C O M M U N I C A T I O N S
transmembrane domain of platelet-derived growth factor receptor)
on the cell membrane were prepared. CATP or FCTP was incubated
with the cells in culture media. Fluorescence microscopy showed
that fluorescent labeling by both probes occurred in the cells
expressing PYP-PDGFRtm (Figures 3b and S5). No fluorescence
was observed in the cells that did not express PYP-PDGFRtm,
demonstrating that PYP is specifically labeled on the cell membrane
by both probes. During the experiments, we noticed that CATP
was cell-permeable. Therefore, intracellular imaging with CATP
was also performed. After the labeling reaction of CATP and the
cells expressing maltose binding protein-fused PYP (MBP-PYP)
in cytosol, fluorescence was observed only in the cells expressing
MBP-PYP, and not in the nonexpressing cells (Figure S5). This
result clearly shows that CATP allows specific labeling of PYP
inside living cells.
In conclusion, we have developed a protein labeling system,
based on a small tag protein, PYP, and its fluorescent probes. The
live-cell imaging and specific labeling of PYP were achieved by
using CATP and FCTP. CATP has dual functions as a fluorescent
probe and a chemical handle for two-step labeling. More impor-
tantly, FCTP shows fluorogenic characteristics, allowing the
identification of the probe bound to its tag protein. These properties
offer a more sophisticated application of this system to protein
imaging studies.
Figure 3. (a) Fluorescence spectra of FCTP (8 µM) in the absence (red
dashed line) or presence (blue line) of PYP (5 µM). (b-e) Fluorescent live-
cell imaging. Images (b, d) or (c, e) represent the cells that expressed or
did not express PYP-PDGFRtm, respectively. The cells after incubation
with CATP (5 µM; b, c) or FCTP (20 µM; d, e) are shown. Scale bars )
10 µm.
a slowly migrating band, which is regarded as FCTP-bound PYP
because of its fluorescence. The binding of PYP and the probes
was also confirmed by MALDI-TOF MS (Figure S1). The addition
of CATP or FCTP to PYP gave the mass value of PYP bound to
the individual probe, in which thiophenyl ester is replaced by
thioester of Cys in the protein. The results indicate that the probes
covalently bind to PYP through transthioesterification.
The binding specificity of the probes toward PYP was investi-
gated. Labeling reactions of purified PYP were carried out in the
lysate prepared from HEK293T cells. Figure 2c and d show that a
single fluorescent band was detected only in the reaction mixture
of PYP and each probe, confirming that PYP is specifically labeled
by CATP or FCTP under this experimental condition. The influence
of free thiols on the labeling reaction was also examined. The
presence of a physiological concentration of glutathione (up to 10
mM) did not affect the labeling reactions (Figure S2).13 Further-
more, the azido moiety of CATP would allow additional labeling
of PYP with the second probe by click chemistry and could expand
the range of the applications of this system. After the reaction of
PYP and CATP in the cell lysate, 6-CFA was added to the reaction
mixture in the presence of Cu2+ and tris(2-carboxyethyl)phosphine.
Electrophoresis revealed a slowly migrating band, as is the case
with the FCTP-bound PYP (Figure 2e). There were no newly
appearing bands, demonstrating that the stepwise labeling reaction
is quite specific.
Acknowledgment. This work was supported by MEXT of
Japan. We thank Prof. Klaas J. Hellingwerf for providing the
plasmid encoding PYP and Dr. Aya Fukuda for giving the plasmid
for mammalian expression.
Supporting Information Available: Experimental procedures and
supplemental results. This material is available free of charge via the
References
(1) (a) Zaccolo, M. Circ. Res. 2004, 94, 866–873. (b) VanEngelenburg, S. B.;
Palmer, A. E. Curr. Opin. Chem. Biol. 2008, 12, 60–65.
(2) (a) Shaner, N. C.; Steinbach, P. A.; Tsien, R. Y. Nat. Methods 2005, 2,
905–909. (b) Pakhomov, A. V.; Martynov, V. I. Chem. Biol. 2008, 15,
755–764.
(3) (a) Zhou, Z.; Koglin, A.; Wang, Y.; McMahon, A. P.; Walsh, C. T. J. Am.
Chem. Soc. 2008, 130, 9925–9930. (b) Chen, I.; Ting, A. Y. Curr. Opin.
Biotechnol. 2005, 16, 35–40.
To examine the fluorogenic properties of FCTP, the fluorescent
spectra of the probes were measured (Figure 3a). In the absence of
PYP, the fluorescence intensity of FCTP is very weak, suggesting
that the coumarin and fluorescein dyes in the probe associate with
each other. On the other hand, the binding of PYP and the probe
leads to a dramatic increase in the fluorescence intensity. This
increase is approximately 20-fold after 24 h of incubation. The result
indicates that the coumarin dissociates from the fluorescein due to
the formation of the complex between PYP and the probe. We then
characterized the binding kinetics of PYP with CATP and FCTP
by size exclusion chromatography and fluorescence measurement,
respectively (Figures S3 and S4; see Supporting Information for
the detailed procedure). The CATP reaction was almost complete
within 2 h, consistent with a previous report, demonstrating the
binding kinetics of a natural ligand to PYP.12 In contrast, the binding
of FCTP to PYP was slow, requiring more than 24 h to complete
the reaction. One probable reason for this difference is that the
intramolecular interaction in FCTP could influence its binding
kinetics.
(4) (a) Los, G. V.; Wood, K. Methods Mol. Biol. 2007, 356, 195–208. (b)
Gautier, A.; Juillerat, A.; Heinis, C.; Correˆa, I. R., Jr.; Kindermann, M.;
Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128–136.
(5) Mizukami, S.; Watanabe, S.; Hori, Y.; Kikuchi, K. J. Am. Chem. Soc. 2009,
131, 5016–5017.
(6) (a) O’Hare, H. M.; Johnsson, K.; Gautier, A. Curr. Opin. Struct. Biol. 2007,
17, 488–494. (b) Wu, P.; Shui, W.; Carlson, B. L.; Hu, N.; Rabuka, D.;
Lee, J.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 3000–
3005. (c) Ferna´ndez-Sua´rez, M.; Baruah, H.; Mart´ınez-Herna´ndez, L.; Xie,
K. T.; Baskin, J. M.; Bertozzi, C. R.; Ting, A. Y. Nat. Biotechnol. 2007,
25, 1483–1487. (d) Nonaka, H.; Tsukiji, S.; Ojida, A.; Hamachi, I. J. Am.
Chem. Soc. 2007, 129, 15777–15779. (e) Adams, S. R.; Campbell, R. E.;
Gross, L. A.; Martin, B. R.; Walkup, G. K.; Yao, Y.; Llopis, J.; Tsien,
R. Y. J. Am. Chem. Soc. 2002, 124, 6063–6076.
(7) Kamiuchi, M.; Hara, M. T.; Stalcup, P.; Xie, A.; Hoff, W. D. Photochem.
Photobiol. 2008, 84, 956–969.
(8) Kyndt, J. A.; Meyer, T. E.; Cusanovich, M. A.; Van Beeumen, J. J. FEBS
Lett. 2002, 512, 240–244.
(9) van der Horst, M. A.; Arents, J. C.; Kort, R.; Hellingwerf, K. J. Photochem.
Photobiol. Sci. 2007, 6, 571–579.
(10) Takakusa, H.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. Anal. Chem.
2001, 73, 939–942.
(11) Anderson, S.; Crosson, S.; Moffat, K. Acta Crystallogr., Sect. D 2004, 60,
1008–1016.
(12) Imamoto, Y.; Ito, T.; Kataoka, M.; Tokunaga, F. FEBS Lett. 1995, 374,
157–160.
(13) Wu, G.; Fang, Y. Z.; Yang, S.; Lupton, J. R.; Turner, N. D. J. Nutr. 2004,
34, 489–492.
Finally, cell labeling experiments were conducted. HEK293T
cells expressing PYP-PDGFRtm (the fusion protein of PYP and a
JA904800K
9
J. AM. CHEM. SOC. VOL. 131, NO. 46, 2009 16611