Turn-on fluorescence switch involving aggregation and elimination processes for β-lactamase-tag

Article abstract of DOI:10.1039/c0cc02432e

The targeted protein of interest is fused with genetically modified β-lactamase enzyme, which reacts with the probe in physiological conditions to break the aggregated interaction between the fluorophore and quencher. This alliance-separation technique is

Full text of DOI:10.1039/c0cc02432e

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Turn-on fluorescence switch involving aggregation and elimination
processes for b-lactamase-tagw
Kalyan K. Sadhu,a Shin Mizukami,ab Shuji Watanabea and Kazuya Kikuchi*ab
Received 8th July 2010, Accepted 13th August 2010
DOI: 10.1039/c0cc02432e
The targeted protein of interest is fused with genetically modified
b-lactamase enzyme, which reacts with the probe in physio-
logical conditions to break the aggregated interaction between
the fluorophore and quencher. This alliance–separation
technique is new for protein labeling and is probed in vitro and
in live cell imaging studies.
physiological conditions. To surmount the restraint in the
FRET process, our new approach is based on the switching
mechanism involving aggregation followed by elimination
processes. In the modified version, the quenching ability of
the fluorescence quencher does not depend upon the emission
wavelength of the fluorophore, as is desired for an effective
FRET process. In this case the quenching phenomenon is the
intrinsic property of the quencher part.
Molecular imaging with the aid of fluorescence microscopy
has become an indispensable means to study the proceedings
in living cells, which would be extensively valuable for the
future of medical science. The practical approach involving
chemistry to control modification and monitoring specific
proteins has proved useful.1 The direct imaging of protein
interactions in single living cells is possible due to the
site-specific labeling of proteins with molecular tags, which is
an authoritative means for studying the structure–function
relationships of proteins.2 The rationale of fluorescence
microscopy in cell biology has been thoroughly tailored with
fluorescence proteins.3 To conquer the shortcomings intrinsic
to proteins due to the lack of robust modification for target
phenomena and nonfluorogenicity, bright and photostable
small fluorophores can be advantageous over their fluorescent
protein counterparts.4 Tetracysteine-tag and its modified
version provided a route for using small molecules in fluoro-
genic labeling and were found to be effective for activation of
G protein-coupled receptors in living cells.5 Specificity towards
labeling was observed with SNAP-tag and its modified version
with semisynthetic fluorescent sensor proteins.6 In our protein
labeling method our aim was to combine fluorogenicity and
specificity of the probe. Several other protein-7 and enzyme-
mediated8 labeling methods have enriched the recent literature.
Among the small fluorogenic molecules, fluorescein is approved
by the U.S. Food and Drug Administration (FDA) for
medical use.9 Herein, we have chosen fluorescein for our
protein labeling experiment.
We have continued our study with the same BL-tag protein.
The reaction of TEM-1 (class A b-lactamases) with b-lactam
rings involves acylation and deacylation steps. Glu166 is
indispensable for the deacylation step,11 and the mutant
version of TEM-1 (E166NTEM or BL) restricts the deacylation
step.12 Our aim was to exploit the properties of BL for
covalent attachment with a fluorescent substrate with a better
versatile technique. Here, we present a rationally designed
fluorogenic probe that takes advantage of the aggregation of
fluorophore and quencher. In the aggregated form, the
interaction between the fluorogenic part and the quencher
restricts the emission of the fluorophore. The viability of the
fluorescently labeled BL-tag under physiological situations has
been explored with the newly designed and synthesized a
cephalosporin-based fluorescent probe (FCDNB, Scheme 1a).
We have used the m-dinitrobenzene (DNB) group as quencher,
whose quenching efficiency is well established.13 The probe
contains 6-carboxyfluorescein as fluorophore and DNB as
quencher and they are connected to different sides of the
b-lactam ring of the central cephalosporin part. A flexible
spacer can control the emission14 and enzyme activity prompts
Recently we have shown10 a protein labeling system that
coalesces genetically modified b-lactamase (BL-tag) with low
molecular weight fluorogenic b-lactam probes. The study
concerned FRET to optimize the emission of the probe.
A shortcoming of the strategy involved the suitable choice of
donor fluorophore, acceptor quencher and their application in
a Division of Advanced Science and Biotechnology, Graduate School of
Engineering, Osaka University, 2-1 Yamadaoka, Suita,
Osaka 565-0871, Japan. E-mail: kkikuchi@mls.eng.osaka-u.ac.jp;
Fax: (+81) 6-6879-7875
b Immunology Frontier Research Center, Osaka University,
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
w Electronic supplementary information (ESI) available: Experimental
details, chemical structures, UV-Visible absorption spectra, and
fluorescence spectra of FCDNB. See DOI: 10.1039/c0cc02432e
Scheme 1 Labeling strategy with (a) structure and (b) labeling state
of the fluorescent probe FCDNB.
c
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Chem. Commun., 2010, 46, 7403–7405 7403

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the fluorescence recovery. This inspired us to introduce poly-
ethylene glycol in the newly developed system for better
aggregation interaction and solubility in physiological
conditions. Incubation with BL-tag protein leads to the
subsequent elimination of quencher to label the covalent
modification of the tag protein with the desired fluorophore.
Using this modification, we can achieve a new and better route
to a turn-on fluorescence protein labeling system compared to
our previous FRET analogue under the same physiological
conditions. The advantage of this design principle is robust-
ness towards fluorescein, whose fluorescence intensity was not
easily modified by the previous design,10 since the quenching
mechanism is due to simple molecular interactions.
Fig. 2 (a) Emission spectra of FCDNB in 100 mM HEPES buffer
(pH 7.4) and methanol (conc. of FCDNB 0.5 mM). (b) Time-dependent
emission spectra (lex = 507 nm) of FCDNB in the presence of WT
TEM in 100 mM HEPES buffer (pH 7.4) containing 0.05% DMSO at
25 1C.
fluorescein emission was sufficiently quenched because of
intramolecular interaction between the fluorophore and
DNB group. 2,4-Dinitroaniline is an efficient intramolecular
fluorescence quencher for fluorescein labeled oligonucleotides.15
The mechanism of fluorescence attenuation can be attributed
to electron-rich aromatic rings that can p-stack with the
electron-poor nitroaromatics.16 The inherent quenching
phenomenon of DNB does not depend upon the nature of
fluorophores, rather it has been found effective in the case of
several fluorogenic probes.17 The quantum yield of FCDNB in
methanol is 0.42. This significant difference of emissions in
these two solutions (Fig. 2a) implies that the aggregation
phenomenon is favorable in the physiological conditions only.
Buffer comparison studies of FCDNB and the DNB-free
version of the probe (FA, Fig. S5w) showed that FCDNB is
sufficiently quenched in each buffer medium (Table S1w). A pH
probe fluorescence assay of FCDNB (Fig. S6w) suggests that
emission from fluorescein is more effective at physiological
conditions compared to in acidic medium.
The labeling mechanism is illustrated in Scheme 1b.
Cleavage of the b-lactam group of FCDNB by Ser70 in BL
produced covalent labeling of the fluorescein to the protein
with concomitant release of DNB. The labeled protein was
detected by irradiating the gel with visible light (l = 470 nm)
in an SDS-PAGE study. A protein band of B29 kDa was
observed, exhibiting green fluorescence (Fig. 1a). To confirm
that the labeling activity is due to the BL-tag, we carried out a
similar experiment with wild-type (WT) TEM-1. In contrast to
BL, incubation of WT TEM with FCDNB ended with no
labeled fluorescence. Labeling in a biological medium with
HEK293T cell lysate was also checked. SDS-PAGE analysis
confirmed competent and selective labeling of FCDNB
(Fig. 1b).
Compound 8 (Fig. S2w) with only DNB quencher showed
maximum absorption at 364 nm and negligible absorption
after 475 nm in HEPES buffer, whereas there is no substantial
emission of fluorescein in this region (Fig. S3w). The
probability of FRET from fluorescein to the DNB group
can be overruled due to the insignificant overlap of fluorescein
emission and DNB absorbance. The absorption peaks of
fluorescein and the DNB group in FCDNB were obtained at
507 nm and 375 nm, respectively, in HEPES buffer. In
methanol, the absorption peaks for both fluorescein and
DNB were shifted by 10–15 nm (Fig. S4w). These positional
shifts suggest a different nature of interaction between the
fluorophore and quencher in the studied media.
The effects of BL-tag and WT TEM on the emission
properties of the probe were studied. The fluorescence
intensity at 520 nm due to fluorescein was monitored after
the incubation of BL with FCDNB. Slow enhancement of the
fluorescence intensity was observed with time (Fig. S7w). This
indicates that the cleavage of the b-lactam of FCDNB
was performed by BL but the disaggregation followed by
elimination of the DNB group is very slow. The process is
significantly different in case of the incubation study with WT
TEM. The DNB group was eliminated by WT TEM with a
much faster rate and the fluorescence signal increased a
considerable amount (Fig. 2b) as expected due to the favorable
deacylation step of the catalytic activity. The change in
fluorescence enhancement was found to be slow after 4 h.
The initial rate of fluorescence enhancement in the case of WT
TEM was found to be B20 times that of the BL-tag. This
noticeable difference in the rate of reaction is also due to the
controlled acylation path as a result of mutation at Glu166.10
In comparison, emission intensities of fluorescein in FA remain
the same with time for both WT TEM and BL-tag.
The fluorescence quantum yield of FCDNB was found to be
0.05 in 100 mM HEPES buffer. This result confirmed that the
The site-specific labeling of target proteins has been probed
by the demonstration of FCDNB on the surface of living cells
(Fig. 3a). For this purpose we have chosen the N-terminus
of epidermal growth factor receptor (EGFR). The target
fluorophore recognizes the transfected protein as a result of
covalent bond formation. The BL-tag fused to the EGFR, and
then it was expressed with HEK293T cells. After treatment
with FCDNB, fluorescence images of the HEK293T cells were
Fig. 1 FCDNB incubated fluorescence (left) and CBB-stained (right)
gel images of (a) BL and (b) BL mixed with HEK293T cell lysate.
c
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support from the Takeda Science Foundation. KKS and
SW acknowledge support from a Global COE Fellowship
of Osaka University. SW acknowledges JSPS Research
Fellowship.
Notes and references
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Fig. 3 (a) Labeling of protein with the probe through the BL-tag.
(b)–(e) Optical microscopic images of FCDNB-labeled HEK293T cells
expressing (b,c) BL-EGFR and (d,e) EGFR. (b,d) DIC images, (c,e)
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This research is supported by the Japan Society for the
Promotion of Science (JSPS) through its ‘‘Funding Program
for World-Leading Innovative R&D on Science and Techno-
logy (FIRST Program). This work was supported in part by
the Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT)
of Japan, the Grant-in-Aid from the Ministry of Health,
Labour and Welfare (MHLW) of Japan, and the New Energy
and Industrial Technology Development Organization
(NEDO) of Japan. KK expresses his special thanks for
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