rapidly labeled engineered eDHFR tags via a reaction
termed affinity conjugation (a half-life of ca. 2 min), which
is one of the top fast chemical probes for protein labeling.
The probe displays 2-fold fluorescence enhancement upon
labeling of proteins. We showed that the probe specifically
labeled intracellular proteins in live cells without and with
washing out the dye. We demonstrated its utility in
visualizing intracellular processes by fluorescence-lifetime
imaging microscopy (FLIM) measurements.
the spectrum of tools used to investigate biological events.
However, intrinsic limitations include cytotoxicity, off-target
labeling, high background staining, and a slow labeling rate.
Many of them rely on either reversible noncovalent labeling or
enzymatic reactions that have limitations of low reactivity in
some organelles and steric hindrance. These limitations
represent a great challenge for the development of chemical
labeling tags in vivo. There remains a high demand for
intracellular labeling reagents with low cytotoxicity, good cell
permeability, fast reaction rates, and low background staining.
Fluorogenicity is a desirable feature in protein labeling
particularly in a complex biological context, as it allows a dye to
“turn-on” after labeling and hence helps to reduce labeling
background and substantially enhances the signal-to-noise ratio.
Therefore, strategies for fluorogenic protein labeling are gaining
enormous interest from the perspective of protein chemistry
and cell biology.7 Affinity probes are an attractive approach,
because selectivity can be conferred by specific ligand binding.
Recently, affinity probes and baits have been specifically
incorporated into proteins via selective chemical conjugations,
which are mediated by inhibitor binding or metal chelating.8
Covalent probes, as opposed to noncovalent ones, resulted in
the permanent labeling of target proteins and are therefore
often more superior in many applications.4,6 Furthermore,
protein labeling kinetics is another essential consideration in
live cell labeling. A fast labeling rate confers better temporal
control, which is a fundamental feature of chemical labeling. A
combination of high selectivity, fast covalent reaction, and
fluorogenicity in an individual chemical probe is profoundly
beneficial for chemical labeling in cells. However, only limited
progress has been achieved in this development.8,9 In this study,
we aimed to develop a rapid and fluorogenic chemical probe for
the covalent labeling of intracellular proteins in live cells.
The trimethoprim (TMP) tag has been introduced by
Cornish’s lab.5b TMP has high affinity (KI = 1nM) for
Escherichia coli dihydrofolate reductase (eDHFR), but a much
lower affinity (KI = 4−8 μM) for a mammalian form of
DHFR.10 eDHFR is small (18 kD, two-thirds of GFP) and
monomeric and marginally perturbs the function of proteins
that it fuses with. Moreover, TMP can be derivatized without
significantly disrupting its binding to eDHFR.5b Here we
tudying protein function in vitro or in the context of live
Scells and organisms is of vital importance in biological
research. Genetic tags such as fluorescent proteins (FPs) are
widely used to detect proteins. However, compared to chemical
tags, FPs have several limitations, including environmental
sensitivity, limited flexibility of modification, and less
amenability for temporal control.1 In contrast, chemical probes
are able to achieve properties that are not readily possible when
using fluorescent proteins, such as fluorophore-assisted light
inactivation, real-time detection of protein synthesis, and
multicolor pulse-chase labeling.1b Many organic dyes are
superior to fluorescent proteins in terms of brightness,
photostability, far red emission, environmental sensitivity, and
potential for modifications to their spectral and biochemical
properties. Altogether, chemical labeling has become an
important strategy for the study of protein function in live
cells and organisms.1
Recent advancements in in vivo chemical labeling techniques
involve the combination of the specificity of genetically
encoded tags and the flexibility of small molecule probes.
Such amino acid sequences include tetracysteine/tetraserine
motifs,2 metal chelation motifs,3 peptide tags for enzymatic
Received: January 7, 2014
Published: March 5, 2014
© 2014 American Chemical Society
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develop a novel probe for chemical protein labeling in live cells,
termed affinity conjugation, using the TMP tag. The general
scheme of this approach is shown in Figure 1.
Second, TMP was demethylated to produce a 4′-phenol
derivative of TMP (1) that was introduced with a carboxylate
group (2), and subsequently coupled with a TEG-like linker,
yielding the intermediate TMP-TEG-NH2 (3). Finally,
AcBOPDIPY (6) was coupled with TMP-TEG-NH2 (3),
affording the target molecule, TMP-AcBOPDIPY (7). The
quantum yield of the TMP-AcBOPDIPY probe was determined
to be Φ 0.31 in 50 mM PBS buffer (pH 7.4) using Fluorescein
as the reference (ΦR = 0.87 at pH 7.4). In PBS buffer (pH 7.4),
TMP-AcBOPDIPY displays maximum absorption at 503 nm (ε
4.9 × 103 M−1 cm−1) and maximum emission at 519 nm
=
(Figure S1).
On the other hand, we designed eDHFR mutants with one
or two cysteine mutations in the vicinity of the TMP binding
site to enable Michael addition with the acrylamide group on
the TMP-AcBOPDIPY probe. The key issue is to identify
mutations that do not influence the tight and specific binding of
TMP to eDHFR. Analysis of the crystal structure identified
several amino acid residues (A19, N23, G51, and R52) located
in two loop regions in the vicinity of the binding site (Figure
2A). These amino acid side chains do not appear to be involved
Figure 1. Principle of labeling of protein via affinity conjugation in live
cells using the TMP-AcBOPDIPY probe.
We first designed a TMP-probe, TMP-AcBOPDIPY,
featuring four modules including a ligand (TMP), a spacer
[TEG (tetraethyleneglycol)-like linker (diethylene glycol bis-
aminopropyl terminated)], a fluorophore (BOPDIPY), and a
mildly thiol-reactive acrylamide group (compound 7) (Scheme
1). To make a fluorogenic probe, we use an environment-
a
Scheme 1. Synthesis of TMP-AcBOPDIPY
Figure 2. Labeling rate and fluorescence enhancement. (A) The TMP-
binding site of eDHFR. The G51, R52, N23, and A19 residues in close
proximity to the ligand binding pocket are highlighted. (B) Kinetic
study of the labeling of eDHFR mutants by TMP-AcBOPDIPY.
Recombinant eDHFR wild type or eDHFR mutant (5 μM) was
labeled with 10 μM probes at 37 °C. At time points, reaction was
quenched by denaturing SDS-sample buffer with 9 M Urea and
resolved by SDS-PAGE. (C) Summary of the first-order reaction
kinetic constants and half-lives. * Reaction was performed in the
presence of 50 μM NADPH. (D) SDS-PAGE analysis of
eDHFR_N23C/G51C reaction with TMP-AcBOPDIPY (upper:
Coomassie stain, lower: fluorescent scan). (E) Emission spectra of
TMP-AcBOPDIPY in the absence (dashed line) and the presence of
eDHFR_N23C/G51C (solid line).
a
(i) BrCH2COOMe, t-BuOK, DMSO, rt; (ii) NaOH, MeOH, rt, 1 h;
(iii) Boc-TEG-NH2, EDC, HOBt, DIEA, DMF, rt, 5 h; (iv) TFA,
DCM, 3 h; (v) neat TFA, rt, 4 h; (vi) DDQ, MePh, rt, 5 min; (vii)
Et3N; (viii) BF3·OEt2, 10 min; (ix) 10% Pd/C, MeOH, rt, overnight;
(x) acryloyl chloride, Et3N, 1,4-dioxane; (xi) LiOH, H2O, THF,
iPrOH; (xii) 3, EDC, HOBt, DIEA, DMF, rt.
sensitive boron phenyldipyrromethene (BOPDIPY) dye.
BOPDIPY can freely rotate around the single bond between
the BODIPY moiety and the phenyl group. The fluorescence
intensity of BOPDIPY increases substantially with increasing
solvent viscosity because of the restricted rotation of the phenyl
group.11 We speculate that BOPDIPY would display dramatic
fluorescence enhancement when it binds to proteins. TMP-
AcBOPDIPY was synthesized via a convergent synthetic route.
First, the BOPDIPY intermediate 4 was prepared through the
condensation reaction between the benzaldehyde derivative and
dimethylpyrrole followed by oxidation.12 The nitro group of 4
was reduced to an amino group via hydrogenation, and the
resulting 5 was acrylated yielding the key intermediate 6.
in essential interactions and are well exposed on the surface.
Moreover, their side chains are close (ca. 8−9 Å) to the 4′-O of
the trimethoxybenzyl group. We generated four eDHFR
mutants (A19C, N23C, N23C/G51C, and R52C) and purified
recombinant proteins from E. coli.
We evaluated the in vitro reactivity of the TMP-AcBOPDIPY
probe with these eDHFR mutants. The reaction was assayed by
denaturing SDS-PAGE. As can be seen from the fluorescent gel,
time-dependent incorporation of TMP-AcBOPDIPY into
eDHFR mutants was observed, but not on the wide type
eDFHR (Figure 2B). The reaction traces can be fitted to a
single exponential function, giving the first-order reaction
kinetic constant of each eDHFR mutant (Figure 2C).
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Apparently, eDHFR N23C and R52C display a very fast
labeling rate with a half time of ca. 2 min. It is worthy to note
that the double mutant, eDHFR N23C/G51C, reacts even
faster with an unprecedented labeling half time of only 1.8 min.
The reaction rate is 10−57-fold faster than those of previous A-
TMP probes8d,e and is also superior to that of most affinity
labeling reactions.13
Next, we investigated fluorescence enhancement upon
labeling. As can be seen from Figure 2E, over 2-fold of
fluorescence enhancement was observed after reaction with
eDHFR_N23C/G51C. The “turn-on” effect seems to solely
originate from the binding of BOPDIPY to eDHFR rather than
covalent conjugation because the similar fluorescence enhance-
ment was also observed when using the wild type eDHFR
(Figure S2). Binding of BOPDIPY to a presumably hydro-
phobic site of eDHFR may also facilitate positioning the
reactive acrylic group to the close proximity of the mutated
cysteine residue and hence substantially enhance the reaction
efficacy. Therefore, the TMP-AcBOPDIPY probe confers the
fast and “turn-on” labeling of proteins.
Thereafter, we proceeded to investigate the labeling of
intracellular proteins in live cells using TMP-AcBOPDIPY. We
fused eDHFR_N23C with the K-Ras C-terminal sequence
(CAAX) which targets the plasma membrane (PM), the
nucleus localization peptide (NUC) which targets the nucleus,
Rab1 protein residing at the Golgi apparatus, and Rab5 protein
which localizes at the endosomes. We added 1 μM TMP-
AcBOPDIPY to the culture medium of Hela cells expressing
one of these fusion proteins. The confocal fluorescence
microscopic images before and after washing out the dye
were both recorded (Figure 3A). TMP-AcBOPDIPY is cell-
permeable and clearly labeled the target proteins in live cells. As
shown in Figure 3A, after washing only the cells expressing the
protein tag are highly fluorescent. Because of the fluorogenic
properties of the probe, images taken before washing out the
probe are analogous to those taken after washing in the
expressed cells (Figure 3A), suggesting that specific labeling
and imaging of intracellular proteins in live cells can be
accomplished under nonwashing conditions. To compare the
covalent labeling with the noncovalent labeling, we labeled the
cells expressing eDHFR wild type (eDHFRwt)-CAAX with
TMP-AcBOPDIPY. As shown in Figure S3, the staining of the
plasma membrane showed a significant decrease after washing,
suggesting that the covalent labeling is more stable than the
noncovalent labeling.
In order to further confirm the specific and covalent labeling
reactions occurred inside live cells, we lysed the labeled cells
and subjected the cell lysate to denaturing SDS-PAGE. The
expression levels of eDHFR wild type (eDHFRwt) or various
eDHFR mutant (eDHFRmt) fusion proteins were confirmed
by Western blot (WB) analysis of the Hemagglutinin (HA) tag
that is fused with each protein. While cell lysates showed a
range of proteins based on Coomassie staining, only a single
fluorescent band was observed in the cells expressing
eDHFRmt fusion proteins, but not in the cells expressing
eDHFRwt or eDHFRwt-CAAX (Figures 3B and S4). This
suggests that TMP-AcBOPDIPY selectively and covalently
labels the proteins of interest inside cells.
In order to demonstrate the utility of the TMP-AcBOPDIPY
probe for visualization of intracellular processes, we measured
the Foster resonance energy transfer (FRET) by fluorescence-
̈
lifetime imaging microscopy (FLIM) in live cells. FRET is
highly useful and can be employed to elucidate many
intracellular processes, such as protein−protein interactions
(PPIs) and substrate−enzyme binding. As a proof of principle,
a red fluorescent mCherry protein was fused N-terminally to
eDHFR_N23C-CAAX to constitute an “interacted” protein
pair. After labeling with TMP-AcBOPDIPY, the FRET between
the donor BOPDIPY and the acceptor mCherry can be
determined by the FLIM measurement (Figure 4). FLIM is an
imaging technique based on measurement of the lifetime of a
fluorophore. Energy transfer from the donor molecule to the
acceptor molecule will lead to a decrease in the lifetime of the
donor, which can be recorded by FLIM. Since the FLIM
Figure 3. Labeling of intracellular proteins in live cells. (A) Confocal
microscopy of live HeLa cells expressing eDHFR_N23C fused with K-
Ras C-terminal sequence (CAAX), Rab1, Rab5, and nucleus localizing
sequence (NUC) locating at the plasma membrane, the Golgi body,
the endosomes, and the nucleus, respectively. Scale bar 10 μm. (B)
Selective and covalent labeling of proteins in cells. HeLa cells
expressing the indicated fusion proteins were lysed and subjected to
denaturing SDS-PAGE analysis.
Figure 4. FLIM measurements in live cells. Fluorescence lifetime
images of BOPDIPY (left panel), fluorescence confocal images of
BOPDIPY and mCherry (middle panels), and phase contrast images
(right panel) of HeLa cells expressing eDHFR_N23C-CAAX (upper
panels) or mCherry-eDHFR_N23C-CAAX (lower panels).
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measurements are insensitive to the concentration of
fluorophores and can thus filter out artifacts resulted from
changes in the concentration and emission intensity, FLIM has
been a very useful technique for monitoring protein
interactions in cells.14 Hela cells expressing eDHFR_N23C-
CAAX or mCherry-eDHFR_N23C-CAAX were treated with
TMP-AcBOPDIPY. In labeled Hela cells expressing
eDHFR_N23C-CAAX, BOPDIPY displayed an average fluo-
rescence lifetime of 4.3 ns. In contrast, in Hela cells expressing
mCherry-eDHFR_N23C-CAAX, the fluorescence lifetime of
BOPDIPY substantially reduced to 3.6 ns, suggesting a
significant energy transfer from BIDIPY to mCherry (Figure
4). Noteworthy, the BOPDIPY fluorescence lifetime is much
higher than that of fluorescent protein donors (ca. 2.5 ns for
EGFP and ca. 3.0 ns for Citrine) and displays a superior
dynamic range in FLIM measurements. This study demon-
strates that the TMP-AcBOPDIPY probe is well suited for
intracellular FRET studies.
In summary, we have developed a rapid (reaction half-lives of
ca. 2 min) and fluorogenic TMP-AcBOPDIPY probe for the
intracellular labeling of proteins fused with an engineered
eDHFR tag. To our knowledge, this is the first affinity probe
which combines all three features (high selectivity, fast covalent
reaction, and fluorogenicity). We expect that the novel probe
can be widely used for the labeling of proteins in cells and for
the elucidation of various cellular processes.
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ASSOCIATED CONTENT
* Supporting Information
General procedures and materials, detailed preparation of
TMP-AcBOPDIPY, fluorescence enhancement of TMP-Ac-
BOPDIPY upon binding to wild type eDHFR, Coomassie
staining of the gel shown in Figure 3B, etc. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
yaowen.wu@mpi-dortmund.mpg.de
■
(10) Kuyper, L. F.; Roth, B.; Baccanari, D. P.; Ferone, R.; Beddell, C.
R.; Champness, J. N.; Stammers, D. K.; Dann, J. G.; Norrington, F. E.;
Baker, D. J.; Goodford, P. J. Med. Chem. 1982, 25, 1120−1122.
(11) Kuimova, M. K.; Yahioglu, G.; Levitt, J. A.; Suhling, K. J. Am.
Chem. Soc. 2008, 130, 6672−6673.
Author Contributions
∥W.L., F.L., X.C., and J.H. contributed equally to this work.
Notes
The authors declare no competing financial interest.
(12) Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891−4932.
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Yasui, R.; Miki, T.; Ojida, A.; Hamachi, I. J. Am. Chem. Soc. 2012, 134,
3961−3964. (c) Levitsky, K.; Boersma, M. D.; Ciolli, C. J.; Belshaw, P.
J. ChemBioChem 2005, 6, 890−899.
ACKNOWLEDGMENTS
■
We thank Philippe Bastiaens for the support in FLIM
measurements. We thank James C. Hu for the kind gift of
the E. coli. DHFR plasmid. We thank Christiane Theiss for the
help on protein purification. This work was supported in part
by DFG grants (Grant No. SPP 1623 and SFB 642 to Y.W.W.).
(14) Wouters, F. S.; Verveer, P. J.; Bastiaens, P. I. Trends Cell Biol.
2001, 11, 203−211.
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