Published on Web 07/21/2006
Oligo-Asp Tag/Zn(II) Complex Probe as a New Pair for
Labeling and Fluorescence Imaging of Proteins
Akio Ojida, Kei Honda, Daisuke Shinmi, Shigeki Kiyonaka, Yasuo Mori, and
Itaru Hamachi*
Contribution from the Department of Synthetic Chemistry and Biological Chemistry, Graduate
School of Engineering, Kyoto UniVersity, Katsura Campus, Nishikyo-ku, Kyoto, 615-8510, Japan
Received March 18, 2006; E-mail: ihamachi@sbchem.kyoto-u.ac.jp
Abstract: To accomplish the selective labeling of a specific protein in complicated biological systems, a
peptide tag incorporated into the protein and a complementary small molecular probe are required. Although
a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the
availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide
tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n )
1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity
of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the
multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between
the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the
labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4
tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.
ies, and quantum dots (QDs),4 labeling with such a tag/small
molecular probe pair is flexibly applicable for a wide variety
Introduction
A pair of a peptide tag incorporated into a protein and a
complementary small molecular probe is now regarded as an
indispensable analytical tool for a wide variety of biological
studies. Some useful peptide tag/probe pairs, such as His tag
with Ni(II)-NTA (nitrilotriacetic acid) and FLAG with the
corresponding antibody, have been developed and are widely
used for protein isolation using affinity column chromatography,
protein/peptide immobilization on a microtiter plate, and sensi-
tive protein detection using Western blotting.1 In recent years,
new types of peptide tag/small-molecule fluorescent probe pairs
have been developed as valuable molecular tools for real-time
imaging of target proteins in living cells.2 Pioneering work was
reported by Tsien and co-workers, describing a tetracysteine
motif (-Cys-Cys-X-X-Cys-Cys-)/bisarsenical ligand (FlAsH)
pair, the usefulness of which has been demonstrated in a number
of bio-imaging experiments in living cells.2e,f Subsequently,
Vogel et al. applied the conventional His tag (-(His)n-)/Ni(II)-
NTA pair for fluorescence labeling of a protein on a cell
surface.2h In contrast to the protein labeling techniques using
green fluorescent proteins (GFPs) as a genetic fusion,3 antibod-
of proteins with little or no interference with the function of
the protein, because of its smaller size (GFPs, antibodies > 27
kDa, and QDs > 2-6 nm in diameter). Another intriguing
feature of the tag labeling system is the interchangeability of
the small molecular probe that enables the sequential labeling
of proteins at different wavelengths (i.e., pulse-chase labeling).
However, the availability of such a useful complementary pair
is still limited, mainly due to the general difficulty in the design
of the recognition pair with a high specificity. Thus, it would
be highly desirable to develop a new pair in order to facilitate
multiple labeling of distinct proteins in living systems, to allow
flexibility for visualization of multiple proteins involved in
complicated cellular events.
We now report a newly designed peptide tag/artificial probe
pair that utilizes multivalent coordination chemistry between a
genetically encodable oligo-aspartate sequence (D4 tag) and
corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs).
This pair is orthogonal to the His tag/Ni(II)-NTA pair and
applicable to the labeling and fluorescence imaging of the tag-
fused proteins on a living cell surface as well as in a test tube
(Figure 1).
(1) Terpe, K. Appl. Microbiol. Biotechnol. 2003, 60, 523.
(2) (a) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13. (b) Chen,
I.; Ting, A. Y. Curr. Opin. Biotechnol. 2005, 16, 35. (c) Goldsmith, C. R.;
Jaworski, J.; Sheng, M.; Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 418.
(d) Lata, S.; Gavutis, M.; Tampe´, R.; Piehler, J. J. Am. Chem. Soc. 2006,
128, 2365. (e) Martin, B. R.; Giepmans, B. N. G.; Adams, S. R.; Tsien, R.
Y.; Nat. Biotechnol. 2005, 23, 1308. (f) Gaietta, G.; Deerinck, T. J.; Adams,
S. R.; Bouwer, J.; Tour, O.; Laird, D. W.; Sosinsky, G. E.; Tsien, R. Y.;
Ellisman, M. H. Science 2002, 296, 503. (g) Howarth, M.; Takao, K.;
Hayashi, Y.; Ting, A. Y. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7583.
(h) Guignet E. G.; Hovius, R.; Vogel, H. Nat. Biotechnol. 2004, 22, 440.
(3) (a) Tsien, R. Y. FEBS Lett. 2005, 579, 927. (b) Miyawaki, A.; Sawano,
A.; Kogure, T. Nat. Cell. Biol. 2003, 5, S1-S7.
Results and Discussion
The metal-ligand interaction is potentially a versatile binding
force for protein recognition since it can work effectively under
physiological aqueous conditions.5 Thus, the combination of a
designed amino acid sequence and a metal complex might serve
(4) Green, M. Angew. Chem., Int. Ed. 2004, 43, 4129.
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J. AM. CHEM. SOC. 2006, 128, 10452-10459
10.1021/ja0618604 CCC: $33.50 © 2006 American Chemical Society