Design of a binuclear Ni(II)-iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag fused proteins

Article abstract of DOI:10.1016/j.bmcl.2014.04.096

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions under live-cell conditions. In this Letter, we report the design of the binuclear Ni(II)-iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag-fused proteins. We found that the Ni(II)-IDA complex 1-2Ni(II) binds to the His6-tag (HHHHHH) with a strong binding affinity (K_d = 24 nM), the value of which is 16-fold higher than the conventional Ni(II)-NTA complex (K_d = 390 nM). The strong binding affinity of the Ni(II)-IDA complex was successfully used in the covalent labeling and fluorescence bioimaging of a His-tag fused GPCR (G-protein coupled receptor) located on the surface of living cells.

Full text of DOI:10.1016/j.bmcl.2014.04.096

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of a binuclear Ni(II)–iminodiacetic acid (IDA) complex for
selective recognition and covalent labeling of His-tag fused proteins
Ikuko Takahira a, Hirokazu Fuchida a, Shigekazu Tabata a, Naoya Shindo a, Shohei Uchinomiya b,
Itaru Hamachi b, Akio Ojida a,
⇑
a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
b Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective protein labeling with a small molecular probe is a versatile method for elucidating protein func-
tions under live-cell conditions. In this Letter, we report the design of the binuclear Ni(II)–iminodiacetic
acid (IDA) complex for selective recognition and covalent labeling of His-tag-fused proteins. We found
that the Ni(II)–IDA complex 1-2Ni(II) binds to the His6-tag (HHHHHH) with a strong binding affinity
(Kd = 24 nM), the value of which is 16-fold higher than the conventional Ni(II)–NTA complex
(Kd = 390 nM). The strong binding affinity of the Ni(II)–IDA complex was successfully used in the covalent
labeling and fluorescence bioimaging of a His-tag fused GPCR (G-protein coupled receptor) located on the
surface of living cells.
Received 26 March 2014
Revised 21 April 2014
Accepted 24 April 2014
Available online xxxx
Keywords:
His tag
Ni complex
Protein labeling
Molecular recognition
Bioimaging
Ó 2014 Elsevier Ltd. All rights reserved.
Selective protein labeling with a synthetic molecular probe is an
important research tool to facilitate detection and functional anal-
ysis of proteins. For this purpose, a variety of protein labeling
methods have been developed in the last few decades to visualize
the target protein in a real time manner under live-cell conditions.1
use of a multinuclear Ni(II) complex (n >2) for bioimaging stud-
ies,4d–f which often causes non-selective labeling and a high back-
ground fluorescence signal. In addition, the synthesis of the
multinuclear Ni(II) complex demands a cumbersome multistep
synthetic procedure, which largely diminishes the availability of
this pair for protein analysis. In this letter, we report the new
design of the binuclear Ni(II)–iminodiacetic acid (IDA) complex
for selective protein labeling of His-tag-fused proteins. The fluores-
cence binding assay revealed that the binuclear Ni(II)–IDA complex
1-2Ni(II) binds to the His6-tag (HHHHHH) with a strong binding
affinity (Kd = 24 nM), the value of which is 16-fold higher than
the conventional Ni(II)–NTA complex (Kd = 390 nM). Taking advan-
tage of the strong binding and facile structural modification of
1-2Ni(II), the fluorescent Ni(II)–IDA derivative 5-2Ni(II) was
successfully applied to the covalent labeling and imaging of a
His-tag fused GPCR (G-protein coupled receptor) located on the
surface of living cells, thereby demonstrating the use of the
Ni(II)–IDA probe in protein analysis.
Our design strategy of the Ni(II) complex to obtain a strong
binding affinity for the His-tag was primarily based on increasing
the Lewis acidity of the Ni(II) ion. We thus employed the tridentate
iminodiacetate (IDA) as a ligand for the Ni(II) ion instead of the
conventional tetradentate NTA ligand (Fig. 1). In the probe synthe-
sis, introduction of the IDA units into a simple aminobenzene scaf-
fold6 gave the ligands 1–3, each of which has a different linker unit
between the scaffold and the IDA units. The ligands were then
Among them, the use of
a complementary recognition pair
between a short peptide-tag and a small molecular probe repre-
sents a viable strategy for selective protein labeling.2 This method
can be used to selectively label a recombinant tag-fused protein
with various functional molecules at appropriate timing. This
method also benefits from the small molecular sizes of both the
peptide-tag and probe (<3 kDa), and thus less likely to perturb pro-
tein functions unlike the protein-based labeling methods involving
GFPs (27 kDa).
The oligo-histidine tag (His-tag) is a representative epitope tag,
which has been used widely in the purification or immobilization
of recombinant proteins.3 Owing to its selective binding property
with a Ni(II) complex of nitriloacetic acid (NTA), several groups
including us have reported the application of this tag-probe pair
to the labeling of His-tag-fused proteins for fluorescence bioimag-
ing under live-cell conditions.4,5 However, the rather weak binding
affinity of the Ni(II)–NTA complex to the His-tag (Kd = 1–10 lM)
results in the requirement of excess amounts of probe and/or the
⇑
Corresponding author. Tel.: +81 92 642 6596.
E-mail address: ojida@phar.kyushu-u.ac.jp (A. Ojida).
http://dx.doi.org/10.1016/j.bmcl.2014.04.096
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Takahira, I.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.096

2
I. Takahira et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
treated with 2 equiv of NiCl2 to afford the binuclear Ni(II)–IDA
complexes 1–3. The Ni(II)–NTA complex 4 was also prepared as a
control compound. The detail synthetic procedure is described in
Supporting information.
strongest binding affinity (Kd = 24 nM) for the hc-His6 peptide,
which is ca. 16-fold stronger than that of 4-2Ni(II). These results
demonstrate the validity of our design strategy of the Ni(II)
complex with a strong binding affinity towards a His-tag.
The binding affinities of the Ni(II)-complexes with the His-tag
were initially evaluated by a fluorescence quenching titration.7
When the Ni(II)–IDA probe 1-2Ni(II) was added to an aqueous
buffer solution of the His6 peptide appended with a fluorescent
7-hydroxycoumarin unit (hc-His6), its fluorescence decreased to
ꢀ40% of its original intensity owing to the paramagnetic quenching
effect of the Ni(II) ions (Fig. 2a). The change in fluorescence inten-
sity at 448 nm was analyzed by curve fitting to afford the apparent
dissociation constant Kd = 24 nM (Fig. 2b). The fluorescence Job’s
plot between 1-2Ni(II) and hc-His6 clearly indicated that their
binding stoichiometry is 1:1 (Fig. S1). The dissociation constants
of the series of the Ni(II) complexes 1–4 for the hc-His6 peptide
are summarized in Table 1. Interestingly, all IDA-type probes
1-2Ni(II)–3-2Ni(II) showed the stronger binding affinities than
the Ni(II)–NTA probe 4-2Ni(II) (Kd = 390 nM, Fig. S2). The binding
affinity of the IDA-type probes was found to be dependent on the
length of the linker units. Among them, 1-2Ni(II) showed the
The Ni(II)–IDA complex was next applied to covalently label the
protein. For this purpose, we prepared a MBP (maltose binding pro-
tein) protein tethered with
a reactive His6-tag (CH6-tag:
CHHHHHH), which contains a nucleophilic cysteine residue to
form a covalent bond with a bound Ni(II) complex.4a The covalent
labeling of MBP-CH6 was examined with the fluorescent BODIPY
derivative 5-2Ni(II), which has an
a-chloroacetyl group reactive
to the Cys residue of CH6-tag. The fluorescent quantum yield (
U
)
of 5-2Ni(II) was determined to be 0.1, a sufficiently high value
for the protein labeling experiment (Table S1). Figure 3 shows
the time-trace of the covalent labeling reaction by in-gel fluores-
cence analysis. The labeling reaction rapidly proceeded and was
almost complete within 20 min under neutral aqueous conditions
(50 mM HEPES, 100 mM NaCl, pH 7.2). The labeled protein was
also identified by MALDI-TOF mass analysis, in which the new
peak (m/z = 45,150) corresponding to the MBP-CH6 labeled with
5-2Ni(II) was observed (Fig. S3). The kinetic analysis, by varying
O
O
H
N
H
N
O
O
O
O
II
II O
H
N
H
O
Ni
Ni
II N
O
N
II O
Ni
N
N
N
O
Ni
O
O
O
O
O
O
O
O
O
NH2
• 4Cl-
• 4Cl-
NHR
2-2Ni(ll)
1-2Ni(ll) : R = H
5-2Ni(ll) : R =
BODIPY
HN
O
O
O
Cl
O
O
N
H
II N
O
N
O
II O
O
O
Ni
O
Ni
• 4Cl-
O
O
O
3-2Ni(ll)
O
O
O
H
H
N
O
N
O
II N
N
O
II O
Ni
Ni
O
O
O
O
O
• 4Cl-
O
NH2
BODIPY
BODIPY =
4-2Ni(ll) : R = H
6-2Ni(ll) : R =
HN
O
N
N
Cl
B
N
H
F
F
O
Figure 1. Structures of the Ni(II) complexes.
a
b
1
600
500
400
300
200
100
0
0.8
0.6
0.4
0.2
0
0.1
0.2
1-2Ni(ll) (µM)
0.3
0.4
400
450
500
550
600
Wavelength (nm)
Figure 2. Fluorescence titration of 1-2Ni(II) to the hydroxy coumarin-appended His-6 peptide (hc-His6). (a) Fluorescence spectral changes of hc-His6 upon addition of
1-2Ni(II). Conditions: [hc-His6] = 0.1 M, [1-2Ni(II)] = 0–0.24 M, 50 mM HEPES, 100 mM NaCl, pH = 7.2. (b) Plot of the fluorescence intensity change at 448 nm. The data
were obtained from the experiment performed in triplicate.
l
l
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I. Takahira et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Summary of the dissociation constants (Kd) of the Ni(II)-complexes with the hc-CH6
peptide
1-2Ni(II)
2-2Ni(II)
3-2Ni(II)
4-2Ni(II)
Kd (nM)
24
97
123
390
the concentration of 5-2Ni(II), revealed that the second-order rate
constant of the labeling reaction between MBP-CH6 and 5-2Ni(II) is
270 MÀ1 sÀ1 (Fig. S4). This value is comparable to the biorothogo-
nal reactions for protein labeling reported to date.8 Conversely,
none of the fluorescent band was detected when using the Ni(II)-
free ligand 5 (Fig. 3), clearly indicating that the labeling reaction
between MBP-CH6 and 5-2Ni(II) is driven by the tag-probe interac-
tion. The labeling reaction was further conducted with MBP-CH6 in
an Escherichia coli crude lysate (Fig. S5). A single fluorescence band
corresponding to the labeled MBP-CH6 was detected by in-gel fluo-
rescence analysis, indicative of the high labeling selectivity of the
Ni(II)–IDA probe for a His-tag-fused protein.
The present protein labeling method was further applied to the
fluorescent bioimaging of GPCR expressing on the surface of living
cells. In this experiment, the bradykinin receptor type2 (B2R) pro-
tein tethered with a CH6 tag (CHHHHHH) at the explasmic N-ter-
minus was transiently expressed in HEK293 cells. When the cells
expressing the tag-fused B2R were treated with 5-2Ni(II) (0.5 lM
in HBS) and then repeatedly washed with a high concentration of
imidazole solution (50 mM in HBS, three times), the clear fluores-
cence owing to BODIPY was undetectable on the cell surface
(Fig. S6). This result indicates that 5-2Ni(II) did not form a covalent
bond with the CH6 tag of B2R and thus be removed readily from
the cell surface by the imidazole washing. However, when the cells
were first treated with the reducing agent tris-(carboxyethyl)phos-
phine (TCEP) to activate the cysteine residue of the CH6 tag,9 fol-
lowed by labeling with 5-2Ni(II), the clear fluorescence of the
BODIPY was detectable on cell surfaces even after repeatedly
applying the imidazole washing step (Fig. 4a). It is noteworthy that
this image overlapped well with that of a B2R antagonist peptide
appended with a rhodamine fluorophore. This result suggests that
5-2Ni(II) selectively forms a covalent bond with the cysteine resi-
due of the CH6 tag to allow the selective visualization of the tag-
fused B2R protein. The trypan blue assay revealed that 5-2Ni(II)
scarcely affects the cell viability under the labeling conditions
(Fig. S9). In the control labeling experiment with the Ni(II)-free
ligand 5, negligible fluorescence of BODIPY was detected on cell
surfaces (Fig. 4e), suggesting that the strong binding between
5-2Ni(II) and the His-tag was crucial for the covalent labeling of
the tag-fused B2R protein. The labeling of the tag-fused B2R was
also conducted with the Ni(II)–NTA probe 6-2Ni(II) under the same
Figure 4. Covalent labeling of the CH6-tag-fused B2R protein on the surface of
HEK293 cells. (upper) Fluorescence visualization of the B2R by labeling with 5-Ni(II)
(a) and the rhodamine-appended B2R antagonist (b), (c) is the overlay image of (a)
and (b), and (d) is the transmission image. (lower) Fluorescence visualization of B2R
by labeling with the ligand 5 (e) and the rhodamine-appended B2R antagonist (f).
(g) is the overlay image of (e) and (f), and (h) is the transmission image.
labeling conditions (Fig. S7). In this case, the fluorescence due to
BODIPY was apparently weak and did not provide a clear fluores-
cent image of the cell surfaces, indicative of the lower labeling
Figure 3. In-gel fluorescence analysis of the covalent labeling of MBP appended with the CH6-tag. (a) Fluorescence (FL) and CBB detection of CH6-MBP labeled with either
5-2Ni(II) (upper) or the Ni(II)-free ligand 5 (lower). (b) Plot of the fluorescence band intensity of CH6-MBP labeled with either 5-2Ni(II) (j) or 5 (d).
Please cite this article in press as: Takahira, I.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.096

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I. Takahira et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
efficiency of 6-2Ni(II) compared to 5-2Ni(II). Although the use of a
higher concentration (5 M) of 6-2Ni(II) was effective for improv-
Supplementary data
l
ing the labeling efficiency, the high background fluorescence owing
to non-selective binding or internalization of the probe inside cells
was apparently problematic for the selective visualization of the
labeled B2R (Fig. S8). These results indicate that the strong binding
affinity of the Ni(II)–IDA probe 5-2Ni(II) gives a clear advantage to
the fluorescence imaging of His-tag-fused proteins over the
conventional Ni(II)–NTA probe.
In conclusion, we have developed the Ni(II)–IDA complex as a
new probe for the selective labeling of a His-tag-fused protein.
The improved strong binding affinity of the Ni(II)–IDA probe com-
pared with the conventional Ni(II)–NTA probe was fully exploited
to covalently label a modified His-tag (CH6-tag) fused protein
under in vitro and in cell conditions. We envision the further appli-
cation of the present tag-probe pair to functional analyses of pro-
teins under various biological conditions. Our research is ongoing
along this line of research.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.
096.
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We appreciate the technical support from the Research Support
Center, Graduate School of Medical Sciences, Kyushu University.
This work was performed under the Cooperative Research Program
of ‘Network Joint Research Center for Materials and Devices’. A.O.
acknowledges Takeda Science Foundation and Toray Science
Foundation for their financial supports. S.T. acknowledges Kyushu
University Interdisciplinary Programs in Education and Projects in
Research Development for its financial support.
Please cite this article in press as: Takahira, I.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.096