DNA-binding hemoproteins tethering polyamine interface

Article abstract of DOI:10.1246/bcsj.20090315

Artificial prosthetic groups tethering a polyamine moiety at the terminal of two peripheral hemepropionate side chains as a molecular interface were inserted into apocytochrome b₅₆₂ to provide positively charged reconstituted hemoproteins, which exhibit a strong binding with double-stranded DNA.

Full text of DOI:10.1246/bcsj.20090315

Short Articles
Bull. Chem. Soc. Jpn. Vol. 83, No. 4, 375–377 (2010)
375
native hemin polyamine-tethering hemin
DNA-Binding Hemoproteins
Tethering Polyamine Interface
rCyt b562
wt-Cyt b562
apo-Cyt b562
(cytochrome b562
)
+
+
Akira Onoda,1,2 Hirokazu Nagai,1 Satoe Koga,1
and Takashi Hayashi*1
NH3
+
NH3
O
O
N
N
H
H
H
N
H
N
NH3
H
H
+
+
O
O
N
N
NH3
O
O
O
O
N
N
N
N
N
N
N
N
Fe
Fe
1Department of Applied Chemistry, Graduate School of
Engineering, Osaka University, Suita, Osaka 565-0871
N
N
NH3
O
N
O
N
H
H
+
+
NH3
H
H
H
H
N
N
O
O
+
NH3
2Frontier Research Base for Global Young Researchers,
Graduate School of Engineering, Osaka University,
Suita, Osaka 565-0871
NH3
1
2
Figure 1. Schematic representation of reconstituted cyto-
chrome b562 with polyamine moiety.
Received November 30, 2009
E-mail: thayashi@chem.eng.osaka-u.ac.jp
Therefore, we next prepared two kinds of cytochrome b562
s
reconstituted with polyamine-tethering hemins, 1 and 2, as a
new DNA-binding hemoprotein (Figure 1). Here we report
the characterization and the binding properties of positively
charged hemoproteins.
Artificial prosthetic groups tethering a polyamine
moiety at the terminal of two peripheral heme-propionate
side chains as a molecular interface were inserted into
apocytochrome b562 to provide positively charged reconsti-
tuted hemoproteins, which exhibit a strong binding with
double-stranded DNA.
The cofactors were prepared as follows: The terminal of
two propionates of the protoporphyrin IX were coupled with
5-amino-1,3-bis(t-butoxycarbonylaminopropyl)carbamoylben-
zene and the Boc groups were then deprotected by TFA/
HCO2H, yielding the positively charged free-base porphyrin.
The corresponding propionate-modified porphyrins were treat-
ed with FeCl2, and purification on a LH-20 column gave the
final products, 1 and 2. The products were characterized by
FAB MS (1: calcd. 1166.5265; found 1166.5273, 2: calcd.
1390.7769; found 1390.7767). From the characteristic absorp-
tion at 360, 396, and 608 nm, both amine-linked hemins
were present as an equilibrium mixture of the Cl-bound
hemin and its ®-oxo dimer in 10 mM HEPES buffer solution
(pH 7.4).
The hemin derivatives were inserted into the apocytochrome
b562 (apo-Cyt b562) by a conventional method15 followed by
purification using a gel filtration column to produce the
reconstituted cyt b562s, rCyt b562(1), and rCyt b562(2). During
the preparation, the reconstituted proteins were isolated as the
ferrous (Fe2+) form with His and Met ligations, providing well-
characterized absorptions at 426, 531, and 562 nm, suggesting
that each heme was located in the normal cyt b562 heme pocket.
The ferric form was then prepared by oxidation with potassium
hexacyanoferrate(III) exhibiting absorptions at 420, 537, and
566 nm.16 Successful installation of the modified hemins was
also determined by ESI-TOF MS experiments of the rCyt
b562(1) and rCyt b562(2). The reconstituted ferric proteins were
used for the following experiments. These reconstituted
proteins were stable for several days at 4 °C.
Biological molecules are attractive building units for nano-
scale assemblies based on bottom-up strategies. In particular,
DNA molecules have been proven to serve as well-defined
nanoscaffolds, where DNA-protein interaction generates fas-
cinating conjugates.1,2 The expanding repertoire of naturally
occurring DNA-protein interaction using artificial surrogates
is a promising target both in the fields of medicinal chemistry
and nanobiotechnology. To this end, a strategy for converting
ubiquitous proteins to DNA-binding ones is required. Espe-
cially, the conjugation of redox-active hemoproteins with DNA
molecules has attracted increasing interest, because a series of
hemoproteins function as an electron-transfer mediator, cata-
lyst, and sensor. One intriguing approach to build a designed
moiety on the hemoprotein surface is cofactor substitution, in
which we can replace the native hemin with an artificial one
having a DNA-binding interface. For example, myoglobin
reconstituted with a modified hemin having an intercalator at
the terminal of a heme-propionate side chain provides a stable
DNA-myoglobin complex.3 Furthermore, the covalent intro-
duction of a single-stranded DNA or DNA-binding peptides to
the heme-propionate side chain leads to DNA-conjugated
hemoproteins.4,5
In contrast, we have recently reported an artificial protein-
protein complexation using negatively or positively charged
myoglobins;6-10 e.g., myoglobin reconstituted with a modified
hemin with a carboxylate cluster at the terminal of two
propionate side chains forms a stable complex with a positively
charged cytochrome c. Our next objective is to create unique
nanobiomaterials involving a hemoprotein-DNA composite.
It is known that various polyamine structures might be
effective as a strong binding interface for the DNA moiety.11-14
The conformational characterization of the reconstituted
proteins was performed by CD measurements (Figure 2).
Characteristic 222- and 208-nm minima, and a crossover point
at 201 nm indicate that the ¡-helical folding of both recon-
stituted ferric cyt b562s are similar to that of the wild type cyt
b562 (wt-Cyt b562).17 Although having the positively charged
moieties, the reconstituted cyt b562s retain mostly monomeric
structure without interaction with the negatively charged region
Published on the web March 18, 2010; doi:10.1246/bcsj.20090315

376
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
© 2010 The Chemical Society of Japan
Figure 4. Frequency changes in the QCM in response to the
sequential (solid line) and the single addition (dashed line)
of rCyt b562(1). The addition of wt-Cyt b562 is shown by the
dotted line. Conditions: 10 mM HEPES, (pH 7.4), 25 °C.
Figure 2. CD spectra of rCyt b562(1) (solid line) and rCyt
562(2) (dashed line). Conditions: 10 ¯M protein, 5 mM
HEPES, 10 mM Tris-HCl, 1 mM EDTA (pH 8.2), 25 °C.
dicated that static binding of wt-Cyt b562 is negligible and that
the tailored amine moiety on the reconstituted protein surface
provides a strong DNA binding.
b
Next, to investigate in detail the binding event between the
reconstituted protein displaying the polyamine moiety and
dsDNA, we performed an electrophoretic mobility shift assay
(EMSA), in which we can obtain the exact ratio of the bound
and free DNA to estimate the binding affinity. The EMSA
analyses using the 32P-labeled 50 bp DNA target showed that
wt-Cyt b562 did not bind to the target at all. In contrast, rCyt
b562(1) and rCyt b562(2) exhibited clearly shifted bands
(Figure 3b), supporting the fact that both polyamine-tethering
reconstituted proteins are potential species for strong DNA
¹7
binding with the dissociation constants of 6.0 © 10 and
4.8 © 10¹8 M, respectively, at 4 °C.19 Positively charged amine
groups with longer alkyl chain in rCyt b562(2) might provide
more effective multivalent electrostatic and/or hydrogen-bond
interaction with the phosphate backbone and the base pair
within the groove. Actually, the DNA binding here was
interrupted in the high salt concentration.
Figure 3. (a) Normalized fluorescent intensity (Imax) as a
function of the cyt b562 concentration. rCyt b562(1) (filled
circle), rCyt b562(2) (filled triangle), and wt-Cyt b562
(filled square). (b) EMSA analyses of DNA binding of
rCyt b562(1) and rCyt b562(2). Protein concentrations: 0-
10 ¯M.).
The binding behavior of the reconstituted proteins on the
dsDNA was also analyzed using a quartz crystal microbalance
(QCM), which has proven to be a useful tool for detecting
the DNA-protein interaction as mass increase on the QCM
electrode.20 The dsDNA with a thiol group on the 5¤-terminus
was immobilized together with 6-mercapto-1-hexanol on the
Au electrode of the 27-MHz QCM. The frequency changes
versus time of the dsDNA-immobilized QCM upon the
additions of the reconstituted proteins revealed the apparent
frequency decrease (¹100 Hz), while wt-Cyt b562 did not
exhibit a significant frequency change as shown in Figure 4.
The sequential addition of rCyt b562(1) exhibited a stepwise
decrease in frequency, revealing that the observed frequency
decay is due to the mass increase by the binding of the
reconstituted protein on the DNA. The calculated Kd value,
1.6 © 10¹7 M, for rCyt b562(1) determined from the sequential
addition decay of the QCM profile is in good accordance with
that from the EMSA analyses. These results suggest that a total
of approximately four positively charged proteins are attached
to the target DNA, which seems to be in coincidence with the
significantly shifted bands in the EMSA.
on the protein surface, supported by size exclusion chromato-
graphic analyses.
We first investigated the qualitative binding ability of the
reconstituted proteins to plasmid DNA by fluorescence assay
using the fluorescent staining dye, GelStar, which exhibits a
strong emission at 534 nm upon intercalation within the base
pairs.18 As shown in Figure 3a, the fluorescence of both
polyamine-displaying cyt b562, rCyt b562(1), and rCyt b562(2),
decreased prominently. This effective quenching could be
explained by two possible reasons. The binding of the
reconstituted proteins expelled the intercalated dyes and/or
the iron-containing protein bound to the double-stranded DNA
(dsDNA) quenched the strong emission derived from the
dyes within the DNA. While in the wt-Cyt b562 without the
polyamine moiety, the fluorescence decreased slightly probably
because only dynamic interaction between the DNA and the
proteins are predominant. Therefore, the results clearly in-
In conclusion, we have successfully prepared the reconsti-
tuted cyt b562 with the hemins tethering the polyamine moiety,

A. Onoda et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
377
bound to the DNA-immobilized QCM cell were removed by
washing with 1.0 M KCl solution for several times.
and our reconstituted proteins provide a strong binding affinity
for dsDNA to afford a DNA complex with multiple hemopro-
teins. The present hemoprotein-DNA conjugate would serve as
a way to create functional nanomaterials such as a protein-
immobilized electrode. A study of the kinetic and thermody-
namic aspects of these complexations is now in progress.
This work was financially supported by MEXT and JSPS
(to T. H.). O. A. acknowledges support from the Special
Coordination Funds for Promoting Science and Technology,
from MEXT. H. N. expresses his special thanks for the Global
COE (center of excellence) Program of Osaka University.
Experimental
Preparation of Cytochrome b562 Reconstituted with Poly-
amine-Tethering Hemins. To an apoprotein solution (10 mM
HEPES, pH 7.4, 500 mM KCl) was slowly added dropwise an
aqueous solution of 1 with gentle shaking at 4 °C and the mixture
was incubated at 4 °C for 12 h. The protein solution was
concentrated and potassium hexacyanoferrate(III) was added to
obtain the ferric protein. The crude protein was purified by
Sephadex G-25 gel filtration with elution of buffered solution
(10 mM HEPES, pH 7.4) to give the reconstituted cyt b562 with 1
(rCyt b562(1)). UV-vis (10 mM HEPES, pH 7.4) -max/nm 420,
537, and 566; ESI-TOF MS m/z calcd for (M + H)+: 12947.8,
found 12948.2. The reconstituted cytochrome b562 with 2 (rCyt
Supporting Information
Synthesis of 1 and 2, UV-vis spectra, ESI-TOF MS, and
fluorometric DNA-binding analyses of reconstituted cyt b562s. This
material is available free of charge on the web at http://www.csj.jp/
journals/bcsj/.
References
1
K. Keren, M. Krueger, R. Gilad, G. Ben-Yoseph, U. Sivan,
E. Braun, Science 2002, 297, 72.
2
16024.
3
C. V. Kumar, M. R. Duff, Jr., J. Am. Chem. Soc. 2009, 131,
H. Takashima, Y. Matsushima, Y. Araki, O. Ito, K.
b562(2)) was obtained according to the same procedure. UV-vis
(10 mM HEPES, pH 7.4) -max/nm 421, 536, and 562; ESI-TOF
MS m/z calcd for (M + H)+: 13172.0, found 13175.1.
Tsukahara, J. Biol. Inorg. Chem. 2008, 13, 171.
Fluorometric Analyses.
The fluorescence spectra were
4
2603.
5
1749.
6
L. Fruk, C. M. Niemeyer, Angew. Chem., Int. Ed. 2005, 44,
measured in a quartz microcell at 25 °C and recorded from 500
to 600 nm with 493 nm excitation. Plasmid DNA pBR322 (4361
bp) (5.0 ¯g mL¹1 = 1.9 nM)) in 10 mM HEPES buffer (pH 7.4).
Protein concentration was varied between 0 to 3.4 ¯M.
S. Sakamoto, K. Kudo, Bull. Chem. Soc. Jpn. 2005, 78,
T. Hayashi, Y. Hisaeda, Acc. Chem. Res. 2002, 35, 35.
T. Hayashi, Y. Hitomi, H. Ogoshi, J. Am. Chem. Soc. 1998,
Electrophoretic Mobility Shift Assay (EMSA). The annealed
50 bp dsDNA (top strand: 5¤-AGCTCTCATGCGGATGCACGT-
CAATAGTTGCTTCG-GAAGATCGACTAGATCCTG-3¤, bottom
strand: 3¤-GAGTACGCCTACGTGCAGTTATCAACGAAGCCT-
TCTAGCTGATCTAGGACTCGA-5¤) was labeled at the 5¤ termi-
nus with [£-32P]ATP by T4 polynucleotide kinase, and then the
32P-labeled DNA fragment was purified by gel filtration using a
buffered solution (10 mM Tris-HCl, 1.0 mM EDTA, pH 8.0) as an
eluent. The different concentrations of reconstituted/wild type
protein (10 mM HEPES, pH 7.4) and the purified 32P-labeled DNA
fragment were incubated at 37 °C for 1 h. The DNA-bound proteins
and free DNA were electrophoretically separated on a 12%
polyacrylamide gel with Tris-borate-EDTA buffer (44.5 mM Tris-
borate, 1.0 mM EDTA) at 230 V and 4 °C. The gel was preelec-
trophoresed for 30 min before running the samples. The gels were
vacuum-dried and visualized by autoradiography using FLA-7000
(FUJIFILM Co., Ltd.).
Quartz Crystal Microbalance (QCM) Measurements. QCM
measurement was performed by AFFINIX QN® (INITIUM INC.).
The cells were equipped with a 27 MHz QCM plate oscillating at
fundamental frequency. To immobilize DNA on the Au electrode,
the cleaned bare Au electrode of the QCM cell was immersed in
50 ¯L of a solution containing 0.5 ¯M 5¤-thiol-modified 50 bp
dsDNA, 10 mM Tris-HCl buffer (pH 8.0) at 25 °C for 30 min.
After being washed with water, the cell was next immersed in
50 ¯L of a solution containing 200 ¯M of 6-mercapto-1-hexanol
and 10 mM of Tris-HCl buffer (pH 8.0) at 25 °C for 30 min and
washed again. The DNA-immobilized QCM cell was filled with
500 ¯L of a buffered solution (10 mM HEPES, pH 7.4) and the
time course frequency changes were observed at different protein
concentrations (99, 200, 290, 380, 480, and 500 nM). The proteins
7
120, 4910.
8
Y. Hitomi, T. Hayashi, K. Wada, T. Mizutani, Y. Hisaeda,
H. Ogoshi, Angew. Chem., Int. Ed. 2001, 40, 1098.
9
T. Matsuo, H. Nagai, Y. Hisaeda, T. Hayashi, Chem.
Commun. 2006, 3131.
10 T. Hayashi, T. Ando, T. Matsuda, H. Yonemura, S. Yamada,
Y. Hisaeda, J. Inorg. Biochem. 2000, 82, 133.
11 M. Saminathan, T. Antony, A. Shirahata, L. H. Sigal, T.
Thomas, T. J. Thomas, Biochemistry 1999, 38, 3821.
12 D. C. Wu, Y. Liu, X. Jiang, L. Chen, C. B. He, S. H. Goh,
K. W. Leong, Biomacromolecules 2005, 6, 3166.
13 M. A. Kostiainen, G. R. Szilvay, D. K. Smith, M. B. Linder,
O. Ikkala, Angew. Chem., Int. Ed. 2006, 45, 3538.
14 D. J. Welsh, S. P. Jones, D. K. Smith, Angew. Chem., Int.
Ed. 2009, 48, 4047.
15 F. Ascoli, M. R. Fanelli, E. Antonini, Methods Enzymol.
1981, 76, 72.
16 E. Itagaki, L. P. Hager, J. Biol. Chem. 1966, 241, 3687.
17 P. A. Bullock, Y. P. Myer, Biochemistry 1978, 17, 3084.
18 The characteristic absorptions of 1 and 2 did not remark-
ably change upon the addition of the dsDNA with the range of ¯M
concentrations, indicating that hemis 1 and 2 are stably inserted
into the apoprotein.
19 R = Ib/(Ib + If), where Ib and If are the intensities of
peptide-bound and free DNA bands, respectively. The Kd values
were evaluated by fitting the experimentally obtained values of R
to the binding isotherm equation (R = [protein]/([protein] + Kd)
assuming that the influence of cooperative binding is absent.
20 Y. Okahata, K. Niikura, Y. Sugiura, M. Sawada, T. Morii,
Biochemistry 1998, 37, 5666.