Quenched ligand-directed tosylate reagents for one-step construction of turn-on fluorescent biosensors

Article abstract of DOI:10.1021/ja902486c

Semisynthetic fluorescent biosensors consisting of a protein framework and a synthetic fluorophore are powerful analytical tools for specific detection of biologically relevant molecules. We report herein a novel method that allows for the construction of

Full text of DOI:10.1021/ja902486c

Published on Web 06/05/2009
Quenched Ligand-Directed Tosylate Reagents for One-Step
Construction of Turn-On Fluorescent Biosensors
Shinya Tsukiji,† Hangxiang Wang,† Masayoshi Miyagawa,† Tomonori Tamura,†
Yousuke Takaoka,† and Itaru Hamachi*,†,‡
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto UniVersity, Katsura, Kyoto 615-8510, Japan, and Core Research for EVolutional Science
and Technology (CREST), Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku,
Tokyo 102-0075, Japan
Received April 4, 2009; E-mail: ihamachi@sbchem.kyoto-u.ac.jp
Abstract: Semisynthetic fluorescent biosensors consisting of a protein framework and a synthetic fluorophore
are powerful analytical tools for specific detection of biologically relevant molecules. We report herein a
novel method that allows for the construction of turn-on fluorescent semisynthetic biosensors in a one-
step manner. The strategy is based on the ligand-directed tosyl (LDT) chemistry, a new type of affinity-
guided protein labeling scheme which can site-specifically introduce synthetic probes to the surface of
proteins with concomitant release of the affinity ligands. Novel quenched ligand-directed tosylate (Q-LDT)
reagents were designed by connecting an organic dye to a conjugate of a protein ligand and a fluorescence
quencher through a tosyl linker. The Q-LDT-mediated labeling directly converts a natural protein to a
fluorescently labeled protein that remains noncovalently complexed with the cleaved ligand-tethered
quencher. The fluorescence of this labeled protein is initially quenched and only in the presence of specific
analytes is the fluorescence enhanced (turned on) due to the expulsion of the ligand-quencher fragment.
Using a single labeling step, this approach was successfully applied to carbonic anhydrase II (CAII) and a
Src homology 2 (SH2) domain to generate turn-on fluorescent biosensors toward CAII inhibitors and
phosphotyrosine peptides, respectively. Detailed investigations revealed that the obtained biosensors exhibit
their natural ligand selectivity. The high target-specificity of the LDT chemistry also allowed us to prepare
the SH2 domain-based biosensor not only in a purified form but also in a bacterial cell lysate. These results
demonstrate the utility of the Q-LDT-based approach to expand the applications of semisynthetic biosensors.
Introduction
as scaffold building blocks (analyte-binding motifs) for the
preparation of biosensors.5 However, the creation of biosen-
Protein-based fluorescent biosensors are now recognized
as indispensable tools in many areas of biological science
and biomedical research because they allow the monitoring
and quantification of specific biological substances both in
vitro and in vivo.1,2 In particular, with the advent of chemical
biology, the need for biosensors for investigating diverse
biological processes has gained considerable momentum.3
Fluorescent biosensors have also attracted much attention as
powerful platforms for drug screening.4 It is fortunate that a
variety of proteins and protein domains are currently available
sors is still not straightforward due to the paucity of general
methodologies to construct such molecules.
The past decade has witnessed significant advances in the
development of genetically encoded biosensors using fluorescent
proteins (FPs).1,6 Several elegant FP-fusion biosensors have been
produced by fusing two distinct FPs, such as cyan and yellow
FPs, to sandwich a protein scaffold so that the analyte can be
detected through the change of fluorescence resonance energy
transfer (FRET) efficiency. This type of biosensor is undoubtedly
powerful, especially for use in living cells. However, the large
size of FPs (27 kDa) and the constraints placed on the fusion
† Kyoto University.
‡ CREST.
(1) Selected reviews of genetically encoded biosensors: (a) Zhang, J.;
Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. ReV. Mol. Cell Biol.
2002, 3, 906. (b) Miyawaki, A. DeV. Cell 2003, 4, 295. (c) Lalonde,
S.; Ehrhardt, D. W.; Frommer, W. B. Curr. Opin. Plant Biol. 2005,
8, 574.
(5) Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry, 6th ed.; Freeman:
New York, 2006.
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J. A.; Ikura, M.; Tsien, R. Y. Nature 1997, 388, 882. (b) Sato, M.;
Hida, N.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2000, 72, 5918. (c)
Fehr, M.; Lalonde, S.; Lager, I.; Wolff, M. W.; Frommer, W. B. I.
Biol. Chem. 2003, 278, 19127. (d) Sato, M.; Ueda, Y.; Takagi, T.;
Umezawa, Y. Nat. Cell Biol. 2003, 5, 1016. (e) Okumoto, S.; Looger,
L. L.; Micheva, K. D.; Reimer, E. J.; Smith, S. J.; Frommer, W. B.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8740. (f) Sato, M.; Ueda, Y.;
Shibuya, M.; Umezawa, Y. Anal. Chem. 2005, 77, 4751. (g) Qiao,
W.; Mooney, M.; Bird, A. J.; Winge, D. R.; Eide, D. J. Proc. Natl.
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P. L.; Hahn, K. M.; Taylor, D. L. Annu. ReV. Biophys. Biomol. Struct.
1995, 24, 405. (b) Hellinga, H. W.; Marvin, J. S. Trends Biotechnol.
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One-Pot Construction of Fluorescent Biosensors
A R T I C L E S
site (i.e., in most cases, restricted to tagging at the N- and
C-terminus of proteins) severely limit the range of scaffold
candidates that the FP-fusion strategy can be applied to. The
construction of FP-based biosensors has so far been successful
only when the fusion constructs induce a large conformational
change upon binding to the analyte. Such cases are unfortunately
rather rare.
of a cysteine residue, followed by the use of thiol chemistry to
attach an organic dye.2,7 However, many proteins are not
amenable to this strategy because of the presence of an inherent
cysteine residue(s). The acquisition of mutant proteins which
retain natural ligand-binding properties (affinity and specificity)
is not always successful. Although we have developed the
post(photo)affinity labeling modification methods as tools for
introducing fluorescent molecules into natural proteins without
genetic manipulation, these approaches require multiple chemi-
cal reactions to be performed on the protein surfaces.8 In
addition, all the prevalent protein modification procedures9
require a careful purification step after the labeling reaction and
cannot be applied to crude protein mixtures. Clearly, the
establishment of a new methodology that can overcome the
aforementioned limitations is of considerable importance for
the progression of semisynthetic biosensors.
Here we describe a novel, rational protein engineering strategy
that allows one-step construction of turn-on fluorescent semi-
synthetic biosensors. The method is based on the recently
developed ligand-directed tosyl (LDT) chemistry, which is
capable of labeling specific proteins with diverse synthetic
probes with high site-specificity and target-selectivity.10 By
designing a new class of labeling reagents, quenched ligand-
directed tosylate (Q-LDT) compounds, it was feasible to convert
natural proteins to fluorescently labeled proteins that can be
directly used as biosensors based on the bimolecular fluores-
cence quenching and recovery (BFQR) mechanism (Figure 1).11
We successfully applied the strategy to two proteins, carbonic
anhydrase II (CAII) and the Src homology 2 (SH2) domain, to
generate turn-on fluorescent biosensors toward the inhibitors
and phosphorylated peptides, respectively, by a single labeling
step not only in a purified form but also in a bacterial cell lysate.
Site-specific chemical modification of proteins with synthetic
fluorescent dyes is another important means to afford semisyn-
thetic, fluorescent biosensors.2,7,8 The major strength of this
chemistry-driven approach is the flexibility in terms of the choice
of fluorophore and the attachment site (not restricted at the
termini of proteins). Consequently, this approach essentially
permits various fluorescence transduction mechanisms to be
integrated into protein frameworks in a tailor-made manner.
During the past decades, a number of semisynthetic biosensors
have been constructed, particularly by installing an environment-
sensitive dye into a protein scaffold at a specific site such as
the periphery of the analyte-binding pocket.7,8 These biosensors
have been shown to transduce the analyte-binding event into a
change in the fluorescence intensity and/or wavelength by
sensing a subtle change of the microenvironment surrounding
the fluorophore, such as pH and solvent polarity. Importantly,
these examples have clearly demonstrated that the semisynthetic
strategy is powerful and has the potential to be applicable in
the conversion of nearly all possible protein scaffolds to
corresponding biosensors. However, there are challenges to be
addressed. First, it remains difficult to rationally predict the
appropriate site and type of fluorophore to be introduced to
achieve the optimal fluorescence response. In this regard,
fluorescence enhancement, rather than reduction, is favorable
for more sensitive and accurate detection. Second, even after
the optimization through a laborious trial-and-error process, in
many cases, the signal change is small to moderate. Third, as a
more fundamental problem, methods for site-specific protein
modification are limited. Most of the semisynthetic biosensors
previously reported were prepared though genetic incorporation
Results and Discussion
General Design of Q-LDT Compounds. Our idea to construct
turn-on fluorescent biosensors is illustrated in Figure 1. The
concept is based on the LDT chemistry that we recently
developed and is a new type of affinity-guided protein surface
labeling scheme.10 In contrast to existing affinity labeling
reactions,12,13 the LDT-based approach is unique in that the
ligand moiety is cleaved concomitantly with the labeling process,
thus enabling covalent, selective attachment of probes of interest
to specific target proteins without irreversibly abolishing the
function of the labeled proteins. By taking advantage of this
strategy, we designed a new class of protein labeling reagents,
Q-LDT compounds, which contain a fluorescent dye and its
quencher together so that the labeling reagent itself is only
weakly fluorescent. The quencher is introduced as a part of the
leaving group. Upon binding of the ligand moiety to the target
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I. ChemBioChem 2005, 6, 1349. (f) Nakata, E.; Koshi, Y.; Koga, E.;
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Takaoka, Y.; Tsutsumi, H.; Kasagi, N.; Nakata, E.; Hamachi, I. J. Am.
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A R T I C L E S
Tsukiji et al.
Figure 1. Molecular configurations of (a) original ligand-directed tosylate
(LDT) reagent and (b) quenched ligand-directed tosylate (Q-LDT) reagent.
(c) Schematic illustration of the strategy for the Q-LDT-mediated construc-
tion of turn-on fluorescent biosensors.
Figure 2. (a) Molecular structures of LDT reagent 1 and Q-LDT reagent
2 designed for CAII labeling. (b) Sulfonamide and nonsulfonamide
derivatives used in this study. ET, ethoxzolamide; NB, p-nitro-benzene-
sulfonamide; SA, 4-sulfamoylbenzoic acid; BS, benzenesulfonamide; PB,
phenylboronic acid; TS, p-toluenesulfonic acid.
protein, the fluorescent dye is covalently transferred to a
nucleophilic amino acid located on the protein surface through
the proximity-induced SN2-type tosyl chemistry. Concomitantly
with this process, the affinity ligand is cleaved off but remains
noncovalently bound to the ligand binding pocket of the protein,
thereby still allowing the efficient quenching of the fluorescence.
The fluorescence can then be efficiently recovered (turned on)
in the presence of specific analytes by expelling the quencher-
tethered ligand from the binding pocket of the labeled protein.
Therefore, the Q-LDT reagents are expected to serve as a unique
tool to label and convert target proteins in one step to generate
semisynthetic, turn-on fluorescent biosensors which function on
the basis of the BFQR scheme.11
One-Step Conversion of CAII to a Turn-On Fluorescent
Biosensor for Sulfonamide Derivatives. We initially tested the
above strategy using human carbonic anhydrase II (CAII) as a
model protein scaffold and sought to convert this protein to a
fluorescent biosensor toward its inhibitors. Carbonic anhydrases
are a group of metalloenzymes involved in numerous physi-
ological and pathological processes such as gluconeogenesis,
ureagenesis, and tumorigenicity.14 We have previously shown
that CAII can be site-specifically labeled with the 7-diethy-
laminocoumarin (DEAC) fluorophore using the LDT reagent 1
(Figure 2a).10 1 contains benzenesulfonamide as a specific ligand
for CAII,15 which is connected with the DEAC dye (probe)
through an electrophilic phenylsulfonate (tosylate) ester bond.13
On the basis of this, Q-LDT reagent 2 was designed. To the
core structure of 1, the 4-dimethylaminophenylazobenzene-4-
carboxylic acid (DABCYL) quencher16 was tethered through a
lysine spacer in a way that the ligand-DABCYL conjugate is
cleaved as a leaving group upon the labeling reaction. The
synthesis of 2 was carried out as shown in Scheme S1.
The reactivity of 2 with CAII was investigated by incubating
the two molecules in a buffer solution. As shown by SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) and fluores-
cence gel imaging (Figure 3a), CAII was covalently modified
with the DEAC fluorophore in a time-dependent manner (lanes
2, 3, and 4). The labeling was completely abolished in the
presence of the strong inhibitor ethoxzolamide15 (ET, Figure
2b) (lane 5), indicating that the labeling occurs by an affinity-
driven proximity effect. Consistent with our previous results,10
no noticeable decrease in the labeling yield was observed even
under a high concentration of reduced glutathione (GSH, lane
6). Using an independently prepared DEAC-CAII (1:1) con-
jugate10 as a standard marker (lane 7), the labeling yield after
48 h of incubation was estimated to be ∼50%. We also
confirmed the site-specificity of this modification by subjecting
the DEAC-labeled CAII to conventional peptide mapping
(Figure S1). Lysyl endopeptidase digestion followed by reversed-
phase HPLC and MALDI-TOF mass spectrometry analysis
revealed that the N-terminal L1 fragment corresponding to
Ser2-Lys9 was selectively modified in accordance with our
previous report.10 This result suggests that the labeling site is
His3. Interestingly, despite the attachment of the relatively large
(14) Supuran, C. T. Nat. ReV. Drug. DiscoV. 2008, 7, 168.
(15) (a) Taylor, P. W.; King, R. W.; Burgen, A. S. V. Biochemistry. 1970,
9, 2638. (b) Innocenti, A.; Zimmerman, S.; Ferry, J. G.; Scozzafava,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 4563. (c)
Winum, J.-Y.; Dogne’, J.-M.; Casini, A.; de Leval, X.; Montero, J.-
L.; Scozzafava, A.; Vullo, D.; Innocenti, A.; Supuran, C. T. J. Med.
Chem. 2005, 48, 2121.
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A R T I C L E S
Figure 3. Construction of the CAII-based turn-on fluorescent biosensor for sulfonamide derivatives. (a) SDS-PAGE analysis of the covalent labeling of
CAII with 2. Reaction conditions: 10 µM CAII, 20 µM 2, 100 µM ET (lane 5), 10 mM GSH (lane 6), 50 mM HEPES, pH 7.2, and 37 °C. In lane 7, an
independently prepared DEAC-CAII (1:1) conjugate was used as a standard marker for the determination of the labeling yields. The gel was analyzed by
in-gel fluorescence imaging (FL, lower), and stained with Coomassie brilliant blue (CBB, upper). (b) Fluorescence spectral change of 2-treated CAII upon
the addition of ET. Labeling conditions: 30 µM CAII, 30 µM 2, 50 mM HEPES, pH 7.2, 37 °C, and 48 h. Measurement conditions: 5 µM 2-treated CAII
(the postlabeling sample was 6-fold diluted with no purification, i.e., a mixture of labeled (48%) and native CAII), 0-75 µM ET, 50 mM HEPES, pH 7.2,
25 °C, and λex ) 427 nm. (Inset) Fluorescence titration curve. λem ) 473 nm. All data points are mean ( SD obtained by three independent experiments.
(c) Photograph of the 2-treated CAII in the absence (left) and presence (right) of ET. Measurement conditions: 25 µM 2-treated CAII, 175 µM ET, 50 mM
HEPES, pH 7.2, 25 °C, and λex ) 365 nm. (d) Fluorescence titration profiles of the 2-treated CAII at the emission wavelength of 473 nm with various
sulfonamide and nonsulfonamide derivatives. Experiments were performed in triplicate under the same conditions as (b). n.i./ET ) the ET titration with no
incubation for the labeling.
DABCYL group, the labeling yield was not significantly reduced
compared with that with 1 and the labeling site was also
identical.
respectively (Figure S2).18 More importantly, as shown in Figure
3b, the fluorescence was significantly recovered (by 5-fold)
when ET was added to the postlabeling solution (without
purification). The fluorescence recovery was so efficient that
the brighter emission in the presence of ET was easily detectable
by the naked eye (Figure 3c). In contrast, almost no fluorescence
change was observed by the addition of p-toluenesulfonic acid
(TS, Figure 2b) which has no affinity toward CAII. Also, a
simple mixture of CAII and 2 without incubation did not induce
a significant fluorescence increase upon addition of ET (Figure
3d). These results are fully consistent with the proposed BFQR
scheme of Figure 1c, in which the fluorescence recovery was
due to the expulsion of the noncovalently bound DABCYL-
containing fragment from the labeled CAII by ET.
The postlabeling solution exhibited an emission with a
wavelength maximum at 473 nm upon excitation at 427 nm
and this emission was ascribed to the DEAC fluorophore. Given
the binding affinity of sulfonamide derivatives (typically >106
M-1),15 the cleaved sulfonamide-DABCYL conjugate should
remain bound in the ligand-binding pocket of the DEAC-labeled
CAII, which may cause considerable fluorescence quenching.17
The formation of this noncovalent complex was confirmed by
simple purification of the labeled CAII by size-exclusion
chromatography. The UV-visible spectrum of the CAII-
containing fraction clearly showed two absorption bands at 427
and 474 nm (broad) due to the DEAC and the DABCYL groups,
(18) After the labeling reaction (30 µM CAII, 30 µM 2), ∼50% of the
total LDT reagent 2 was used for the labeling, 20% of that was
hydrolyzed, and 30% remained intact. Since the affinity of the
sulfonamide ligand is high, only the free and small DEAC fragment
generated by hydrolysis can be removed by a short gel column we
used (that is, other sulfonamide-containing components including the
cleaved sulfonamide-DABCYL conjugate and intact reagent 2
remains bound to CAII). In agreement with this, the absorbance ratio
at 427 and 474 nm (427/474) was decreased from 2.00 to 1.76 after
the gel filtration, as shown in Figure S2.
(17) As described in the caption of Figure 3b, we performed the labeling
reaction and fluorescence titration under the conditions in which the
concentrations of the LDT reagent (1 equiv of CAII) and CAII are
sufficiently higher than the dissociation constant of the sulfonamide
ligand to maintain the complex formation. We also confirmed that
when the labeling is carried out in the presence of an excess amount
of CAII, the cleaved sulfonamide-DABCYL conjugate is transferred
from the DEAC-labeled CAII to unmodified CAII (data not shown).
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Tsukiji et al.
Table 1. Apparent Binding Constants (Kapp, M-1) of the
CAII-Based Biosensor to Various Inhibitors and the Relative
Fluorescence Intensities (F/F0) against the Initial Statea
Construction of
a
SH2 Domain-Based Biosensor for
Phosphotyrosine Peptides. Due to the valuable modular feature
of the (Q-)LDT chemistry, it is rationally expected that the
present strategy can be applied to other proteins or protein
domains by changing the affinity ligand. The construction of a
fluorescent biosensor toward phosphorylated peptides by ex-
ploiting a phosphopeptide-recognition domain was our next
target. Since protein phosphorylation plays a central role in the
regulation of cell functions, the development of fluorescent
(bio)sensors for specific phosphorylated peptides/proteins rep-
resents an important area of research.22,23 Here, we chose an
N-terminal SH2 domain of the p85R subunit of human phos-
phatidylinositol-3-kinase (PI3K) (p85-nSH2) as a scaffold,24
which recognizes peptides containing a pYZXM (pY, phos-
photyrosine; Z, Met or Val; X, any amino acid) motif.25
Accordingly, by adopting a pYVPM sequence derived from the
platelet-derived growth factor receptor (PDGFR) as an affinity
ligand,25 LDT reagent 3 and its quenched version 4 were
designed (Figure 4a). The syntheses were conducted as shown
in Scheme S2 by using thiol-maleimide ligation to incorporate
the DEAC fluorophore via a tosylate linker to peptides prepared
by solid-phase peptide synthesis. The p85-nSH2 domain cor-
responding to residues 328-435 of the human p85R was
overexpressed in bacteria as a fusion construct with a glutathione
S-transferase (GST). After purification by glutathione-Sepharose
chromatography, the GST tag was removed by thrombin
proteolytic digestion.
c
)
e
)
inhibitorsb
Kapp (M-1
Ka for native CAII (M-1
F/F0g
ET
NB
SA
BS
PB
TS
8.0 × 105
8.2 × 104
6.5 × 103
9.9 × 103
n.d.d
1.3 × 108
1.6 × 107
3.7 × 106
6.5 × 105
9.4 × 101
n.a.f
5.0 (75 µM)
1.9h (0.5 mM)
3.6 (3.5 mM)
4.4 (5 mM)
2.2 (50 mM)
1.1 (50 mM)
n.d.d
a Measurement conditions: 5 µM 2-treated CAII (a mixture of labeled
(48%) and native CAII) in 50 mM HEPES, pH 7.2, λex ) 427 nm, and
25 °C. b The structures of the inhibitors are shown in Figure 2b.
c Determined by a nonlinear curve-fitting analysis. d Not determined due
to the low affinity. e Reported values.15 f No literature value is available.
g F/F0 indicates the relative fluorescence intensities (F) at 473 nm in the
presence of the inhibitor at the highest concentration used in the assay,
which is shown in parentheses, against those of the initial state (F0).
h The relatively low F/F0 value is due to the nitrobenzene compound
which also has fluorescence-quenching properties.21
The analyte-induced fluorescence increase (turn-on mode) is
a beneficial feature of the present fluorescent biosensor. We next
investigated in more detail the fluorescence response of the
2-treated CAII toward various sulfonamide and nonsulfonamide
derivatives with different binding affinities. As shown in the
inset of Figure 3b, the fluorescence titration curve for ET showed
the typical saturation behavior with an apparent binding constant
of 8.0 × 105 M-1. The titration curves and the apparent binding
affinities are summarized in Figure 3d and Table 1. The relative
order of the affinity of the tested compounds for the semisyn-
thetic biosensor was clearly shown to be practically identical
with that for native CAII. This demonstrated that the natural
ligand specificity is retained even after the labeling. In addition,
the obtained apparent affinity values for each of the sulfonamide
inhibitors are ∼2 orders of magnitude smaller than those
reported for native CAII. This is because the present system is
a competitive assay. In other words, the present system can set
an appropriate threshold to eliminate weak binding ligands,
which is generally advantageous for the screening of more potent
binders.19 On the basis of the competitive inhibition model, the
net affinities of each tested CAII inhibitor were estimated to be
3.3 × 108 M-1 for ET, 3.4 × 107 M-1 for NB, 2.7 × 106 M-1
for SA, and 4.1 × 106 M-1 for BS.20 These values are almost
identical to the reported binding constants of the inhibitors to
native CAII (Table 1), indicating that the original molecular
recognition ability of CAII is fully retained.
We first carried out the labeling of the recombinant SH2
domain. As shown in Figure 5a, by incubating the SH2 domain
with 3 or 4 for 44 h, the protein domain was labeled by the
DEAC dye with an approximate yield of 20% or 30%,
respectively (lane 3 and 8). In the presence of an excess amount
of phosphopeptide ligand 5 (Figure 4b), almost no fluorescence
band was observed. This indicated that the labeling was driven
by the proximity effect induced by the recognition of the
phosphopeptide moiety of the reagents by the SH2 domain. The
DEAC-labeled SH2 domain was also characterized by MALDI-
TOF mass analysis. In addition to a peak at 13131.4 Da
corresponding to the intact SH2 domain, a peak at 13460.2 Da
was detected (data not shown). This peak corresponds to the
molecular weight of the singly DEAC-labeled SH2 domain.26
Conventional peptide mapping using 3 and subsequent tandem
mass analysis showed that the solvent exposed His407 located
(22) (a) Sato, M.; Ozawa, T.; Yoshida, T.; Umezawa, Y. Anal. Chem. 1999,
71, 3948. (b) Ohiro, Y.; Ueda, H.; Shibata, N.; Nagamune, T. Anal.
Biochem. 2007, 360, 266.
Overall, the intact CAII was converted in one-step to a turn-
on fluorescent biosensor for its inhibitors (sulfonamide deriva-
tives) by the Q-LDT-mediated labeling. Notably, following the
incubation of the protein and the Q-LDT reagent, the resulting
solution could be used directly for the fluorescence assay without
the need of any additional step. Since carbonic anhydrases are
recognized as important therapeutic targets to treat a range of
disorders such as obesity, cancer, and glaucoma,14 the present
biosensor platform might be useful for the future development
of new potent CA inhibitors.
(23) (a) Ojida, A.; Mito-oka, Y.; Inoue, M.; Hamachi, I. J. Am. Chem.
Soc. 2002, 124, 6256. (b) Ojida, A.; Inoue, M.; Mito-oka, Y.; Hamachi,
I. J. Am. Chem. Soc. 2003, 125, 10184. (c) Ojida, A.; Mito-oka, Y.;
Sada, K.; Hamachi, I. J. Am. Chem. Soc. 2004, 126, 2454. (d) Anai,
T.; Nakata, E.; Koshi, Y.; Ojida, A.; Hamachi, I. J. Am. Chem. Soc.
2007, 129, 6232.
(24) Skolnik, E. Y.; Margolis, B.; Mohammadi, M.; Lowenstein, E.; Fischer,
R.; Drepps, A.; UIIrich, A.; Schlessinger, J. Cell 1991, 65, 83.
(25) (a) Songyang, Z.; Shoelson, S. E.; Chaudhuri, M.; Gish, G.; Pawson,
T.; Haser, W. G.; King, F.; Roberts, T.; Ratnofsky, S.; Lechleider,
R. J.; Neel, B. G.; Birge, R. B.; Fajardo, J. E.; Chou, M. M.; Hanafusa,
H.; Schaffhausen, B.; Cantley, L. C. Cell 1993, 72, 767. (b) Piccione,
E.; Case, R. D.; Domchek, S. M.; Hu, P.; Chaudhuri, M.; Backer,
J. M.; Schlessinger, J.; Shoelson, S. E. Biochemistry 1993, 32, 3197.
(c) Ladbury, J. E.; Lemmon, M. A.; Zhou, M.; Green, J.; Botfield,
M. C.; Schlessinger, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3199.
(26) The calculated molecular weights of the p85-nSH2 domain and its
singly DEAC-labeled derivative are 13141.68 and 13472.06, respectively.
(27) Nolte, R. T.; Eck, M. J.; Schlessinger, J.; Shoelson, S. E.; Harrison,
S. C. Nat. Struct. Biol. 1996, 3, 364.
(19) Hemmila¨, I. A.; Hurskainen, P. Drug Disc. Today 2002, 7, S150.
(20) For the calculations, we used an inhibition constant of a reported
compound (p-H2NSO2-C6H4-CO-Ala-Phe-OH, Ki ) 12 nM) whose
structure is similar with the cleaved sulfonamide derivative. Mincione,
F.; Starnotti, M.; Menabuoni, L.; Scozzafava, A.; Casinid, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2001, 11, 1787.
(21) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum
Press: New York, 1983.
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One-Pot Construction of Fluorescent Biosensors
A R T I C L E S
Figure 4. (a) Molecular structures of LDT reagent 3 and Q-LDT reagent
4 designed for p85-N-SH2 domain labeling. (b) Sequences of peptide ligands
used in this study. pYZXM (Z ) Met or Val, X ) any amino acid) motifs
are shown in red. CSF-1R, colony-stimulating factor 1 receptor; IRS-1,
insulin receptor substrate 1; hmT, hamster polyoma middle tumor antigen.
(c) Crystal structure of p85-nSH2 complexed with its phosphopeptide ligand
(the DpYVPML region is presented as a stick model).27
in the RB helix of the SH2 domain was predominantly modified
(Figure 4c and Figure S3).27,28
The reaction mixture after the labeling using 4 was passed
through a short gel filtration column, and the eluent was used
for evaluating the function of the fluorescent biosensor.29 This
brief purification procedure removes only molecules unbound
on the SH2 domain, i.e., the eluted SH2 domain still complexes
with the DABCYL-carrying fragment.30 Addition of phospho-
rylated peptide 5, a natural ligand for the SH2 domain, to this
Figure 5. Construction of the SH2 domain-based turn-on fluorescent
biosensor for specific phosphotyrosine peptides. (a) SDS-PAGE analysis
of the covalent labeling of the SH2 domain with 3 and 4. Reaction
conditions: 5 µM p85-N-SH2, 10 µM 3 or 4, 1 mM peptide 5 (lane 4
and 9, Figure 4b), 10 mM GSH (lane 5 and 10), 50 mM HEPES, pH
8.0, and 37 °C. Lane 11, DEAC-CAII (1:1) conjugate. (b) Fluorescence
spectral change of 4-treated p85-N-SH2 upon the addition of phospho-
tyrosine peptide 5. Measurement conditions: 1 µM 4-treated SH2 domain
(the labeled sample shown in lane 8 of (a) was passed through a gel
column and diluted, i.e., a mixture of labeled (30%) and unlabeled SH2
domain), 0-120 µM 5, 50 mM HEPES, pH 7.4, 25 °C, and λex ) 427
nm. (Inset) Fluorescence titration curve, λem ) 473 nm. All data points
are mean ( SD obtained by three independent experiments. (c)
Fluorescence titration profiles of the 4-treated p85-N-SH2 at the emission
wavelength of 473 nm with various peptides. Experiments were
performed in triplicate under the same conditions as (b).
(28) According to the sequence alignment of various SH2 domains, many
SH2 domains possess a His residue at the same (or almost same)
location with the His407 of the p85-nSH2 domain. Thus, it is
reasonable to expect that the present strategy can be efficiently applied
to the labeling of other SH2 domains by simply modifying the
recognition sequence. Kuriyan, J.; Cowburn, D. Annu. ReV. Biophys.
Biomol. Struct. 1997, 26, 259.
(29) This purification step is not essential. However, we have found that it
can reduce the initial background fluorescence caused by partial
hydrolysis of the tosyl linkage of 4 and increase the relative
fluorescence intensity (F/F0).
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A R T I C L E S
Tsukiji et al.
Table 2. Apparent Binding Constants (Kapp, M-1) of the SH2
Domain-Based Biosensor to Various Peptide Ligands and the
Relative Fluorescence Intensity (F/F0) against the Initial Statea
d
)
peptidesb pYZXMc Kapp ( × 105 M-1
ID50 (µM) for native SH2e
F/F0h
5
+
-
-
+
+
+
-
-
1.1
1.8 (0.47)f
n.a.g
n.a.g
1.1
2.7 (120 µM)
1.2 (200 µM)
1.7 (200 µM)
2.4 (120 µM)
2.6 (120 µM)
2.1 (120 µM)
1.7 (200 µM)
2.5 (150 µM)
6
7
0.02
0.06
2.0
8
9
1.9
0.5
10
11
12
1.1
0.08
0.13
1.1
n.a.g
n.a.g
a Measurement conditions: 1 µM 4-treated SH2 domain (a mixture of
labeled (30%) and unlabeled p85-nSH2) in 50 mM HEPES, pH 7.4, λex
) 427 nm, and 25 °C. b The sequences of the peptide ligands are shown
in Figure 4b. c This indicates whether the peptide contains the pYZXM
motif. d Determined by the nonlinear curve-fitting analysis. e Reported
half-maximal inhibition values determined by the competition
experiments using
a resin-immobilized GST-fusion p85-nSH2 and
peptides shown.25b f The value in parentheses is the actual dissociation
constant (µM) of a phosphotyrosine peptide (H2N-SVDpYVDMSK-
CO2H) to p85-nSH2, which was determined by isothermal titration
calorimetry (ITC).25c g No literature value is available. h F/F0 indicates
the relative fluorescence intensities (F) at 473 nm in the presence of the
peptide at the highest concentration used in the assay, which is shown in
parentheses, against those of the initial state (F0).
solution induced an increase in the intensity of the fluorescence
by ca. 3-fold with a typical saturation profile. In contrast, the
fluorescence scarcely changed upon the addition of the non-
phosphorylated peptide 6 (Figure 4b) because this peptide has
no affinity to the SH2 domain. The apparent binding affinity of
peptide 5 to the SH2 domain was determined to be 1.1 × 105
M-1 from the titration curve. This value is smaller than that
reported for intact p85-nSH2. This is because the remaining
cleaved quencher-ligand conjugate acts as a competitive binder
for the labeled SH2 domain. These observations are in good
agreement with the sensing mechanism (BFQR) proposed in
the case of CAII discussed above.
The SH2 domain-based fluorescent biosensor thus prepared
was further tested for its ability to distinguish specific phos-
phorylated sequences from other derivatives. Figure 5c and
Table 2 summarize the titration curves and the apparent binding
affinities for various peptides. Clearly, peptides 5, 8, 9, and 10
bearing the pYZXM motif, which is essential for the high-
affinity interaction with p85-nSH2,25 can be fluorescently
detected in a concentration range of 1-100 µM. In contrast,
replacement of the pY residue in 5 with phosphoserine (peptide
7) greatly reduced the sensing response. More importantly,
neither phosphotyrosine peptide 11 nor 12 containing pYTDL
and pYEEI sequences, respectively, were detected efficiently.
Both these peptides are natural ligands for other members of
the SH2 domain family.25a The observed sensing selectivity
agrees well with the affinity of natural p85-nSH2. Overall, these
results indicate that the SH2 domain was successfully converted
to a biosensor equipped with a turn-on fluorescent switch that
retains the natural recognition selectivity for phosphopeptides.
Generation of a Turn-On Biosensor in a Crude Bacterial
Cell Lysate. The Q-LDT strategy was finally applied to construct
a turn-on fluorescent biosensor for direct use in bacterial cell
lysates. After expression of the GST-fused p85-nSH2 (GST-
SH2) in Escherichia coli, 4 was incubated with the crude lysate
Figure 6. One-pot construction of a SH2 domain-based turn-on fluorescent
biosensor in a bacterial lysate. (a) SDS-PAGE analysis of the labeling of
GST-SH2 in the bacterial lysates with 4. Reaction conditions: 40 µM 4, 4
mM 5 (lane 3), lysate of GST-SH2-expressing E. coli containing ∼8 µM
GST-SH2 (50 mM HEPES, pH 8.0), 37 °C, and 40 h. Lane 4, DEAC-CAII
(1:1) conjugate. (b) Fluorescence spectral change of the 4-treated bacterial
lysate upon the addition of peptide 5. Measurement conditions: 4-treated
bacterial lysate (the postlabeling sample was diluted with no purification
such that the concentration of the GST-SH2 is ∼3 µM), 0-300 µM 5 or 6,
50 mM HEPES, pH 8.0, 25 °C, and λex ) 427 nm. (Inset) Fluorescence
titration curve. λem ) 473 nm, peptide 5 (circle), 6 (square).
of the cells. In-gel fluorescence analysis of the reaction mixture
showed a single fluorescent band corresponding to the DEAC-
labeled GST-SH2, which was significantly diminished by the
excess amount of phosphopeptide ligand 5 (Figure 6a). These
results confirm the high target-selectivity of the 4-mediated
affinity labeling even in the presence of many other contaminat-
ing proteins in the lysate. More remarkably, the fluorescence at
473 nm was intensified in a typical saturation manner when
phosphotyrosine peptide 5 was added to the postlabeling lysate
without any purification step (Figure 6b). In contrast, no change
in fluorescence occurred when the nonphosphorylated peptide
6 was added. This clearly demonstrates that the Q-LDT strategy
is widely applicable to convert an intact protein to a turn-on
fluorescent biosensor even under crude heterogeneous conditions.
Conclusions
While there is a growing recognition of the importance of
semisynthetic fluorescent biosensors in biological research and
drug discovery, engineering strategies for generating such
functionalized proteins remain very limited. In this work, we
have developed a new methodology using modular, Q-LDT
reagents to convert target proteins to ‘turn-on’ fluorescent
biosensors in a one-step labeling procedure. The approach was
successfully applied to two different protein scaffolds, CAII and
an SH2 domain, without genetic mutations, by adopting
(30) We also confirmed that the UV-visible spectrum of the fraction
containing the SH2 domain after the gel filtration showed absorption
bands of both the DEAC and DABCYL groups as with the case of
the CAII experiments.
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One-Pot Construction of Fluorescent Biosensors
A R T I C L E S
membrane (MWCO: 10 000) (Spectrum Laboratories). The resulting
solution was concentrated using a Centricon Ultracel YM-10
(Millipore). To this solution, urea (at a final concentration of 2 M)
and lysyl endopeptidase (LEP) (LEP/substrate ratio ) 1:50 (w/w))
were added. Native (unlabeled) CAII was also subjected to LEP
digestion. After incubation at 37 °C for 48 h, the digested peptides
were separated by RP-HPLC using an YMC-Pack ODS-A column
(5 µm, 4.6φ × 250 mm). The peaks were monitored by UV
absorbance at 220 nm and fluorescence at 473 nm (λex ) 427 nm).
The collected fractions were analyzed by MALDI-TOF MS
(CHCA).
appropriate affinity ligands. We have also demonstrated that
the high target-selectivity of the Q-LDT-mediated labeling
allows us to prepare biosensors not only in test tubes using
purified proteins but also in a cell lysate. To our knowledge,
this work represents the first demonstration of the construction
of a semisynthetic biosensor in a crude mixture. Since we have
previously proved that the LDT chemistry is applicable to the
labeling of specific proteins in living cells or tissues,10 the
present strategy may be extended to create fluorescent biosensors
directly in such environments. Moreover, it is envisioned that
new LDT reagents containing a FRET pair of fluorophores
(donor and acceptor) can be designed along the same lines to
provide an approach to a one-pot construction of FRET-based
ratiometric biosensors.22a Such efforts will significantly enhance
the prospect of semisynthetic biosensors and are currently
underway in our laboratory.
Fluorescence Titration of the CAII-Based Biosensor. Labeling
was performed under the following conditions: 30 µM CAII, 30
µM 2, 50 mM HEPES, pH 7.2, and 37 °C. After 48 h of incubation,
the labeling solution was diluted 6-fold with no purification.
Titration experiments with various sulfonamide and nonsulfonamide
derivatives were carried out using the solution containing a mixture
of labeled (48%) and native CAII (the total concentration of CAII
) 5 µM, 50 mM HEPES, pH 7.2) in a quartz cell (500 µL) at 25
°C. The fluorescence spectral change (λex ) 427 nm) was monitored
upon addition of a solution of the analyte with a microsyringe. All
the titration experiments were performed in triplicate on each
analyte to obtain mean and standard deviation values of the relative
fluorescence intensities (F/F0). Fluorescence titration curves at 473
nm were analyzed with the nonlinear least-squares curve-fitting
method to evaluate the apparent binding constants (Kapp, M-1).
Evaluation of the SH2 Domain Labeling. Recombinant human
p85-nSH2 was obtained by bacterial expression as a fusion protein
with GST, followed by cleavage of the GST tag using thrombin
(the plasmid encoding GST-p85-nSH2 was kindly provided by Prof.
Teruyuki Nagamune). The concentration of p85-nSH2 was deter-
mined by measuring the absorbance at 280 nm using a molar
absorption coefficient of 21 300 M-1 cm-1. Purified p85-nSH2 (5
µM) was incubated with 3 or 4 (10 µM) in HEPES buffer (50 mM,
pH 8.0) at 37 °C. Control reactions with 5 (1 mM) or GSH (10
mM) were also included. Aliquots were taken at 20 and 44 h and
mixed with an equal volume of 2× SDS-PAGE loading buffer.
The samples were applied to a 15% SDS-PAGE and the labeled
p85-nSH2 was detected by in-gel fluorescence imaging. The 1:1
DEAC-CAII conjugate was used as a standard marker to determine
the labeling yields on the basis of the relative fluorescence
intensities. After fluorescence imaging, the gel was stained with
CBB.
Peptide Mapping of the Labeled SH2 Domain. Labeling was
performed under the following conditions: 22 µM p85-nSH2, 110
µM 3, 50 mM HEPES, pH 8.0, and 37 °C. After 40 h of incubation,
the labeled p85-nSH2 was purified by semipreparative RP-HPLC
using an YMC-Pack ODS-A column (5 µm, 10φ × 250 mm). The
peaks were monitored by UV absorbance at 220 nm and fluores-
cence at 473 nm (λex ) 427 nm). The fraction containing the labeled
p85-nSH2 was collected and dialyzed against HEPES buffer (50
mM, pH 8.0) with a Spectra/Por dialysis membrane (MWCO:
3000). The resulting solution was concentrated using a Centricon
Ultracel YM-3 (Millipore). To this solution, urea (at a final
concentration of 2 M) and trypsin (trypsin/substrate ratio ) 1:10
(w/w)) were added. Unlabeled, intact p85-nSH2 was also subjected
to trypsin digestion. After incubation at 37 °C for 40 h, the digested
peptides were separated by RP-HPLC using an YMC-Pack ODS-A
column (5 µm, 4.6φ × 250 mm). The collected fractions were
analyzed by MALDI-TOF MS (CHCA) and the labeled fragment
was further characterized by MALDI-QIT-TOF MS/MS analysis.
Experimental Section
Synthesis. All synthetic procedures and compound characteriza-
tions are described in the Supporting Information.
General Materials and Methods. Unless otherwise noted, all
proteins/enzymes and reagents were obtained from commercial
suppliers (Sigma, Aldrich, Tokyo Chemical Industry (TCI), or Wako
Pure Chemical Industries) and used without further purification.
UV-visible spectra were recorded on a Shimadzu UV-2550
spectrophotometer. Fluorescence spectra were recorded on a
PerkinElmer LS 55 fluorescence spectrometer. SDS-PAGE was
carried out using a Bio-Rad Mini-Protean III electrophoresis
apparatus. Fluorescence gel images were acquired using a Bio-
Rad ChemiDoc XRS system with a 480BP70 filter, and analyzed
with Quantity One 1-D Analysis Software (Bio-Rad Laboratories).
Reversed-phase HPLC (RP-HPLC) was carried out on a Hitachi
LaChrom L-7100 system equipped with LaChrom L-7400 UV and
L-7485 fluorescence detectors. All runs used linear gradients of
acetonitrile containing 0.1% TFA (solvent A) and 0.1% aqueous
TFA (solvent B). Matrix-assisted laser desorption/ionization time-
of-flight mass spectrometry (MALDI-TOF MS) spectra were
recorded on an Applied Biosystems Voyager Elite mass spectrom-
eter using R-cyano-4-hydroxycinnamic acid (CHCA) or sinapinic
acid (SA) as the matrix. Matrix-assisted laser desorption/ionization
quadrupole ion trap time-of-flight mass spectrometry/mass spec-
trometry (MALDI-QIT-TOF MS/MS) analysis was performed by
Dr. Masaki Yamada (Shimadzu Corporation) with a Shimadzu
Biotech AXIMA QIT mass spectrometer.
Evaluation of the CAII Labeling. The concentration of human
CAII (Sigma) was determined by measuring the absorbance at 280
31
nm using a molar absorption coefficient of 54 000 M-1 cm-1
.
CAII (10 µM) was incubated with 2 (20 µM) in HEPES buffer (50
mM, pH 7.2) at 37 °C. Control reactions with ethoxzolamide (ET,
100 µM) or reduced glutathione (GSH, 10 mM) were also included.
Aliquots were taken at 6, 24, and 48 h and mixed with an equal
volume of 2× SDS-PAGE loading buffer. The samples were applied
to a 12.5% SDS-PAGE and the labeled CAII was detected by in-
gel fluorescence imaging. The 1:1 DEAC-CAII conjugate was
prepared as previously described10 and used as a standard marker
to determine the labeling yields on the basis of the relative
fluorescence intensities. After fluorescence imaging, the gel was
stained with CBB.
Peptide Mapping of the Labeled CAII. Labeling was per-
formed under the following conditions: 50 µM CAII, 50 µM 2, 50
mM HEPES, pH 7.2, and 37 °C. After 72 h of incubation, the
labeled CAII was purified by size-exclusion chromatography using
a TOYOPEARL HW-40F (Tosoh Corporation) and dialyzed against
HEPES buffer (50 mM, pH 8.5) with a Spectra/Por dialysis
Fluorescence Measurement of the SH2 Domain-Based Bio-
sensor. Labeling was performed under the conditions mentioned
in the Evaluation of the SH2 Domain Labeling section. After 44 h
of incubation, the labeling solution was briefly purified by size-
exclusion chromatography using a PD-10 column (GE Healthcare)
(Note that, after this procedure, the eluted SH2 domain still
complexes with the DABCYL-tethered ligand). Titration experi-
ments with various peptides were carried out using the solution
(31) Supuran, C. T.; Briganti, F.; Tilli, S.; Chegwidden, W. R.; Scozzafava,
A. Bioorg. Med. Chem. 2001, 9, 703.
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A R T I C L E S
Tsukiji et al.
containing a mixture of labeled (30%) and unmodified p85-nSH2
(the total concentration of SH2 domain ) 1 µM, 50 mM HEPES,
pH 7.4) in a quartz cell (500 µL) at 25 °C. The fluorescence spectral
change (λex ) 427 nm) was monitored upon addition of a solution
of the analyte with a microsyringe. All the titration experiments
were performed in triplicate on each analyte to obtain mean and
standard deviation values of the relative fluorescence intensities
(F/F0). Fluorescence titration curves at 473 nm were analyzed with
the nonlinear least-squares curve-fitting method to extract the
apparent binding constants (Kapp, M-1).
Generation of a SH2 Domain-Based Biosensor in a Bacte-
rial Cell Lysate. Bacterial cells (E. coli BL21(DE3)) transformed
with the plasmid encoding GST-p85-nSH2 were grown at 37 °C
in TB medium containing ampicillin to an O.D. (600 nm) of 0.8,
at which time expression of the protein was induced by the addition
of isopropyl-ꢀ-D-thiogalactopyranoside (IPTG) to a final concentra-
tion of 1 mM. The cells were further grown at 25 °C overnight.
The cells were harvested and lysed by sonication in Tris buffer
(100 mM, 50 mM EDTA, 0.2 M NaCl, pH 8.0). The lysate was
dialyzed against HEPES buffer (50 mM, pH 8.0) with a Spectra/
Por dialysis membrane (MWCO: 3000) and used for the following
experiments. The labeling was performed by incubating the lysate
with 4 (40 µM) in the absence or presence of 5 (4 mM) at 37 °C
for 40 h. The labeling was analyzed by SDS-PAGE followed by
in-gel fluorescence imaging as described above. The concentration
of the GST-p85-nSH2 in the lysate was also roughly estimated by
SDS-PAGE/CBB staining using purified and quantified GST-p85-
nSH2 as a standard. The fluorescence titration experiments with 5
and 6 were carried out as described above with a lysate solution
containing ∼3 µM of total GST-p85-nSH2.
Acknowledgment. We thank Prof. Teruyuki Nagamune (The
University of Tokyo) for the plasmid encoding GST-p85-nSH2.
We thank Dr. Masaki Yamada (Shimadzu Corporation) for measur-
ing the tandem mass spectra (MALDI-QIT-TOF MS/MS). We also
thank Guanfan Liu for assistance with peptide synthesis. Y.T.
acknowledges the JSPS Research Fellowships for Young Scientists.
Supporting Information Available: Figures S1-S3, Schemes
S1-S2, and experimental details of the synthesis. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA902486C
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9054 J. AM. CHEM. SOC. VOL. 131, NO. 25, 2009