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Self-Organization of Oriented Calcium CarbonatePolymer Composites Effects of a Matrix Peptide Isolated from the Exoskeleton of a Crayfish.

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DOI: 10.1002/anie.200503800
Self-Organization of Oriented Calcium Carbonate/Polymer Composites: Effects of a Matrix
Peptide Isolated from the Exoskeleton of a
The exoskeletons of crustaceans[10] are fascinating biominerals in the view of materials science, because they are
ductile, tough, and lightweight. The exoskeleton of crayfish
mainly consists of frameworks of a-chitin/protein microfibrils
and calcium carbonate that is closely associated with the
fibrils. Figure 1 a shows the scanning electron microscopy
Ayae Sugawara, Tatsuya Nishimura, Yuya Yamamoto,
Hirotaka Inoue, Hiromichi Nagasawa, and
Takashi Kato*
Biological minerals are inorganic/organic composites that
function as structural supports, protection, and mineral
stores.[1] The cooperation of biomacromolecules such as
proteins and polysaccharides plays a determining role in
morphological control in the formation of biominerals.[1] A
number of in vitro experiments for the nucleation and growth
of biominerals demonstrated that their morphologies are
influenced by the extracted organic biomacromolecules of
bone and dentin,[2] mollusk shells,[3] skeletons,[4] and larval
spicules[5] of sea urchin, ascidian spicules,[6] and coralline alga
skeletons.[7] However, studies on individual proteins are
limited because of difficulties in the isolation and purification
of these biomacromolecules.[3g] An understanding of the
structure–function relationships of individual proteins for
biomineralization would offer not only deeper insight into
biomineralization, but also ideas for the design of novel
inorganic/organic composite materials.[1a, 8] Our strategy is to
develop new inorganic/organic composites that exhibit controlled morphologies and properties. We have prepared thinfilm CaCO3 crystals by using a combination of polymer thin
films and simpler acidic polymers such as poly(acrylic acid)
and poly(glutamic acid).[9] For further control of the morphologies of such composite materials, more careful design of
the organic matrix is essential.
[*] Dr. A. Sugawara, Dr. T. Nishimura, Y. Yamamoto, Prof. Dr. T. Kato
Department of Chemistry and Biotechnology
School of Engineering
The University of Tokyo
Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)
Fax: (+ 81) 3-5841-8661
E-mail: [email protected]
Dr. H. Inoue, Prof. Dr. H. Nagasawa
Department of Applied Biological Chemistry
Graduate School of Agricultural and Life Sciences
The University of Tokyo
Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)
[**] This study was partially supported by a Grant-in-Aid for the 21st
Century COE Program for Frontiers in Fundamental Chemistry (T.K.
and A.S.) and for Exploratory Research (17655048; T.K.) from the
Ministry of Education, Culture, Sports, Science, and Technology.
T.N. expresses thanks for a JSPS Research Fellowship for Young
Scientists (No. 10624).
Supporting information for this article is available on the WWW
under or from the author.
Figure 1. a) Photograph of Procambarus clarkii and SEM image of the
fractured surface of its exoskeleton. b) Amino acid sequence of CAP-1.
pS = phosphoserine.
(SEM) image of a fracture surface of the exoskeleton of a
crayfish, Procambarus clarkii. X-ray studies revealed that
both calcite and amorphous calcium carbonate are present in
crayfish exoskeletons.[10a] If we could prepare biomineralization-inspired materials with self-organized structures similar
to that of the exoskeleton, new soft composite synthetic
materials that are environmentally friendly would be
obtained. For this purpose, it is important to examine the
role of natural peptides in the formation of biominerals.
In 2001, Inoue et al.[11a] isolated a peptide named calcification-associated peptide (CAP-1) from the exoskeleton of a
crayfish (Figure 1 a). They showed that CAP-1 has calciumbinding ability and it inhibits the precipitation of calcium
carbonate. They also showed that CAP-1 interacts with
chitin.[11] CAP-1 is expressed in the epidermal tissue during
the postmolt stage where and when calcification takes
place,[12] which indicates that CAP-1 is involved in the
formation of the exoskeleton. CAP-1 has three specific
features (Figure 1 b): 1) it is a highly acidic peptide—in
particular, it has a hexameric aspartic acid sequence in the
C-terminal section; 2) it has a “Rebers–Riddiford (R&R)
consensus sequence”, which is conserved in cuticle proteins of
many arthropods[13]—it was postulated that this motif might
be involved in the protein–chitin interaction;[14] and 3) it has a
phosphoserine residue at the 70th position.
Herein, we report the formation of oriented thin-film
crystals of calcium carbonate by the specific combination of
chitin and CAP-1 as the matrix for crystallization. We also
describe the effects of the recombinant peptide rCAP-1
(Scheme 1 a) and synthetic peptides (Scheme 1 b and c) on the
morphologies of calcium carbonate. These peptides have
been designed and prepared based on the chemical structure
of CAP-1 to understand the specific function of organic
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 2876 –2879
Scheme 1. a) Amino acid sequence of the recombinant peptide rCAP-1.
Red circles indicate the amino acids that are different from those of
CAP-1. b, c) Chemical structures of the synthetic peptides pSSED6 and
The CaCO3 crystallization from a supersaturated solution
of calcium carbonate was conducted on a chitin substrate in
the presence of CAP-1 for 20 hours. The chitin matrix for the
crystallization was prepared by spin-coating the solution onto
glass substrates. SEM observation of the matrix revealed that
microfibrils of about 10-nm diameter are formed (see
Supporting Information). The X-ray diffraction (XRD)
pattern of the matrix shows that its crystalline structure is
assigned to a-chitin, the same crystalline form as that in the
exoskeleton of the crayfish (see Supporting Information).[15]
In the absence of CAP-1, rhombohedral calcite crystals form
on the chitin matrix, which shows that the matrix alone has no
significant effect on the crystallization of calcium carbonate.
In the presence of 3.0 C 10 3 wt % of CAP-1, thin crystalline
films of calcium carbonate grow on the chitin matrix. The size
of these crystals is about 10 mm and the thickness is about
1 mm (Figure 2 a). The detailed observation of these crystalline films by SEM has clarified that they are composed of
assemblies of nanocrystals (Figure 2 b). The crystals intimately associate with chitin fibrils through CAP-1, and form
calcium carbonate/chitin nanocomposites.
Observations by polarizing optical microscopy revealed
that the chitin surface is not fully covered with CaCO3 thin
films (Figure 2 c). This observation suggests that CAP-1 has
an inhibitory effect on CaCO3 crystallization. Figure 2 c shows
that when the samples are rotated under crossed polarizers at
458, the optical images of some crystals change from bright to
dark. This switching is observed for all crystals on 458 rotation
from a proper angle. The fact that such optical change is
observed for all of these crystals indicates that each film is
uniaxially oriented (Figure 2 c). The direction of the orientation of each film is random on the chitin matrix. Transmission
electron microscopy (TEM) and electron diffraction studies
of these films showed that they have single-crystallographic
orientation such that the c axis is unidirectionally oriented in
these calcite films (Figure 2 d and e). The same electron
diffraction pattern was observed for the whole of one calcite
film (see Supporting Information).
To understand the formation process of the unidirectionally oriented calcite films, we performed a real-time observation of calcium carbonate deposition by using polarizing
optical microscopy under crossed polarizers. About 5 minutes
Angew. Chem. Int. Ed. 2006, 45, 2876 –2879
Figure 2. a) SEM image of CaCO3 crystals grown on a chitin matrix in
the presence of CAP-1 (3.0 E 10 3 wt %). b) Magnified image of the
crystal surface in (a). c) Polarizing optical microscopic images of the
uniaxially oriented crystals. The right-hand image is a 458 rotation of
the sample in the image on the left. d) TEM image and e) the
corresponding selected-area electron diffraction image of the uniaxially
oriented crystal grown on a chitin matrix in the presence of CAP-1.
after mixing the solutions of CAP-1 and calcium carbonate,
oriented crystalline films 10 mm in size suddenly appeared on
the chitin matrix. No further growth was observed during
incubation for 20 hours. The sudden appearance of the
crystalline films can be attributed to the transformation
from the amorphous to the crystalline state of calcium
carbonate. It is expected that CAP-1, which was isolated
from the exoskeleton of crayfish containing amorphous
calcium carbonate (ACC), can interact with the ACC and
lead to its stabilization.[5, 6, 16]
We consider the process of formation of the unidirectionally oriented crystals over 10 mm in size to be as follows (see
Supporting Information). ACC interacting with CAP-1 may
initially form on the chitin matrix. Then nucleation starts on
the nanoscale region of the ACC on the oriented crystalline
chitin fibril that is 10 nm in width. This transformation might
be induced by the aligned functional groups of CAP-1 that
bind to the chitin fibril with the R&R consensus sequence. For
cuticle proteins of insects containing this sequence, it was
proposed that they bind to a-chitin with the R&R consensus
sequence, which adopts an antiparallel b-pleated sheet
conformation.[14] Amorphous-to-crystalline transformation
of calcium carbonate has often been observed in biominer-
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
alization[16] and in crystal growth in biologically inspired
systems.[17a, 18] Our assumption on the formation process is also
supported by some work on single-crystalline films obtained
through ACC precursors.[17, 18]
One of the characteristics of the chemical structure of
CAP-1 is the phosphoserine residue at the 70th position. To
examine the role of this residue, we performed CaCO3
crystallization in the presence of rCAP-1 in which the
phosphate group is absent from the 70th position (Scheme 1 a). rCAP-1 was expressed in Escherichia coli cells as
reported previously.[12] Figure 3 a shows the SEM image of the
Figure 3. a) SEM image of CaCO3 crystals grown on a chitin matrix in
the presence of rCAP-1. b) Magnified image of the crystal surface in
CaCO3 crystals formed on the chitin matrix in the presence of
rCAP-1 (3.0 C 10 3 wt %). Although the surface of the crystals
is not as flat as those obtained in the presence of CAP-1,
uniaxially oriented crystals are also formed in the presence of
rCAP-1 (see Supporting Information). Figure 3 b reveals that
the surface of the oriented crystals is composed of an
assembly of block-shaped calcite crystals about 0.2 mm in
size, unlike the crystals formed in the presence of CAP-1
which are finer (tens of nanometers; Figure 2 b). Phosphate
groups at serine residues were reported to have calciumbinding ability.[19] CAP-1 may also bind to calcium ions more
strongly than rCAP-1 because of the existence of the
phosphate group, which leads to disturbance of the crystal
growth and to the stabilization of the amorphous state. Such
effects of CAP-1 on the crystallization may give rise to the
smaller size of crystals.
To examine the effects of the size of acidic molecules and
of the R&R consensus sequence on the crystallization, we
prepared peptides pSSED6 and S2ED6 (Scheme 1 b) by solidphase synthesis. These synthetic peptides are the C-terminal
acidic domains of CAP-1 and rCAP-1, respectively. No thinfilm crystals were obtained by using these synthetic peptides
(Figure 4). Half-dome structures are formed in the presence
of pSSED6 (Figure 4 a), while peanut-shaped crystals are
observed with S2ED6 (Figure 4 c). Crystallographic orientation is not unidirectional for these crystals, while oriented
crystals are obtained in the presence of CAP-1 and rCAP-1.
These results demonstrate that the R&R consensus sequence
interacts with chitin and affects the crystallographic orientation of CaCO3 on the chitin matrix. The phosphate group of
pSSED6 also exerts effects on the surface morphology of the
crystals. The size of the block-shaped crystals formed with
Figure 4. SEM images of CaCO3 crystals grown on chitin matrices in
the presence of the synthetic peptides a, b) pSSED6 and c, d) S2ED6.
b, d) Magnified images of the crystal surface in (a) and (c), respectively.
pSSED6 is much smaller than that of the crystals formed with
S2ED6, which has no phosphate group (Figure 4 b and d). The
observation that no thin films are formed in the presence of
the shorter peptides reveals that longer chains of acidic
structures may be needed for thin-film formation of CaCO3
In conclusion, we have succeeded in preparing uniaxially
oriented CaCO3 thin-film crystals on chitin matrices by using
a natural peptide (CAP-1) isolated from the exoskeleton of a
crayfish. The peptide exhibits several functions: 1) deposition
of CaCO3 on the surface; 2) arrangement of its acidic groups
by specific interaction with chitin; and 3) stabilization of
amorphous CaCO3. The combination of these functions has
led to the formation of the uniaxially oriented CaCO3 films.
The structure–function relationship of CAP-1 has been
examined by comparison with the functions of analogous
synthetic peptides. These results have provided us with
important information on the design of organic matrices for
the preparation of inorganic/organic hybrid materials that
exhibit highly controlled morphologies and significant properties.
Received: October 27, 2005
Revised: January 24, 2006
Keywords: biomineralization · calcium · crystal growth ·
materials science · peptides
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crayfish, effect, matrix, exoskeleton, self, isolated, compositum, calcium, organization, oriented, peptide, carbonatepolymer
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