COMMUNICATION
Ratiometric fluorescence detection of a tag fused protein using the
dual-emission artificial molecular probe{
Kei Honda, Eiji Nakata, Akio Ojida and Itaru Hamachi*
Received (in Cambridge, UK) 20th June 2006, Accepted 25th July 2006
First published as an Advance Article on the web 17th August 2006
DOI: 10.1039/b608684e
We have successfully developed a ratiometric detection system
for protein of interest using the complementary recognition pair
of the tetra-aspartate peptide tag and the SNARF-appended
Zn(II)–DpaTyr probe.
The sensing and imaging of a protein of interest is of great
importance for the understanding of protein functions in
complicated biological events.1 Unlike the conventional protein
labeling method using antibodies or the genetic fusion of a
fluorescent protein such as green fluorescent proteins (GFPs),2 a
pair of peptide tags fused to a protein and a complementary
fluorescent binding probe is now being recognized as a promising
labeling technique.3 Thus, several tag–probe pairs have been
actively developed.4 In protein labeling using an artificial probe, it
Fig. 1 New ratiometric fluorescence detection system of the D4-tagged
protein.
is highly desirable to couple the binding event of the probe with a
fluorescence change in its intensity or wavelength, because such a
change significantly contributes to reduce the background signal
due to the unbound probe, thereby facilitating precise detection or
imaging of the target protein even under complicated biological
conditions.5 Although Tsien and coworkers reported an exclusive
pair of tetra-cysteine tag–FlAsH probe that showed a large
fluorescence enhancement in the labeling process,6 useful pairs that
exhibit a ratiometric emission change have been poorly developed,7
irrespective of its versatility for quantitative analysis in bio-imaging
experiments.
pH indicator that shows a dual-emission change in the neutral pH
region, was appended to the Zn(II)–DpaTyr. 1–2Zn(II) was
synthesized by the simple condensation reaction of the DpaTyr
unit with a carboxy SNARF, followed by Zn(II) complexation
with two equivalents of ZnCl2 (Scheme S1{). The compound
characterizations of the SNARF ligand 1 and 1–2Zn(II) were
1
performed by H-NMR and high resolution mass spectroscopy.
We recently proposed a peptide tag–artificial probe pair as a
new protein labeling system.8 This system is composed of the
sequential tetra-aspartate tag (D4-tag) and the fluorescent
binuclear Zn(II) complex (Zn(II)–DpaTyr) as a complementary
binding pair, being applicable to labeling a cell surface membrane
protein. In this communication, we describe that a fluorescence
ratiometric detection mode is successfully equipped to the D4-tag–
Zn(II)–DpaTyr system by utilizing the binding-induced micro pH
change (Fig. 1). The ability of this ratiometric sensing was clearly
demonstrated by the fluorescence detection of D4-tagged RNase in
a homogeneous neutral aqueous solution.
Fluorescence titration of 1–2Zn(II) with a D4-tag model peptide
(D4: Boc-DDDD-NH2) was conducted, prior to application of the
present system to proteins. Fig. 2 showed the fluorescence spectral
change of 1–2Zn(II) upon addition of D4 peptide under neutral
aqueous conditions (50 mM HEPES, pH 7.2). Typical ratiometric
fluorescence change immediately occured in response to D4
peptide addition (within 5 s), in which the emission at 628 nm due
to the basic phenolate form of the SNARF decreased and the
emission at 586 nm due to its acidic phenol form concurrently
increased with an emission isosbestic point at 610 nm. The plot of
the emission intensity ratio R (586 nm/628 nm) showed a good
saturation behavior (DR = 0.57), and the curve-fitting analysis
afforded the binding constant of 1.1 ¡ 0.1 6 106 M21 (Fig. S1{).
This value is in good agreement with that of the 2–2Zn(II) (i.e.,
without SNARF unit) with D4 peptide determined by ITC
It seems reasonable to assume that the local pH in the proximity
to the D4-tag is rather acidic than the bulk pH due to its accumu-
lated carboxylate residues. On the basis of this assumption, we
designed a new probe 1–2Zn(II), which possesses a pH-responsive
dual-emission fluorophore as a sensory unit. Among various
fluorophores, seminaphthorhodafluor (SNARF),9 a representative
Department of Synthetic Chemistry and Biological Chemistry, Graduate
School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku,
Kyoto, 615-8510, Japan. E-mail: ihamachi@sbchem.kyoto-u.ac.jp;
Fax: +81-75-383-2759; Tel: +81-75-383-2757
{ Electronic supplementary information (ESI) available: Experimental.
See DOI: 10.1039/b608684e
4024 | Chem. Commun., 2006, 4024–4026
This journal is ß The Royal Society of Chemistry 2006