Self-assembly of one- And two-dimensional hemoprotein systems by polymerization through heme-heme pocket interactions

Article abstract of DOI:10.1002/anie.200804006

Supramolecular protein polymers: When a heme moiety was introduced to the surface of an apo-cytochrome b₅₆₂(H63C) mutant, supramolecular polymers formed through noncovalent heme-heme pocket interactions. The incorporation of a heme triad as a p

Full text of DOI:10.1002/anie.200804006

Angewandte
Chemie
DOI: 10.1002/anie.200804006
Protein Self-Assembly
Self-Assembly of One- and Two-Dimensional Hemoprotein Systems by
Polymerization through Heme–Heme Pocket Interactions**
Hiroaki Kitagishi, Yasuaki Kakikura, Hiroyasu Yamaguchi, Koji Oohora, Akira Harada, and
Takashi Hayashi*
The construction of supramolecular polymers has recently
become one of the principal challenges in nanoscience based
on chemistry and biology.[1–5] For biomedical applications
such as drug delivery[6,7] and tissue engineering[8] biomole-
cules have great potential as structural units in supramolec-
ular polymers because of their biocompatibility and biode-
gradability. In addition, the design of highly ordered struc-
tures of biomacromolecules by self-assembly should shed
light on the principles required for the development of
“bottom-up” nanobiotechnology.[9–11] We have recently
reported a system for the self-assembly of linear hemopro-
teins composed of subunits in which an externally introduced
heme moiety is attached to the surface of a H63C single
mutant of cytochromeb562 (cytb562(H63C)). This system
displays an interprotein heme–heme pocket interaction after
the native heme is removed from the modified protein.[12,13]
The next attractive target for the development of further
applications would be a supramolecular polymer with more
highly ordered and/or higher-dimensional structures.[14–16] We
demonstrate herein the construction of a supramolecular
polymer composed of the genetically modified cytb562(H63C)
protein and the synthetic heme analogue 2 (Scheme 1). The
morphology of this polymer was investigated by atomic force
microscopy (AFM). This linear hemoprotein self-assembly
system has now been developed further into two-dimensional
Scheme 1. Structures of synthetic heme analogues. These compounds
have regioisomers with respect to the substitution position in the two
heme propionate side chains of protoheme IX.
network structures by the introduction of the novel heme
triad 3 as a pivot molecule. The strategy of this approach is
illustrated in Scheme 2.
In our previous work, the synthetic heme 1, which has an
iodoacetamide-derivatized thiol-reactive group at the termi-
nus of one of the heme propionate side chains, was prepared
and used to obtain the heme-appended protein 1-cytb562
-
(H63C).[12] However, for completion of the conjugation
between 1 and cytb562(H63C), alkaline conditions (pH 9.0)
and long reaction times (over 7 h) were required because of
the low reactivity of 1 to the thiol group of Cys63. Therefore,
we prepared the synthetic heme 2; its maleimide group is
more reactive than iodoacetamide towards thiols.[17–19] The
surface modification of cytb562(H63C) with 2 was carried out
in aqueous 0.05m Tris-HCl buffer at pH 7.3. After a mixture
of cytb562(H63C) was gently stirred with an excess of 2 for
1.5 h at room temperature, the solution was acidified to
pH 1.9 and the native heme was removed from cytb562(H63C)
by using conventional extraction with 2-butanone.[20] The
aqueous phase was neutralized by dialysis against a 0.05m
Tris-HCl buffer solution at pH 7.3. The UV/Vis spectrum of
the resulting 2-apo-cytb562(H63C) exhibits characteristic
[*] Dr. H. Kitagishi,[+] Y. Kakikura, K. Oohora, Prof. Dr. T. Hayashi
Department of Applied Chemistry
Graduate School of Engineering, Osaka University
2-1 Yamadaoka, Suita 565-0871 (Japan)
Fax: (+81)6-6879-7930
E-mail: thayashi@chem.eng.osaka-u.ac.jp
Dr. H. Yamaguchi, Prof. Dr. A. Harada
Department of Macromolecular Science
Graduate School of Science, Osaka University
Toyonaka 560-0043 (Japan)
[+] Present address:
Department of Molecular Chemistry and Biochemistry
Doshisha University, Kyotanabe, Kyoto 610-0321 (Japan)
[**] This work was supported by Grants-in-Aid for Scientific Research
from MEXT. T.H. acknowledges a research grant from the Asahi
Glass Foundation. H.K. was financially supported as an Inoue
Fellow of the Inoue Foundation for Science.
Soret (418 nm) and Q (530 and 564 nm) bands consistent
[21]
with those of wild-type cytb562
.
This finding indicates that
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200804006.
the heme externally attached on the cytb562(H63C) surface is
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Figure 1. a) Native- and b) SDS-PAGE of cytb562(H63C) and 2-apo-
cytb562(H63C). Lane 1: marker proteins; lane 2: cytb562(H63C); lane 3:
2-apo-cytb562(H63C).
The size and morphology of the hemoprotein assemblies
were characterized by tapping-mode atomic force microscopy
(TMAFM) measurements.[23,24] Samples were prepared for
measurements by immersing a freshly cleaved, highly ori-
ented pyrolytic graphite (HOPG) substrate in the protein
solution for a few seconds; the substrate was then washed with
deionized water to remove buffer salts and subsequently
dried.[25] The AFM image of immobilized 2-apo-cytb562
-
(H63C) on the substrate shows uniformly dispersed linear
objects (Figure 2a). The height of the objects ranges from 2.5
to 5.0 nm (see the Supporting Information), which is in good
agreement with the size of the cylindrically shaped cytb562
protein (height 5.0 nm; diameter 2.5 nm).[12,26] The length of
the observed protein fiber in Figure 2b is approximately
200 nm, which corresponds to an approximately 80-mer
protein chain. Remarkably, a cyclic protein assembly struc-
ture is also detected (Figure 2c). This structure could be
formed by self-termination of linear polymerization. The
doughnut-shaped polymer, which has a height of 5 nm, should
comprise over 100 protein units, as estimated from its
diameter (ca. 80 nm).
Scheme 2. Formation of the supramolecular assembly 3/2-apo-cytb562
(H63C).
-
incorporated into the heme pocket within the protein matrix
in a manner similar to that observed for 1-apo-cytb562
-
(H63C).[22] The native-PAGE analysis of 2-apo-cytb562(H63C)
exhibits a protein band distributed over a wide range of
molecular weights, while the cytb562(H63C) mutant without
the external heme has a single protein band at the expected
position (Figure 1a). In contrast, the 2-apo-cytb562(H63C)
protein exhibits a single band by SDS-PAGE analysis
(Figure 1b), indicating that dissociation of the self-assembled
protein cluster can be achieved by incubation with SDS. These
results suggest that 2-apo-cytb562(H63C) forms a large
supramolecular protein assembly through interprotein
heme–heme pocket interactions. The formation of the
supramolecular polymer is also indicated by size-exclusion
chromatography (SEC) (see the Supporting Information).
Figure 2. a)–c) AFM images of 2-apo-cytb562(H63C) assemblies on a
HOPG substrate. d) The height profile along the green line in (c).
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Next, to introduce a branching point into the linear
protein fiber, we have designed and synthesized the heme
triad 3 with a 1,3,5-trisubstituted benzene core (Scheme 1).[27]
The addition of a small amount of the heme triad 3 to the
linear 2-apo-cytb562(H63C) assembly resulted in a dramatic
change in the AFM images. Surprisingly, a premixed solution
of 3 and 2-apo-cytb562(H63C) (molar ratio 1:40) gives rise to
highly detailed network structures on the HOPG substrate
(Figure 3a). The uniform height of the network (Figure 3b,c)
Figure 3. a),b) AFM images of 3/2-apo-cytb562(H63C) assemblies at a
molar ratio of 1:40 on a HOPG substrate. c) The height profile along
the green line in (b).
is clearly indicative of protein monolayer assemblies in the
absence of multiple deposits or unselective aggregations of
protein. Figure 4 shows the morphological changes in the
assembly as a function of the molar ratio of 3 and 2-apo-
cytb562(H63C). In the absence of 2-apo-cytb562(H63C) (Fig-
ure 4a) only point particles were observed resulting from the
aggregation of 3. At a 3/2-apo-cytb562(H63C) molar ratio of
1:1 (Figure 4b), a network was not formed, although several
rodlike objects were evident. At a molar ratio of 1:10
Figure 4. AFM images of 3/2-apo-cytb562(H63C) assemblies at differ-
ent molar ratios: a) 1:0, b) 1:1, c) 1:10, and d) 1:100 on a HOPG
substrate. e) An enlarged image of (d). f) The height profile along the
green line in (d).
(Figure 4c),
a localized protein network structure was
obtained which had many junctions relative to the structure
obtained from a molar ratio of 1:40 (Figure 3). As expected, at
a molar ratio of 1:100, a low-density network was observed
(Figure 4d). An enlarged image of this network is shown in
Figure 4e. The height profile (Figure 4 f) of the structure
formed at the 1:100 molar ratio clearly shows the branched
protein chain structures that have a uniform height corre-
sponding to the monolayer protein assembly. These morpho-
logical changes further indicate that formation of the network
from the 2-apo-cytb562(H63C) protein assembly is induced by
addition of the heme triad 3.
A plausible mechanism for the formation of the network
is shown in Scheme 3. When the heme triad 3 is incorporated
into the 2-apo-cytb562(H63C) linear assemblies, a trident
hemoprotein assembly is first formed. The termini of the
assembly should be the externally attached hemes on the
protein surfaces. The exposed hemes in aqueous solution
associate with each other.[30] Therefore, the protein assembly
further extends and diverges through the interprotein heme–
heme interactions, thereby providing the unique two-dimen-
sional hemoprotein networks.
Scheme 3. A plausible mechanism for the formation of the hemopro-
tein network assembly.
In conclusion, the present methodology for the construc-
tion of a supramolecular hemoprotein polymer using succes-
sive heme–heme pocket interactions should serve as a new
method for creating not only linear protein fibers but also
multidimensional protein networks in the form of uniform-
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[13] To prepare a reaction site for the heme analogues 1 and 2, His63
height monolayers on HOPG substrates. One series of our
was replaced with cysteine, because there is no cysteine residue
AFM studies suggests that the two-dimensional morphology
of protein networks is controlled by the molar ratio of the
protein and the pivot molecule. We believe that such
advanced supramolecular architectures hold promise in the
development of protein-based nanobiomaterials. Further
investigations on the detailed structure and function of
supramolecular hemoprotein polymers in solution and on
substrates are in progress.
in cytb562
.
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[22] The surface-modified hemoprotein 2-apo-cytb562(H63C) was
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[26] The size was estimated from crystal structure of wild-type cytb562
(PDB ID: 1QPU).
Received: August 13, 2008
Revised: October 14, 2008
Published online: December 3, 2008
Keywords: cytochromeb562 · heme proteins ·
.
protein modifications · self-assembly · supramolecular chemistry
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