Binuclear NiII-DpaTyr complex as a high affinity probe for an oligo-aspartate tag tethered to proteins

Article abstract of DOI:10.1002/asia.200900362

A complementary recognition pair of a short-peptide tag and a small molecular probe is a versatile molecular tool for protein detection, handling, and purification, and so forth. In this manuscript, we report that the binuclear Ni^II-DpaTyr (DpaTyr = bis((dipicolylamino)methyl)-tyrosine) complex serves as a strong binding probe for an oligo-aspartate tag tethered to a protein. Among various binuclear metal complexes of M-DpaTyr (M = Zn ^II, Ni^II, Mn^II, Cu^II, Cd ^II, Co^III, and Fe^III), we have found that Ni^II-DpaTyr (1-2Ni^II) displays a strongbinding affinity (apparent binding constant: K_app≈10⁵M^-1) for an oligo-aspartate peptide under neutral aqueous conditions (50 mM HEPES, 100 mM NaCl, pH 7.2). Detailed isothermal-titration calorimetry (ITC) studies reveal that the tri-aspartate D3-tag (DDD) is an optimal sequence recognized by 1-2Ni^II in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for Ni^II-and Zn^II- DpaTyr, show a negligible binding affinity for the oligo-aspartate peptide. The binding affinity was greatly enhanced in the pair between the dimer of Ni ^II-DpaTyr and the repeated D3 tag peptide (D3x2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K_app 10 ⁹M^-1) was achieved between the Ni^II-DpaTyr dimer 4-4Ni^II and the D3 x 2 tag peptide (DDDNGDDD). This affinity is ≈fold stronger than that observed in the binding pair of the Zn ^II-DpaTyr (4-4Zn^II) and the D4x2 tag (DDDDGDDDD), a useful tagprobe pair previously reported by us. The recognition pair of the Ni ^II-DpaTyr probe and the D3 x 2 tag can also work effectively on a protein surface, that is, 4-4Ni^II is strongly bound to the FKBP12 protein tethered with the D3 x 2 tag (DDDNGDDD) with a large K_app value of 5 x 10⁸ m^-1. Taking advantage of the strong-binding affinity, this pair was successfully applied to the selective inactivation of the tagfused (β-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.

Full text of DOI:10.1002/asia.200900362

DOI: 10.1002/asia.200900362
Binuclear NiII-DpaTyr Complex as a High Affinity Probe for an Oligo-
Aspartate Tag Tethered to Proteins
Akio Ojida,[a, b] Sho-hei Fujishima,[a] Kei Honda,[a] Hiroshi Nonaka,[a]
Sho-hei Uchinomiya,[a] and Itaru Hamachi*[a, c]
Abstract: A complementary recogni-
tion pair of a short-peptide tag and a
small molecular probe is a versatile
molecular tool for protein detection,
handling, and purification, and so
forth. In this manuscript, we report
that the tri-aspartate D3-tag (DDD) is
an optimal sequence recognized by 1-
2NiII in a 1:1 binding stoichiometry. On
the other hand, other metal complexes
of DpaTyr, except for NiII- and ZnII-
DpaTyr, show a negligible binding af-
finity for the oligo-aspartate peptide.
The binding affinity was greatly en-
hanced in the pair between the dimer
of NiII-DpaTyr and the repeated D3
tag peptide (DDDNGDDD). This af-
finity is ꢀ100-fold stronger than that
observed in the binding pair of the
ZnII-DpaTyr (4-4ZnII) and the D4ꢀ2
tag (DDDDGDDDD), a useful tag-
probe pair previously reported by us.
The recognition pair of the NiII-DpaTyr
probe and the D3ꢀ2 tag can also work
effectively on a protein surface, that is,
4-4NiII is strongly bound to the
FKBP12 protein tethered with the
that
the
binuclear
NiII-DpaTyr
(DpaTyr=bis((dipicolylamino)methyl)-
tyrosine) complex serves as a strong
binding probe for an oligo-aspartate
tag tethered to a protein. Among vari-
ous binuclear metal complexes of M-
DpaTyr (M=ZnII, NiII, MnII, CuII, CdII,
CoIII, and FeIII), we have found that
NiII-DpaTyr (1-2NiII) displays a strong-
binding affinity (apparent binding con-
stant: Kapp ꢀ105 mꢁ1) for an oligo-aspar-
tate peptide under neutral aqueous
conditions (50 mm HEPES, 100 mm
NaCl, pH 7.2). Detailed isothermal-ti-
tration calorimetry (ITC) studies reveal
tag
peptide
(D3ꢀ2),
such
as
DDDXXDDD, on the basis of the
multivalent coordination interaction
between them. Most notably, a remark-
ably high-binding affinity (Kapp =2ꢀ
109 mꢁ1) was achieved between the NiII-
DpaTyr dimer 4-4NiII and the D3ꢀ2
D3ꢀ2 tag (DDDNGDDD) with a
large Kapp value of 5ꢀ108 mꢁ1. Taking
advantage of the strong-binding affini-
ty, this pair was successfully applied to
the selective inactivation of the tag-
fused b-galactosidase by using the
chromophore-assisted light inactivation
(CALI) technique under crude condi-
tions, such as cell lysate.
Keywords: molecular recognition ·
peptides · probe · proteins
Introduction
Molecular recognition of proteins with small molecules has
long been a central interest for protein researchers. Regard-
less of their biological activities, the small molecular probes
with a strong and selective binding affinity for a certain pro-
tein are useful as a versatile molecular tool in a wide range
of protein research. For example, biotin is well known as a
specific ligand for (strept)avidin with an extremely strong-
binding affinity (apparent binding constant: Kapp ꢀ1015 mꢁ1),
so that this recognition pair is now widely used as an invalu-
able tool in detection, isolation, as well as immobilization of
proteins.[1] Other specific binding pairs, which include me-
thotrexate and DHFR (dihydrofolate reductase),[2] and rapa-
mycin and FKBP (FK506 binding protein) or FRB (FKBP-
rapamycin binding protein),[3] have also been used for selec-
tive protein labeling and function analyses of cells.
[a] Dr. A. Ojida, S.-h. Fujishima, Dr. K. Honda, H. Nonaka,
S.-h. Uchinomiya, Prof. I. Hamachi
Department of Synthetic Chemistry and Biological Chemistry
Graduate School of Engineering
Kyoto University
Katsura Campus, Nishikyo-ku, Kyoto, 615-8510 (Japan)
Fax : (+81)75-383-2759
E-mail: ihamachi@sbchem.kyoto-u.ac.jp
[b] Dr. A. Ojida
JST, PRESTO (Life Phenomena and Measurement Analysis)
Sanbancho, Chiyodaku, Tokyo, 102-0075 (Japan)
[c] Prof. I. Hamachi
JST, CREST (Creation of Next-generation Nanosystems through Pro-
cess Integration)
Sanbancho, Chiyodaku, Tokyo, 102-0075 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900362.
Chem. Asian J. 2010, 5, 877 – 886
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
877

FULL PAPERS
A complementary recognition pair consisting of a small
molecular probe and a short-peptide tag is an artificial rec-
ognition system to realize a desirable specific binding with a
protein of interest. In contrast to the binding systems em-
ploying the above-mentioned natural proteins, the artificial
tag-probe system is generally useful to achieve a specific
binding just by genetically incorporating a short-peptide tag
into a target protein with minimal perturbation of protein
functions, owing to its small size. Owing to these general
utilities and advantages, several tag-probe pairs have been
developed as a tool to facilitate protein research.[4] A repre-
sentative pair of NiII-NTA (nitrilotriacetic acid) and oligo-
histidine tag (His-tag)[5] is now available for protein purifica-
tion by using affinity-column chromatography, protein im-
mobilization on a microtiter plate, as well as cell-bioimaging
study by using fluorescence microscopy.[6] However, despite
the apparent versatility, available complementary tag-probe
pairs are still limited, mainly owing to the general difficulty
in de novo design of a molecular recognition system com-
posed with a small molecular probe and a short-peptide tag.
Therefore, it is a challenging task in the field of molecular
recognition to develop a new tag-probe pair with a high spe-
cificity, as well as an orthogonality against other protein rec-
ognition systems, which would contribute to expand the ver-
satility of the tag-probe system and provide a molecular tool
useful for protein analysis and engineering.[7]
Figure 1. Molecular structures of the synthetic metal-DpaTyr complexes.
Results and Discussion
We have recently reported that the binuclear ZnII-com-
plex 1-2ZnII (ZnII-DpaTyr, (DpaTyr=bis((dipicolylamino)-
methyl)tyrosine), Figure 1) serves as a specific binding
probe for a tetra-aspartate peptide tag (DDDD, D4-tag)
under the neutral aqueous conditions.[8] Based on this find-
ing, a strong-binding affinity (Kapp =1.8ꢀ107 mꢁ1) was ach-
ieved in the pair between ZnII-DpaTyr dimer such as 4-4ZnII
and a D4ꢀ2 (DDDDGDDDD) tag-fused protein, so that
this tag-probe pair was successfully applied for the fluores-
cence imaging of a protein on a living-cell surface.[8a] In this
manuscript, we describe a new molecular recognition pair
composed of the NiII-complex of DpaTyr (NiII-DpaTyr) and
an oligo-aspartate tag. Among various metal complexes of
DpaTyrs, we found that Ni-(II)-DpaTyr 1-2NiII serves as an
effective binding probe for the tri-aspartate tag (DDD, D3-
tag). Most interestingly, the dimer of NiII-DpaTyr (4-4NiII)
showed a remarkably strong-binding affinity (Kapp =2.0ꢀ
109 mꢁ1) for a short D3ꢀ2 tag peptide (DDDNGDDD), the
value of which is ꢀ100-fold larger than that of the previous
tag-probe pair of 4-4ZnII and D4ꢀ2 tag (Kapp =1.8ꢀ107 mꢁ1).
Taking advantage of the strong
Evaluation of the Binding Affinity of the Metal-DpaTyrs
toward Oligo-Aspartate Peptide
To perform screening of a metal complex that displays a
strong-binding affinity towards an oligo-aspartate peptide,
we initially prepared a series of the binuclear metal com-
plexes of the DpaTyr 1-2M (M=ZnII, NiII, MnII, CuII, CdII,
CoIII, and FeIII, Figure 1) and evaluated their binding affini-
ties for the tetra-aspartate D4 peptide (Boc-DDDD-NH2)
by isothermal-titration calorimetry (ITC). The results of the
ITC experiments are summarized in Table 1. We previously
revealed, by means of an ITC study, that 1-2ZnII binds to
the D4 peptide with a moderate-binding affinity (4.87ꢂ
0.13ꢀ104 mꢁ1, n=0.76) under neutral aqueous conditions
(50 mm HEPES, 100 mm NaCl, pH 7.2) through an exother-
mic binding process (DH=ꢁ5.10ꢂ0.11 kcalmolꢁ1, TDS=
1.30 kcalmolꢁ1, (Figure 2).[8a] Among the other metal com-
plexes, we have found that 1-2NiII is able to interact with
the D4 peptide through an endothermic entropy driven pro-
cess (DH=6.18ꢂ0.09 kcalmolꢁ1, TDS=13.96 kcalmolꢁ1)
binding of NiII-DpaTyr, we
Table 1. Summary of the ITC titration experiments between various metal complexes of DpaTyr 1-2m and D4
successfully applied this pair
for the selective deactivation
of D3ꢀ2 tag-fused b-galactosi-
dase by using the CALI (chro-
mophore-assisted light inacti-
vation) technique under crude
conditions, such as in the cell
lysate of E. coli.
peptide.[a,b,c]
1-2ZnII
1-2NiII
1-2MnII
1-2CuII
1-2CdII
1-2CoIII
1-2FeIII
n
0.76ꢂ0.01
(4.87ꢂ0.13)ꢀ104
ꢁ5.10ꢂ0.11
1.30
0.53ꢂ0.06
(5.18ꢂ0.41)ꢀ105
6.18ꢂ0.09
13.96
Kapp
DH
TDS
<103
–
<103
–
<103
–
<103
–
<103
–
[d]
[d]
[d]
[d]
[d]
[a] n=stoichiometry, Kapp =binding constant (mꢁ1), DH=enthalpy (kcalmolꢁ1), TDS=entropy (kcalmolꢁ1).
[b] D4 peptide=Boc-(Asp)4-NH2. [c] Measurement conditions: [1-2M]=50 mm, 50 mm HEPES, 100 mm NaCl,
pH 7.2, 258C. [d] Heat formation was scarcely detected.
878
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Chem. Asian J. 2010, 5, 877 – 886

A Nickel Probe for Tagged Proteins
with a large Kapp value of 5.18ꢂ0.41ꢀ105 mꢁ1, although their
binding stoichiometry is apparently not 1:1 (n=0.53). On
the other hand, negligible heat formation was observed in
the titration of other metal complexes, such as 1-2MnII, 1-
2CuII, 1-2CdII-, 1-2CoIII-, and 1-2FeIII, indicating that their
The binding property of 1-2NiII was further evaluated by
the ITC measurements with a series of the oligo-aspartate
peptides. These data are summarized in Table 2, which also
includes the binding data of 1-2ZnII for comparison. We
found that 1-2NiII displays a 1:1 binding stoichiometry (n=
0.91) for D3 peptide (DDD), wherein the binding affinity
(Kapp =1.5ꢀ105 mꢁ1) is larger by ꢀ20- and 3-fold than the
corresponding binding pair of 1-2ZnII with D3 (Kapp =8.54ꢀ
103 mꢁ1, n=0.93) and D4 peptide (Kapp =4.87ꢀ104 mꢁ1, n=
0.76), respectively. The binding affinities of 1-2NiII with the
other tri-aspartate peptides (DDD, DADD, DDAD) were
greater than those with the di-aspartate peptides (DD,
DAD, and DAAD), in all cases the binding stoichiometry
was evaluated to be 1:1 (n=0.83–1.07). Given the data that
the binding stoichiometry of 1-2NiII with the tetra-aspartate
D4 peptide was estimated to be 2:1 (n=0.51, Table 1), we
can conclude that 1-2NiII interacts with up to three aspartate
residues in a short oligo-aspartate peptide.[9] The ITC data
also revealed that the interaction of 1-2NiII with the oligo-
aspartate peptides is an endothermic entropy driven process
(DH>0, TDS>0) in all the cases. This is a good contrast to
the results of ZnII-DpaTyr for which the binding to the
oligo-aspartate peptides is an exothermic process (DH<0,
Tables 1 and 2). These results imply that the binding of 1-
2NiII involves a larger number of the released hydration
waters compared to the case of 1-2ZnII, so that the positive
enthalpy change was induced, which is in turn favorably
compensated by the positive entropy change in the binding
processes.[10]
binding affinities for the D4 peptide are very weak (Kapp
<
103 mꢁ1). This screening suggested that 1-2NiII is a promising
new metal complex as a probe for the oligo-aspartate tag
with a potentially strong-binding affinity.
The binding selectivity of 1-2NiII was also evaluated by
using ITC experiments with other peptides and biological
relevant anions (Table 3). We found that the binding of 1-
2NiII to His-tag peptide (His6; HHHHHH) was very weak
(Kapp <103 mꢁ1).[5] Since significant interaction was not also
observed between the oligo-aspartate peptide and NiII-
NTA,[8a] the D3 tag/NiII-DpaTyr pair is orthogonal to the
conventional His tag/NiII-NTA pair. 1-2NiII was able to bind
to E3 peptide (EEE) with a moderate binding affinity
(Kapp =1.8ꢀ104 mꢁ1). However, this binding is weaker by
ꢀ10-fold than the corresponding binding with D3 tag
(Kapp =1.5ꢀ105 mꢁ1), indicating that D3 tag is better than E3
tag. We also found that the binding affinity of 1-2NiII with
the biological relevant anions, such as inorganic phosphate
and sulfate is negligible (Kapp <103 mꢁ1). These results sug-
gest that 1-2NiII not only strongly binds to D3 tag, but also
has a sufficient binding selectivity among the biological sub-
stances.
Enhancement of the Binding Affinity by Multivalent
Binding Effect
The exploitation of multivalent binding is an effective strat-
egy to enhance the binding affinity of a recognition pair.[11]
On the basis of this strategy, we designed several dimers of
the NiII-DpaTyr complex, 2-4NiII, 3-4NiII, and 4-4NiII, which
possess a different number of glycine linker units (n=0–2,
Figure 1). We evaluated their binding affinities for the three
Figure 2. ITC titration curve (upper) and processed data (lower) in the ti-
tration of 1-2NiII with a) D4 peptide and b) D3 peptide. Measurement
conditions: [1-2NiII]=50 mm, [D4 or D3]=2 mm (5 mLꢀ48 injections),
50 mm HEPES, 100 mm NaCl, pH 7.2, 258C.
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879

I. Hamachi et al.
FULL PAPERS
Table 2. Summary of the ITC titration experiments of 1-2NiII or 1-2ZnII with various oligo-aspartate peptides.[a,b,c]
DDD (D3)
DADD
DDAD
DD
DAD
DAAD
n
0.91ꢂ0.01
(1.56ꢂ0.05)ꢀ105
4.91ꢂ0.05
11.98
0.93ꢂ0.10
(8.54ꢂ0.36)ꢀ103
ꢁ3.75ꢂ0.43
1.61
0.83ꢂ0.01
(7.80ꢂ0.22)ꢀ104
6.80ꢂ0.01
13.46
0.84ꢂ0.00
(3.37ꢂ0.06)ꢀ105
4.60ꢂ0.02
12.1
0.96ꢂ0.06
(9.43ꢂ0.25)ꢀ103
5.81ꢂ0.41
11.22
0.91ꢂ0.01
(5.88ꢂ0.15)ꢀ104
5.90ꢂ0.09
12.40
1.07ꢂ0.24
(6.32ꢂ0.47)ꢀ103
3.15ꢂ0.76
8.33
Kapp
DH
TDS
n
Kapp
DH
TDS
1-2NiII
1-2ZnII
[d]
[d]
[d]
[d]
[d]
–
–
–
–
–
[a] n=stoichiometry, Kapp =binding constant (mꢁ1), DH=enthalpy (kcalmolꢁ1), TDS=entropy (kcalmolꢁ1). [b] DDD (D3)=Boc-(Asp)3-NH2, DADD=
Boc-Asp-Ala-Asp-Asp-NH2, DDAD=Boc-Asp-Asp-Ala-Asp-NH2, DD=Boc-Asp-Asp-NH2, DAD=Boc-Asp-Ala-Asp-NH2, DAAD=Boc-Asp-Ala-
Ala-Asp-NH2. [c] Measurement conditions: [1-2NiII]=50 mm, 50 mm HEPES, 100 mm NaCl, pH 7.2, 258C. [d] Not measured.
Table 3. Summary of the ITC titration experiments of 1-2NiII with vari-
ous peptides and anions.[a,b]
[d]
HHHHHH[c] (His6) EEE[c] (Glu3)
Na2SO4[d] Na2HPO4
n
1.11ꢂ0.07
Kapp
DH
TDS
<103
–
(1.77ꢂ0.11)ꢀ104 <103
<103
–
[e]
[e]
[e]
4.17ꢂ0.29
–
9.96
[a] n=stoichiometry, Kapp =binding constant (mꢁ1), DH=enthalpy (kcal
molꢁ1), TDS=entropy (kcalmolꢁ1). [b] HHHHHH (His6)=Ac-Tyr-
(His)6-NH2, EEE=Boc-(Glu)3-NH2, [c] Measurement conditions: [1-
2NiII]=50 mm, 50 mm HEPES, 100 mm NaCl, pH 7.2, 258C. [d] Measure-
ment conditions: [1-2NiII]=50 mm, 50 mm Tris-HCl, 100 mm NaCl, pH 7.2,
258C. [e] Heat formation was barely detected.
types of the poly-aspartate D3ꢀ2 peptides composed of the
repeated D3 units (DDD-XX-DDD, XX=AA, PG, or NG).
The synthesis and characterization of the NiII complexes are
described in Figure S1 in the Supporting Information. It is
well known that paramagnetic NiII complexes display a
strong fluorescence quenching effect.[12] Thus, we evaluated
the binding affinity between the NiII-DpaTyrs with D3ꢀ2
peptides by using fluorescence quenching titration. We ini-
tially examined the titration between the monomer complex
1-2NiII and AMCA-D3 peptide having a fluorescent coumar-
in unit (AMCA) at the adjacent Lys residue (Figure 3).
When 1-2NiII was added to the aqueous solution of AMCA-
D3 peptide (1 mm in 50 mm HEPES, 100 mm NaCl, pH 7.2),
the fluorescence assigned to the AMCA unit decreased to
ꢀ30% of its original intensity (Figure 4a). The change of
the fluorescence intensity at 450 nm was evaluated by curve
fitting analysis to afford Kapp =3.1ꢀ105 mꢁ1. This value is
almost the same as that observed in the ITC experiment be-
tween 1-2NiII and D3 peptide (Kapp =1.5ꢀ105 mꢁ1), this vali-
Figure 4. Fluorescence quenching titration of the AMCA-labeled oligo-
aspartate peptide with the NiII-DpaTyr complex. a) Fluorescence spectral
change of AMCA-D3 peptide (1 mm) upon addition of 1-2NiII (0–20 mm).
Inset: curve-fitting analysis of the fluorescence emission change at
450 nm. b) Fluorescence spectral change of AMCA-D3-NG-D3 peptide
(0.1 mm) upon addition of 4-4NiII (0–1.0 mm) in the presence of 10 mm of
4-4ZnII. Inset: curve-fitting analysis of the fluorescence emission change
at 450 nm. Measurement conditions: 50 mm HEPES, 100 mm NaCl,
pH 7.2, 258C.
dates the fluorescence quenching titration for the binding
analysis. A significant fluorescence quenching was also ob-
served in the titration between the NiII-DpaTyr dimer (4-
4NiII) and AMCA-D3-NG-D3 peptide. However, the sharp
decrease of fluorescence intensity prevented the precise
evaluation of the binding affinity (data not shown). Thus,
the titration was next carried out in the presence of an
excess amount of 4-4ZnII as a competitive binder, the bind-
Figure 3. Sequences of the AMCA-labeled oligo-aspartate peptides.
880
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Chem. Asian J. 2010, 5, 877 – 886

A Nickel Probe for Tagged Proteins
ing affinity of which, for the AMCA-D3-NG-D3 peptide,
was readily determined by non-competitive titration under
the same conditions (Table 4, Figure S1 in the Supporting
Information). When 4-4NiII was added to the aqueous solu-
tion of AMCA-D3-NG-D3 peptide (0.1 mm in 50 mm
HEPES, 100 mm NaCl, pH 7.2) in the presence of a large
excess of 4-4ZnII (10 mm), a significant fluorescence decrease
prepared by the Michael-type reaction between fluorescein
5-maleimide and a cysteine introduced at the neighboring
site of the D3ꢀ2 tag. When 4-4NiII was added to the PBS
buffer solution (10 mm phosphate buffer, 138 mm NaCl,
2.7 mm KCl, pH 7.4) of Flu-D3ꢀ2-FKBP12 (0.05 mm), a
sharp and significant decrease of the fluorescence took
place (Figure 5 a,b). The change of the fluorescence intensi-
ty was analyzed by curve-fitting analysis to afford 5ꢀ108 mꢁ1
as a binding constant, the value of which is comparable to
that with the AMCA-appended peptides (Table 4). As a
control experiment, the titration using the fluorescein-ap-
Table 4. Summary of the binding constants (Kapp, mꢁ1) of 1-2NiII or 1-
2ZnII for various oligo-aspartate peptides determined by the fluorescence
titration.[a,b]
2-4NiII
3-4NiII
4-4NiII
4-4ZnII
AMCA-D3-NG-D3
AMCA-D3-PG-D3
AMCA-D3-AA-D3
8.5ꢀ108
1.7ꢀ109
2.0ꢀ109
1.5ꢀ107
A
A
A
1.1ꢀ109
(9.5ꢀ106)
4.9ꢀ108
(4.4ꢀ106)
1.6ꢀ109
(1.3ꢀ107)
7.7ꢀ108
(6.9ꢀ106)
1.8ꢀ109
(1.6ꢀ107)
8.9ꢀ108
(8.1ꢀ106)
1.1ꢀ107
1.1ꢀ107
N
N
ACHTUNGTRENNUNG
E
E
ACHTUNGTRENNUNG
[a] Measurement conditions: 50 mm HEPES, 100 mm NaCl, pH 7.2, 258C.
[b] The binding constants in the parentheses are the apparent values di-
rectly obtained by the competitive titration in the presence of 10 mm of 4-
4ZnII.
was again observed as shown in Figure 4b. The titration
curve showed a typical saturation manner, and the curve fit-
ting analysis afforded Kapp =1.3ꢀ107 mꢁ1. This value was fur-
ther analyzed based on the competitive binding equation to
afford Kapp =2.0ꢀ109 mꢁ1 as a binding constant between 4-
4NiII and AMCA-D3-NG-D3.[13] This value is ꢀ104-fold
larger than the binding between the monomeric 1-2NiII and
D3 peptide (Kapp =3.1ꢀ105 mꢁ1 determined from the fluores-
cence titration), clearly indicating that the multivalent coor-
dination between the polyaspartate residues of the tag and
the tetranuclear NiII centers of the probe works effectively
to enhance the binding affinity. It is of particular importance
that this binding affinity (Kapp =2.0ꢀ109 mꢁ1) is ꢀ130-fold
greater than that of the corresponding binding between the
dimeric ZnII complex 4-4ZnII (Kapp =1.8ꢀ107 mꢁ1) and D4ꢀ2
peptide (DDDDGDDDD), which is an optimal tag-probe
pair previously reported by us.[8a] The binding constants of
the series of the dimer complexes towards the three differ-
ent D3ꢀ2 peptides are summarized in Table 4. It is general-
ly shown that the NiII complexes are stronger by ꢀ50–160-
fold in the binding affinity, relative to the corresponding
ZnII complex in all cases. Among them, the interaction be-
tween 4-4NiII and AMCA-D3-NG-D3 is the best pair, dis-
playing the strongest binding affinity (Kapp =2.0ꢀ109 mꢁ1),
though the binding constant is not significantly affected by
the structure of the NiII complex and the tag sequence
(Kapp =2.0–0.49ꢀ109 mꢁ1). This might be ascribed to the rela-
tively flexible structure of both tag and probe, which allows
them to adopt suitable conformations for the binding.
Figure 5. a) Fluorescence spectral change of the fluorescein labeled
FKBP12 protein tethered with a D3ꢀ2 tag (Flu-D3ꢀ2-FKBP12, 0.05 mm)
upon addition of 4-4NiII (0–0.25 mm). b) Plot of the fluorescence emission
*
change at 518 nm observed in the titration of Flu-D3ꢀ2-FKBP12 ( ) or
II
&
Flu-FKBP12 lacking a D3ꢀ2 tag ( ) with 4-4Ni . Measurement condi-
tions: PBS buffer (10 mm phosphate buffer, 138 mm NaCl, 2.7 mm KCl,
pH 7.4), 258C.
pended FKBP12 protein lacking a D3ꢀ2 tag (Flu-FKBP12)
was conducted, showing only a slight decrease of the fluo-
rescence (Figure 5b). These results clearly demonstrate that
the pair of 4-4NiII and D3-NG-D3 tag works effectively in
the selective binding of a D3ꢀ2-tag-fused protein.
The validity of the pair of 4-4NiII and D3ꢀ2 tag was fur-
ther evaluated on a protein surface by using FKBP12
(FK506 binding protein 12) that has
a D3ꢀ2 tag
(DDDNGDDD) at its N-terminal. To determine the binding
affinity by the fluorescence quenching experiment, a fluoro-
phore-appended FKBP12 protein (Flu-D3ꢀ2-FKBP12) was
Chem. Asian J. 2010, 5, 877 – 886
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881

I. Hamachi et al.
FULL PAPERS
diiodo-BODIPY 12[15] to yield the ligand 5. The complexa-
tion of 5 with 4 equiv of NiIICl2 in aqueous solution provided
the CALI probe 5-4NiII. We employed b-galactosidase as a
model target protein, which was fused with a D3ꢀ2 tag
(DDDNGDDD) at its N-terminal. The mixture of the lysate
Inactivation of a Tag-Fused Protein by Using the CALI
Technique
CALI is a technique that enables the selective inactivation
of a protein of interest for elucidation of its biological func-
tion through a loss of function process.[14] Strong light irradi-
ation to the chromophore (photosensitizer) generates short-
lived reactive oxygen species, such as singlet oxygen that
can inactivate a protein in the immediate vicinity of the
chromophore. Recently, several successful examples of
CALI studies have been reported by using various protein-
labeling techniques.[15] To demonstrate the utility of the
present tag/probe pair in biological research, we examined
the CALI experiment by using a photosensitizer-appended
NiII-DpaTyr probe. Figure 6 illustrates the schematic repre-
sentation of the CALI experiment, in which the selective
binding of the photosensitizer-appended NiII-DpaTyr to a
D3NGD3 tag should facilitate an inactivation of the tag-
fused protein by the action of light irradiation.
of E.coli BL21 starTM
ACTHNUTRGNEU(GN DE3) expressing the D3ꢀ2-tag-fused
b-galactosidase and 5-4NiII was irradiated with a mercury
lamp (510–550 nm), and the CALI efficiency was evaluated
based on the hydrolysis activity of the b-galactosidase by
using ONPG (o-nitrophenyl-b-galactopyranoside) as a sub-
strate. As shown in Figure 7a, the rapid inactivation of the
tag-fused b-galactosidase took place by the presence of 5-
4NiII, wherein the inactivation rate increased by the elonga-
tion of the irradiation time to reach to 73% after 5 min.
This is a good contrast to the CALI experiment using the b-
galactosidase lacking a D3ꢀ2 tag, wherein the inactivation
rate was only 27% after 5 min. These results clearly indicate
that the tag-probe interaction largely enhances the CALI ef-
ficiency, as a result of the immediate vicinity of the diiodo-
BODIPY unit to the tag-fused b-galactosidase. Several con-
trol experiments were further carried out to confirm that
the inactivation of b-galactosidase is actually induced by the
CALI process (Figure 7b). First, negligible inactivation rate
(3%) was observed without light irradiation. Second, the in-
activation rate significantly decreased to 9% and 25% when
the irradiation was conducted without 5-4NiII and with 4-
4NiII lacking a diiodo-BODIPY unit, respectively. Finally,
the addition of 20 mm sodium azide, a well-known singlet
oxygen quencher, largely suppressed the inactivation rate to
29%, further supporting that the light-induced formation of
the reactive oxygen species
A new NiII-DpaTyr probe (5-4NiII) was designed for the
CALI experiment, as shown in Figure 6. 5-4NiII possesses
diiodo-BODIPY
(4,4’-difluoro-4-bora-3a,4a-diaza-s-inde-
cene) as a photosensitizer unit,[15] which was separated from
the NiII-DpaTyr unit through a rigid linker unit to prevent
unfavorable intramolecular interactions between the two
units. The synthesis of 5-4NiII is outlined in Scheme 1. Brief-
ly, the sequential conjugation reactions between the linker
unit 6 and 7, and between 9 and 10 gave the DpaTyr dimer
11. The hydrogenation of the azide group of 11 was followed
by the conjugation reaction with the activated ester of
mainly contributes to the dis-
ruption of the b-galactosidase
activity. It is also pointed out
that the ZnII complex 5-4ZnII
is less effective (55%) for the
enzyme inactivation compared
to the corresponding NiII com-
plex 5-4NiII (Figure 7a). Since
the inactivation activity of
both the NiII- and ZnII-DpaTyr
probe was almost the same
when the CALI experiment
was conducted using the puri-
fied b-galactosidase in the
HEPES buffer (80–90% after
5 min irradiation), this result
suggests an advantage of the
strong binding ability of 5-4NiII
towards the tag-fused protein,
which works more effectively
than 5-4ZnII under crude lysate
conditions.
Figure 6. a) Schematic illustration of the CALI (chromophore-assisted light inactivation) experiment using the
specific binding pair between 5-4NiII and the D3ꢀ2-tag-fused protein. b) Structure of the photosensitizer-ap-
pended CALI probe 5-4NiII.
882
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Chem. Asian J. 2010, 5, 877 – 886

A Nickel Probe for Tagged Proteins
Scheme 1. Synthesis of the CALI probe 5-4NiII.
Conclusions
the refinement of the probe structure, such as employment
of a longer linker unit[6c] or a fluorophore insusceptible to
the quenching effect,[17] would facilitate further biological
applications as a result of the improvement of this short-
coming. Our efforts are now ongoing along this line.
In summary, we have demonstrated that NiII-DpaTyr is an
effective binding probe for the oligo-aspartate tag-fused pro-
teins. The binding constant of the optimized tag-probe pair,
i.e., NiII-DpaTyr dimer 4-4NiII and D3ꢀ2 tag
(DDDNGDDD), reaches to 5ꢀ108–2ꢀ109 mꢁ1 under neutral
aqueous conditions. This binding fully exploits the multiple
coordination interaction between the polyaspartate residues
of the tag and the tetra-nuclear NiII centers of the probe, by
which the strong affinity was achieved in the molecular rec-
ognition between the relatively small molecular probe and
the short-peptide tag. The utility of this pair has been suc-
cessfully demonstrated by the selective deactivation of the
D3ꢀ2 tag-fused b-galactosidase in the CALI experiment
under the conditions of the crude lysate of E.coli cells. We
envision further biological application using the present tag-
probe pair such as in analysis of protein–protein interaction
and protein structure formation. Although the fluorescence
quenching character of the NiII ion may limit the utility of
the present tag-probe pair in luminescence-based studies,
Experimental Section
Syntheses of the Metal Complexes of DpaTyr
The metal complexes of DpaTyr 1--2m (M=ZnII, NiII, MnII, CuII, CdII,
CoIII, FeIII) were prepared by the treatment of the ligand 1 with an aque-
ous solution of MCl2 (2 equiv) in MeOH at RT, for 30 min. After remov-
al of the solvent by evaporation, the residue was dissolved in distilled
H2O and then lyophilized. The solid was collected by filtration and
washed with AcOEt. The solid was dried in vacuo to give a powder. In
the case of 1–2CoIII, a complex of DpaTyr with 2 equiv of CoCl2 was oxi-
dized by O2 bubbling at RT for 1.5 h.[18] The resultant solution was treat-
ed by the same procedure to give a purple solid.
Data for 1-2ZnII: Yield: 91%; FAB-MS: m/e: 918 [Mꢁ2Cl]+; elemental
analysis (%) calcd for C41H46N7O5·2Zn·3Cl·HCl·2H2O: C 48.02, H 4.91,
N 9.56; found: C 47.81, H 4.91, N 9.56.
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883

I. Hamachi et al.
FULL PAPERS
Syntheses of the Metal Complexes of the DpaTyr Dimers
The tetranuclear NiII or ZnII complexes of DpaTyr 2-4NiII, 3-4NiII, 4-
4NiII, and 4-4ZnII were prepared from the corresponding ligands of
DpaTyr dimers 2, 3, or 4 by complexation with 4 equiv of NiCl2 or ZnCl2
as the same procedure for the synthesis of 1-2NiII or 1-2ZnII. The synthe-
sis of the ligand 2 and 3 was described in the Supporting Information,
and the synthesis of 4 was previously reported in the literature.[8a]
Data for 2-4NiII: Yield: 94%; FAB-MS: m/e: 1713 [Mꢁ3Cl]+; elemental
analysis (%) calcd for C76H82N14O7·4Ni·6Cl·4H2O·2HCl: C 48.23, H 4.95,
N 10.34, found: C 48.19, H 4.79, N 10.35.
Data for 3-4NiII: Yield: 94%; FAB-MS: m/e: 1770 [Mꢁ3Cl]+; elemental
analysis (%) calcd for C78H83N15O8·4Ni·6Cl·4H2O·2HCl: C 48.02, H 4.80,
N 10.77; found: C 48.22, H 4.87, N 10.97.
Data for 4-4NiII: Yield: 92%; FAB-MS: m/e: 1827 [Mꢁ3Cl]+; elemental
analysis (%) calcd for C80H86N16O9·4Ni·6Cl·4H2O·2HCl: C 47.85, H 4.82,
N 11.16; found: C 47.87, H 4.84, N 11.26.
Data for 4-4ZnII: Yield: 87%; FAB-MS: m/e: 1845 [Mꢁ3Cl]+; elemental
analysis (%) calcd for C80H88N16O9·4Zn·6Cl·4H2O·2HCl: C 47.22,
H 4.76, N 11.01; found: C 47.00, H 4.53, N 10.91.
Synthesis of the CALI Probe 5–4NiII
The synthesis was carried out according to the synthetic route shown in
Scheme 1. The preparation of 6 is described in the Supporting Informa-
tion.
Synthesis of 8: To an ice-cooled solution of 6 (240 mg, 0.68 mmol) in an-
hydrous CH2Cl2 (5 mL) was added dropwise trifluoroacetic acid (5 mL),
and the mixture was stirred for 30 min at RT. After removal of the sol-
vent in vacuo, the residue was diluted with water. The aqueous solution
was neutralized with aq. NH3 solution in an ice-bath and then extracted
with CH2Cl2 (ꢀ2). The combined organic layers were dried over Na2SO4
and concentrated in vacuo to give the crude amino derivative of 6 as a
colorless solid. This crude material used for the next reaction without fur-
ther purification.
A
mixture of the crude amine, 7[8a] (619 mg,
0.88 mmol), EDCI·HCl (EDCI=N,N-diisopropylethylamine) (196 mg,
1.02 mmol), HOBt·H2O (156 mg, 1.02 mmol), and DIEA (DIEA=1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide) (0.71 mL, 4.08 mmol) in
anhydrous DMF (10 mL) was stirred for 5 h at RT. After dilution with
AcOEt, the mixture was washed with sat. NaHCO3 (ꢀ2) and brine fol-
lowed by drying over Na2SO4. After removal of the solvent by evapora-
tion, the residue was purified by flash column chromatography on SiO2
(CH2Cl2/MeOH/NH3; 300:10:1!200:10:1) to give 8 (631 mg, 99%) as a
Figure 7. Evaluation of the CALI efficiency for b-galactosidase. a) Time-
dependent inactivation of b-galactosidase upon light irradiation. The
CALI experiment was performed in the combination of the D3ꢀ2 tag-
II
fused b-galactosidase with 5-4NiII
(
), 5-4Zn
(
), or without probe (&),
*
^
and of the b-galactosidase lacking the D3ꢀ2 tag with 5-4NiII
(
). b) Sum-
~
mary of the inactivation rate of the b-galactosidase after 5 min of light ir-
radiation under various conditions. The error bars represent standard de-
viations obtained from at least triplicate experiments. The inactivation
rate was defined as (kint,0ꢁkint,irr)/kint,0, where kint,0 and kint,irr is the initial
rate of the enzyme reaction observed with the non-irradiated and the ir-
radiated sample, respectively. The detailed experimental conditions are
described in the experimental section.
1
colorless amorphous powder. H NMR (400 MHz, CHCl3): d=11.12 (brs
1H), 8.58 (dd, J=4.8, 0.8 Hz, 4H), 8.39 (brs, 1H), 7.64 (ddd, J=8.0, 7.4,
1.6 Hz, 4H), 7.44 (d, J=8.0 Hz, 4H), 7.16 (ddd, J=7.4, 4.8, 1.2 Hz, 4H),
6.99 (s, 2H), 6.85 (brd, J=8.0 Hz, 1H), 6.08 (d, J=8.0 Hz, 1H), 5.28 (d,
J=5.6 Hz, 1H), 4.35–4.30 (m, 1H), 3.98–3.89 (m, 4H), 3.95 (s, 2H), 3.89
(d, J=14.6 Hz, 4H), 3.82 (d, J=14.6 Hz, 4H), 3.75–3.60 (m, 2H), 3.68 (d,
J=14.0 Hz, 2H), 3.27 (br d, J=13.2 Hz, 1H), 2.96 (dd, J=13.6, 6.0 Hz,
1H), 2.86 (dd, J=13.6, 9.2 Hz, 1H), 2.00–1.90 (m, 3H), 1.82–1.79 (m,
1H), 1.42 (s, 9H), 1.39–1.15 ppm (m, 4H); FAB-MS: m/e: 941 [M+H]+.
Data for 1-2NiII: Yield: 89%; FAB-MS: m/e: 902 [Mꢁ2Cl]+; elemental
analysis (%) calcd for C41H46N7O5·2Ni·3Cl·HCl·2H2O: C 48.61, H 5.07,
N 9.68; found: C 48.49, H 5.13, N 9.73 .
Data for 1-2MnII: Yield: 87%; FAB-MS: m/e: 896 [Mꢁ2Cl]+; elemental
analysis (%) calcd for C41H46N7O5·2Mn·3Cl·HCl·2H2O: C 50.79, H 4.89,
N 10.11; found: C 51.03, H 4.73, N 10.15.
Data for 1-2CuII: Yield: 94%; FAB-MS: m/e: 912 [Mꢁ2Cl]+; elemental
analysis (%) calcd for C41H46N7O5·2Cu·3Cl·HCl·2H2O: C 49.90, H 4.80,
N 9.94; found: C 50.08, H 4.56, N 9.92.
Data for 1-2CdII: Yield: 87%; FAB-MS m/e1012 [Mꢁ2Cl]+; elemental
analysis (%) calcd for C41H46N7O5·2Cd·3Cl·HCl·2H2O: C 45.41, H 4.37,
N 9.04; found: C 45.49, H 4.34, N 9.00.
Data for 1-2CoIII: Yield: 70% in 2 steps; FAB-MS: m/e: 904 [Mꢁ2Cl]+;
elemental analysis (%) calcd for C41H46N7O5·2Co·4Cl·H2O·OH: C 49.51,
H 4.86, N 9.86; found: C 49.90, H 4.76, N 9.98.
Data for 1-2FeIII: Yield: 73%; FAB-MS: m/e: 898 [Mꢁ2Cl]+; elemental
analysis (%) calcd for C41H46N7O5·2Fe·5Cl·HCl: C 47.25, H 4.55, N 9.41;
found: C 47.31, H 4.65, N 9.13.
Synthesis of 9: Azide 8 (400 mg, 0.425 mmol) was used as a starting mate-
rial. According to the same synthetic procedure described for the depro-
tection of 6, azide 9 was obtained as a colorless amorphous powder
(343 mg, 90%). 1H NMR (400 MHz, CHCl3): d=11.08 (brs, 1H), 8.52
(ddd, J=5.0, 2.0, 0.8 Hz, 4H), 8.02 (t, J=5.8 Hz, 1H), 7.62 (ddd, J=7.6,
7.6, 2.0 Hz, 4H), 7.47 (d, J=7.6 Hz, 4H), 7.13 (ddd, J=7.4, 5.0, 1.4 Hz,
4H), 7.05 (s, 2H), 6.24 (d, J=8.0 Hz, 1H), 6.14 (d, J=8.0 Hz, 1H), 3.96
(s, 2H), 3.86 (s, 6H), 3.82 (d, J=13.6 Hz, 4H), 3.77 (d, J=13.6 Hz, 4H),
3.74–3.69 (m, 2H), 3.58 (dd, J=9.0, 4.6 Hz, 1H), 3.11 (dd, J=13.8,
4.6 Hz, 1H), 2.60 (dd, J=13.4, 9.0 Hz, 1H), 1.99 (brd, J=6.8 Hz, 4H),
1.34–1.21 ppm (m, 4H); FAB-MS: m/e: 1540 [M+H]+.
Synthesis of 11:
A mixture of 9 (150 mg, 0.18 mmol), 10 (175 mg,
0.21 mmol), EDCI·HCl (51 mg, 0.27 mmol), HOBt·H2O (41 mg,
0.27 mmol), and DIEA (93 mL, 0.53 mmol) in anhydrous DMF (5 mL)
was stirred for 4 h at RT. After dilution with AcOEt, the mixture was
washed with sat. NaHCO3 (ꢀ2) and brine followed by drying over
Na2SO4. After removal of the solvent by evaporation, the solid was
884
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Chem. Asian J. 2010, 5, 877 – 886

A Nickel Probe for Tagged Proteins
washed with AcOEt/hexane (1:2) and collected by filtration to give 11
(262 mg, 90%) as a colorless powder. 1H NMR (400 MHz, CHCl3): d=
11.10 (brs, 2H), 8.88 (brs, 1H), 8.55–8.53 (m, 8H), 8.47 (brs, 1H), 8.29
(brs, 1H), 7.64 (dd, J=7.6, 2.0 Hz, 4H), 7.60 (dd, J=7.6, 2.0 Hz, 4H),
7.48 (d, J=8.0 Hz, 4H), 7.44 (d, J=8.0 Hz, 4H), 7.19–7.12 (m, 10H), 7.00
(s, 2H), 6.98 (brs, 1H), 6.18 (d, J=8.0 Hz, 1H), 5.88 (brs, 1H), 4.47 (dd,
J=14.2, 7.4 Hz, 1H), 4.44–4.38 (brm, 1H), 3.99–3.63 (m, 33H), 3.55
(brd, J=12.8 Hz, 1H), 3.40 (br d, J=12.8 Hz, 1H), 3.21 (dd, J=13.8,
7.4 Hz, 1H), 3.04 (dd, J=13.6, 8.4 Hz, 1H), 2.98–2.88 (m, 2H), 1.95 (brd,
J=10.4 Hz, 4H), 1.49–1.18 (m 4H), 1.32 ppm (s, 9H); FAB-MS: m/e:
1640 [M+H]+.
K2
Kapp
¼
ð1Þ
1 þ K1½4 ꢁ 4 ZnIIꢃ
In which K1 is the binding constants between 4–4ZnII and the peptide
which was separately obtained by the non-competitive fluorescence titra-
tion experiment.
Fluorescence Titration with the Fluorescein-Appended FKBP12
The titration experiments were performed at 258C using a solution of
Flu-(D3ꢀ2)-FKBP12 or Flu-FKBP12 in PBS buffer (10 mm phosphate
buffer, 138 mm NaCl, 2.7 mm KCl, pH 7.4) in a quartz cell. The concen-
tration of the FKBP was determined in SDS-PAGE by comparison of the
CBB band intensity with a standard protein. The fluorescence emission
spectra (excitation wavelength; lex =492 nm) were measured immediately
after addition of an aqueous solution of 4-4NiII with a micro syringe.
Fluorescence titration curves (lem =518 nm) were analyzed using nonlin-
ear least-square curve-fitting assuming 1:1 binding to evaluate the bind-
ing constant (Kapp, mꢁ1).
Synthesis of 5: A solution of 11 (34 mg, 21 mmol) and 10% Pd–C ethyle-
nediamine complex (10 mg) in MeOH–CH2Cl2 (1:1, 4 mL) was vigorously
stirred for 8 h under H2 atmosphere at RT. Pd–C was filtered off and
washed with MeOH. The filtrate was concentrated in vacuo to give the
amino derivative of 11 (31 mg) as a pale yellow amorphous powder. This
material was used for the next reaction without further purification;
FAB-MS: m/e: 1614 [M+H]+.
A
solution of the crude amine (11 mg, 6.8 mmol), 12[15] (7.6 mg,
Selective Inactivation of the Tag-Fused b-Galactosidase by CALI
9.9 mmol), and DIEA (10 mL, 57 mmol) in anhydrous DMF (1 mL) was
stirred for 10 hr at RT. The reaction mixture was directly purified by
using reverse-phase HPLC (YMC-pack ODS-A, 20ꢀ250 mm) with
CH3CN (0.1% TFA)/H2O (0.1% TFA) solvent system (linear gradient)
mode to give 5 (9.9 mg, 63%) as a reddish powder. 1H NMR (400 MHz,
CDCl3OD): d=8.63 (m, 8H), 7.93 (m, 8H), 7.53–7.48 (m, 17H), 7.17–
7.14 (m, 7H), 4.46–4.38 (m, 1H), 4.37 (s, 8H), 4.34 (s, 8H), 4.27 (dd, J=
9.0 5.8 Hz, 1H), 4.23–4.09 (m, 6H), 4.03 (t, J=6.0 Hz, 2H), 3.88 (dd, J=
16.8, 6.8 Hz, 1H), 3.82–3.56 (m, 11H), 3.07 (dd, J=14.0, 5.2 Hz, 2H),
2.91 (dd, J=14.0, 8.8 Hz, 1H), 2.75 (dd, J=13.8, 9.4 Hz, 1H), 2.54 (s,
6H), 2.10 (t, J=7.2 Hz, 2H), 1.87–1.79 (m, 4H), 1.66–1.60 (m, 2H), 1.52–
1.46 (m, 8H), 1.38–1.25 (m, 4H), 1.18 ppm (s, 9H); FAB-HRMS: m/e:
calcd for C112H128BF2I2N22O12 [M+H]+ 2287.8227, found 2287.8213.
The crude cell lysate of E. coli BL21 starACTHUNRGTNE(NUG DE3) expressing the b-galacto-
sidase fused with a D3ꢀ2 tag (DDDNGDDD) was mixed with a DMSO
solution 5-4NiII (3 mm in final concentration), which was prepared by in
situ complexation of 5 with 4 equivalent of NiII ions by using an aqueous
solution of NiCl2 (10 mm). The mixture (55 mL, containing 3% DMSO)
was spotted on a 96-well glass-base plate and irradiated for appropriate
time with a mercury lamp (100 W) through a bandpass filter (510–
550 nm) on the stage of a fluorescence microscope (IX-71, OLYMPUS).
The irradiated solution (50 mL) was replaced on a 96-well polystyrene-
base plate, and mixed with a solution (50 mL) of o-nitrophenyl-ß-galacto-
pyranoside (ONPG) (1.33 mgmLꢁ1 in 200 mm sodium phosphate buffer
(pH 7.3), 2 mm MgCl2, 100 mm b-mercaptoethanol). The absorbance
change at 420 nm was monitored for 2 min at 378C using a plate reader
(Infinite M200, Tecan) to obtain the initial rate of the enzyme reaction.
The inactivation rate was defined as the following Equation (2):
Isothermal Titration Calorimetry (ITC) Measurements
ITC titration was performed by using an isothermal titration calorimeter
(MicroCal Inc). All measurements were conducted at 298 K. A solution
of the peptide (2.0 mm) in a buffer solution (50 mm HEPES, 100 mm
NaCl, pH 7.2) was injected stepwise (5 mLꢀ48 times) to a solution of the
metal complex of DpaTyr (50 mm) in the same solvent system. The mea-
sured heat flow was recorded as function of time and converted into en-
thalpies (DH) by integration of the appropriate reaction peaks. Dilution
effects were corrected for by subtracting the result of a blank experiment
with an injection of the HEPES solution into cell under identical experi-
mental conditions. The binding parameters (Kapp, DH, DS, n) were evalu-
ated by applying one site model using the software Origin (MicroCal
Inc.).
kint;0 ꢁ kint;irr
Inactivation rate ¼
ð2Þ
kint;0
In which kint,0 and kint,irr is the initial rate of the enzyme reaction observed
with the non-irradiated and the irradiated sample, respectively.
Acknowledgements
S.F. thanks JSPS for his predoctoral fellowship.
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1996, 2, 1028–1032.
Fluorescence Titration with the AMCA-Appended Peptides
The fluorescence spectra were recorded by using a Perkin–Elmer LS55
spectrofluorophotometer. The titration experiments of NiII or ZnII-Dpa-
Tyrs were performed at 258C using a solution of the AMCA-appended
oligo-aspartate peptide in buffer solution (50 mm HEPES, 100 mm NaCl,
pH 7.2) in a quartz cell. The concentration of the AMCA-appended pep-
tide was determined based on the reported extinction coefficient of
AMCA-X, SE (e=19,000 cmꢁ1 mꢁ1, Invitrogen). The competitive titra-
tions of the tetranuclear NiII-complex were performed in the presence of
10 mm of 4–4ZnII in a buffer solution (50 mm HEPES, 100 mm NaCl,
pH 7.2) of the AMCA-appended peptide (0.1 mm). The fluorescence
emission spectra (excitation wavelength; lex =350 nm) were measured
immediately after addition of a freshly prepared aqueous solution of NiII-
DpaTyrs with
a micro syringe. Fluorescence titration curves (lem =
450 nm) were analyzed using nonlinear least-square curve-fitting assum-
ing 1:1 binding to evaluate the apparent binding constant (Kapp, mꢁ1). The
binding constants (K2) of the tetranuclear NiII-complexes with a series of
oligo-aspartate peptides (Table 4) were calculated based on the following
Equation (1):
Chem. Asian J. 2010, 5, 877 – 886
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
885

I. Hamachi et al.
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Received: August 11, 2009
Revised: October 5, 2009
Published online: February 8, 2010
886
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Chem. Asian J. 2010, 5, 877 – 886