Fluorogenic protein labeling through photoinduced electron transfer-based BL-tag technology

Article abstract of DOI:10.1002/asia.201100647

Keeping quenchers on a short leash: Shortening of the linker chain length combined with modification of the quencher moiety was found to improve fluorogenic probes useful for protein labeling by the mutant β-lactamase-tag (BL-tag) technology. The most eff

Full text of DOI:10.1002/asia.201100647

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DOI: 10.1002/asia.201100647
Fluorogenic Protein Labeling through Photoinduced Electron Transfer-Based
BL-Tag Technology
Kalyan K. Sadhu,[a] Shin Mizukami,[a, b] Carolyn R. Lanam,[a] and Kazuya Kikuchi*[a, b]
In recent years, protein labeling has been routinely per-
formed with various fluorescent markers.[1] The labeling of
proteins allows monitoring of the specific location, move-
ment, and interaction of proteins with other intracellular
components using fluorescence microscopy.[2] For several de-
cades, the labeling strategy mostly dealt with green fluores-
cent protein (GFP) and its variants. In order to overcome
the unaltered fluorescent property and uncontrolled expres-
sion time of GFP, small molecular probe-based labeling ap-
proaches for live-cell imaging methods have been devel-
oped.[3] However, most of the reported small-molecule label-
ing probes do not show different fluorescence properties for
the labeled and unlabeled states. Fluorescein-based arsenical
hairpin binder (FlAsH) and resorufin-based arsenical hair-
pin binder (ReAsH) are the pioneer techniques for fluoro-
genic protein labeling with small molecular probes.[4]
the synthesized probes such as CCDNB, which carries a cou-
marin fluorophore moiety, a cephalosporin moiety, and
DNB (Figure 1). The necessary incubation time for a cell
imaging experiment using CCDNB was 60 minutes. To
In our earlier studies, we developed a site-specific protein
labeling technique that employs a genetically modified b-
lactamase (BL-tag).[5] Mutation at a specific position in
TEM-1 (class A b-lactamases) provides turn-on fluorogenic
biosensors involving a b-lactam ring. The reaction of wild-
type TEM-1 (WT TEM) with the b-lactam moiety involves
acylation and deacylation steps.[6] We have utilized the BL-
tag protein for covalent attachment with a substrate. Despite
the similar molecular weights of the BL-tag and GFP, fluo-
rogenicity can be introduced only through the BL-tag tech-
nology.
We have developed a fluorogenic mechanism based on ag-
gregation–elimination processes for highly selective protein
labeling with a fluorophore of desired color using the BL-
tag technology. We have also shown a broad applicability of
dinitrobenzene (DNB) as a quencher.[5c,d] However, the limi-
tation of the technology is a slow fluorogenic response of
Figure 1. Chemical structures of the synthesized fluorescent probes used
for protein labeling.
reduce the incubation time for the imaging experiments, we
aimed at developing more sophisticated probes that possess
the DNB quencher. Herein, we report two newly designed
probes with shorter linkers compared to those of CCDNB.
In addition, we modified slightly the quencher in one probe.
The probe with the modified quencher showed a compara-
tively fast fluorogenicity in vitro and in live-cell imaging
studies. Fluorescence lifetime measurements indicated a dif-
ferent quenching mechanism for the most active probe. A
detailed analysis of this new fluorogenic mechanism re-
vealed a photoinduced electron transfer (PET) process[7]
from the fluorophore donor to the quencher acceptor.
The two new probes CC2DNB and CC3DNB that have a
comparatively short or no linker between the b-lactam and
quencher, respectively, are depicted in Figure 1. 2-(2-Amino-
ethoxy)ethanamine was used as the linker between the
cephalosporin part and the DNB quencher in CC2DNB. A
coumarin derivative was attached to the opposite side of
cephalosporin through a glycine linker to synthesize the
CC2DNB probe. On the other hand, the synthesis of
CC3DNB did not require any linker between the cephalo-
sporin part and the quencher. 2,4-Dinitrothiophenol was di-
rectly introduced as a quencher to the cephalosporin part to
synthesize CC3DNB.
[a] Dr. K. K. Sadhu, Dr. S. Mizukami, C. R. Lanam, Prof. Dr. K. Kikuchi
Division of Advanced Science and Biotechnology
Graduate School of Engineering
Osaka University
2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)
Fax : (+81)6-6879-7875
E-mail: kkikuchi@mls.eng.osaka-u.ac.jp
[b] Dr. S. Mizukami, Prof. Dr. K. Kikuchi
Immunology Frontier Research Center
Osaka University
3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100647.
272
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Chem. Asian J. 2012, 7, 272 – 276

Absorption spectra of CC2DNB and CC3DNB were re-
corded in 100 mm HEPES buffer (pH 7.4, Figure 2a) and
methanol (Figure 2b), and compared with those of CCDNB
under similar conditions. The reported absorption maxima
of 348 nm for free N-ethyl-substituted 2,4-dinitroaniline[8]
The fluorogenicities of the synthesized probes were stud-
ied by measuring the enhancement of the fluorescence in-
tensity of each probe in the presence of the BL-tag and WT
TEM. The reaction rates in the presence of WT TEM were
faster for all three probes as compared to the rates in the
presence of the BL-tag (Figure 3). In our earlier study, we
found that the quencher part was eliminated from the
CCDNB probe in the presence of the BL-tag.[5d] To modu-
late the elimination kinetics, the CC2DNB probe was syn-
Figure 2. Absorbance spectra of CCDNB, CC2DNB, and CC3DNB.
a) Spectra in 100 mm HEPES buffer (pH 7.4). b) Spectra in methanol.
and of 362 nm for 1-(methylthio)-2,4-dinitrobenzene[9] in
methanol are quite similar to the observed absorption peaks
at 350 nm in methanol for the synthesized probes containing
the DNB part. In aqueous buffer, the absorption maxima
for the coumarin and DNB parts of both CCDNB and
CC2DNB were observed at 407 nm and 375 nm, respective-
ly. Under the same conditions, the corresponding absorption
peaks in CC3DNB were detected at 402 nm and 356 nm
(shoulder band), respectively. The observed shift of the
latter peak was due to the different DNB linkage in this
probe. The slight blue shift in the absorption peak of cou-
marin in CC3DNB suggested a change in the interaction be-
tween the coumarin and DNB parts within this probe. Com-
parative measurements with the previously reported probe
CA[5b] (Figures S3 and S4 in the Supporting Information)
confirmed that the peaks around 356 nm or 375 nm in the
new probes were due to the quencher DNB parts.
Next, we measured the fluorescence emission spectra of
the free probes in 100 mm HEPES buffer of pH 7.4 (physio-
logical pH) and compared them with those in methanol[10]
(Figure S5 in the Supporting Information). The fluorescence
signals of all the probes were sufficiently quenched in the
aqueous buffer (Table 1). The strength of the interaction be-
tween the fluorophore and the quencher was comparable
for CCDNB and CC2DNB due to the similar nature of the
interacting parts. However, maximum fluorescence quench-
ing was observed in the case of CC3DNB. The measured
emission spectrum of CA in aqueous buffer (Figure S4 in
the Supporting Information) confirmed the quenched state
of the fluorophore in the newly synthesized probes.
Figure 3. Change in the fluorescence intensities of CCDNB, CC2DNB,
and CC3DNB (1 mm each) with time in the presence of WT TEM or BL-
tag (0.5 mm each) in 100 mm HEPES buffer (pH 7.4) containing 0.1%
DMSO at 258C. Marker and color codes, circles: CCDNB, squares:
CC2DNB, triangles: CC3DNB; black: WT TEM, dark grey: BL-tag,
light grey: free probe.
thesized with a much shorter linker compared to the previ-
ous polyethylene glycol spacer. However, there was only a
slight enhancement in the reaction rate in the presence of
the BL-tag. The rate of fluorescence enhancement with the
BL-tag was the fastest for CC3DNB (Figure 3).
The difference in the rates of fluorescence enhancement
indicates that the removal of the quencher in CC3DNB is
faster compared to the removal of the quencher in the other
quenched probes. The overall stability of the eliminating
groups partly controls the removal of quenchers from the
probes. In the case of CC3DNB, the eliminating thiopheno-
late anion is sufficiently stabilized due to the presence of
two highly electron-withdrawing nitro groups at the 2- and
4-positions. On the other hand, such stabilization was less
pronounced in the CCDNB and CC2DNB probes owing to
the less electron-withdrawing monosubstituted amide func-
tional group at the 4-position of the thiophenol parts (Fig-
ure S6 in the Supporting Information).
Table 1. Fluorescence quantum yields of the synthesized probes in buffer
of physiological pH and in methanol.
The fluorescence enhancements of the different probes
were not the same, even in the presence of WT TEM. Ap-
proximately 36% and 45% of the reactions were completed
within 1 minute during the incubation of WT TEM with
CCDNB[5d] and CC2DNB, respectively. The highest rate of
Solvent
Fluorescence quantum yield (F)
CCDNB[5d] CC2DNB CC3DNB
100 mM HEPES buffer (pH 7.4) 0.04
0.04
0.19
0.02
0.19
Methanol
0.19
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273

K. Kikuchi et al.
COMMUNICATION
fluorescence enhancement was observed for the CC3DNB
in the presence of WT TEM, with 88% of the catalytic reac-
tion completed within 1 minute.
The labeling efficiency with each probe in the presence of
the BL-tag was determined in vitro using SDS-PAGE. Incu-
bation with the BL-tag protein for 30 minutes led to the
elimination of the quencher in each probe and labeling of
the tag protein with the same coumarin fluorophore through
covalent linkage. Successful labeling of protein was verified
for all three probes by visualization of protein bands in the
gel upon irradiation with UV light (lex =350 nm, see
Figure 4). In each case, a fluorescent band at approximately
Figure 5. Confocal microscopic images of CCDNB-labeled (top),
CC2DNB-labeled (middle), and CC3DNB-labeled (bottom) HEK293T
cells expressing BL-tag–EGFR fusion protein after 15 min of incubation.
a) Phase-contrast microscopic images, b) fluorescence microscopic
images, and c) merged images. For fluorescence microscopic images, the
cells were excited at 405 nm. Scale bars: 20 mm.
Figure 4. Gel images of BL-tag incubated with CCDNB (left), CC2DNB
(middle), and CC3DNB (right) upon irradiation with UV light (FL) or
after staining with Coomassie brilliant blue (CBB), as indicated on the
top of each gel.
trast, the other fluorogenic probes exhibited biexponential
decays due to the phenomenon of fluorescence quenching
(Table S1 in the Supporting Information). The calculated
average fluorescence lifetimes (t) of the CCDNB and
CC2DNB probes were very close to that of CA (Table 2).
29 kDa was observed. Similar experiments carried out with
WT TEM did not result in labeled protein. Because the rel-
atively high rate of labeling with the CC3DNB probe com-
pared to that with the other probes could not be completely
understood from the in vitro analysis, further studies with
living cells were performed.
Table 2. Radiative and nonradiative rate constants of the synthesized
probes in 100 mm HEPES buffer (pH 7.4) at 258C.
For live-cell imaging experiments, we performed site-spe-
cific labeling of the transmembrane protein epidermal
growth factor receptor (EGFR) fused with the BL-tag at the
N-terminus, and observed labeling by confocal microscopy.
To check the efficiencies of the synthesized probes, specific
labeling of the cell membranes expressing the BL–EGFR
fusion protein was assessed after 15 minutes of incubation
with each probe (Figure 5). In the case of CC3DNB, suffi-
cient protein labeling at the cell membrane was observed
after this incubation period (Figure 5). However, for
CCDNB and CC2DNB, cell membrane labeling was not suf-
ficient after 15 minutes. Fluorescence images taken at 15-
minute intervals confirmed sufficient labeling after 60 mi-
nutes of incubation with these probes (Figure S7 in the Sup-
porting Information).
Probe
F
Avg t [ns]
kr [sÀ1
]
knr [sÀ1
]
CA
0.4[5b]
0.04[5d]
0.04
3.25
3.60
3.00
0.39
1.2ꢁ108
1.1ꢁ107
1.3ꢁ107
5.1ꢁ107
1.9ꢁ108
2.6ꢁ108
3.2ꢁ108
2.5ꢁ109
CCDNB
CC2DNB
CC3DNB
0.02
The radiative decay rate constants (kr) were significantly
lower in CCDNB and CC2DNB compared to that of the
CA. This can be attributed to an aggregation between the
coumarin fluorophore and the DNB quencher of these two
probes, a phenomenon quite similar to static aggregation-
based quenching,[11] which is probably due to the p–p stack-
ing interaction between the fluorophore and quencher.
In the case of the CC3DNB probe, both the average fluo-
rescence lifetime and quantum yield decreased significantly
compared to those of CA. This is due to the dynamic
quenching process[11] operating in the CC3DNB probe. The
radiative (kr) and nonradiative (knr) decay rate constant
values (Table 2) confirmed the existence of a different
quenching mechanism in CC3DNB compared to that in
To check the origin of the different labeling efficiency
among the probes, fluorescence decay profiles of the synthe-
sized probes were obtained (Figure S9 in the Supporting In-
formation). The data were compared with the fluorescence
decay profile of CA. The CA probe, without any quencher,
exhibited a single-exponential fluorescence decay. By con-
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Chem. Asian J. 2012, 7, 272 – 276

PET-Based BL-Tag Technology
CCDNB and CC2DNB. The radiative decay rate constant
of CC3DNB was slightly increased in comparison to those
of the other quenched probes. This result confirmed that the
aggregation between the quencher and the fluorophore in
CC3DNB was not affected too much. However, the nonra-
diative decay rate constant in CC3DNB was much higher
compared to those in all other probes. This dynamic quench-
ing can be explained by an effective photoinduced electron
transfer (PET) process from the coumarin donor to the
DNB acceptor. This phenomenon is well known to occur
with coumarin probes.[12]
Acknowledgements
This research was supported by the Japan Society for the Promotion of
Science (JSPS) through its “Funding Program for World-Leading Innova-
tive R&D on Science and Technology (FIRST Program).” This work was
supported in part by the CREST funding program from the Japan Sci-
ence and Technology Agency (JST); a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of Japan; and a Grant-in-Aid from the Ministry of
Health, Labour and Welfare (MHLW) of Japan. K.K.S. acknowledges
support from a Global COE Fellowship of Osaka University. C.R.L. ac-
knowledges a JASSO Fellowship.
According to the Rehm–Weller equation,[7] the rate of the
PET reaction from coumarin to DNB is governed by the
energy of the excited state species (DG00), the ground state
oxidation potential of the coumarin donor (ED+/D), the re-
duction potential of the DNB acceptor (EA/A-), and the cou-
lombic attraction energy (Ecoul). On the basis of the experi-
mental data for the excited state energy (DG00 =2.93 eV)
and the available data for the oxidation potential of 7-hy-
droxycoumarin[12b] (ED+/D =0.67 V vs. SCE) and for the re-
duction potential of DNB,[13] (EA/A-=À0.88 V vs. SCE), the
Gibbs energy (DG) for the electron transfer in a polar envi-
ronment[7] (Ecoul =0.03 eV) was found to be approximately
À1.35 eV. This value was sufficiently negative for operating
the diffusion-controlled electron-transfer process at ambient
temperature. The shortening of the linker chain length in
CC3DNB led to an effective PET process. The quencher
did not directly interact with the fluorophore in the PET
process like it did in the previous aggregation mode. This
phenomenon was reflected in the faster recognition of the
tag protein in the new PET-based probe. The probability of
fluorescence resonance energy transfer (FRET) from cou-
marin to the DNB group can be ruled out due to the small
overlap integral (J=2ꢁ10À23 mÀ1 cm3, Figure S10 in the Sup-
porting Information) between the coumarin donor emission
in CC3DNB and the acceptor DNB absorbance of com-
pound 12 (probe without coumarin, Scheme S2 in the Sup-
porting Information).
In summary, we have developed new fluorogenic probes
valuable for protein labeling through our developed BL-tag
technology. We reduced the linker chain length and slightly
modified the quencher with regard to the probe previously
reported by us, CCDNB. The probe with the shortest linker,
CC3DNB, was found to be most effective, resulting in fast
kinetics. The quenching mechanism in this probe was dem-
onstrated as a PET process, which is different from the
FRET- or static aggregation-based quenching occurring in
the other probes. Thus, the new probe overcomes the limita-
tion of the previous coumarin probes that required a long
incubation period. In principle, such PET-based probe
design could lead to the development of multi-colored fluo-
rogenic probes for successful use in next-generation live-cell
imaging technologies.
Keywords: biotechnology · electron transfer · fluorescence
spectroscopy · fluorescent probesprotein labeling
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Published online: December 20, 2011
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