Biomedical Engineering Reference
In-Depth Information
knowledge of the structure-function relationship. Since current knowledge on
the structure-function relationship of proteins is incomplete, it is more practical
to take a combinatorial approach [
68
-
70
]. Using a motif programming method
[
71
], Tsuji et al. generated a combinatorial library of proteins containing peptides
ESQES and QESQSEQDS in various numbers and different orders. Eighteen
genes were synthesized in vitro, expressed in
E. coli
, and purified using affinity
chromatography. Proteins with the ability to accelerate HAP crystal formation
without immobilization were then identified. A calcium phosphate solution with an
adjusted pH of 7.4 did not produce deposits within a 10-day reaction time. Adding
1.6
g/mL of artificial protein (#64) led to the formation of spherical aggregates
of OCP within 5 days. In crystallization experiments using supersaturated solution
with a high pH (8.0), #64 protein accelerated HAP crystal formation. The process of
HAP crystal formation in the presence of #64 protein was analyzed using TR-SLS
measurements [
66
].
As a control, a crystallization experiment was performed in the absence of the
protein. Upon mixing of phosphate and calcium solutions, particles with a gyration
radius of 350 nm soon formed. The fractal dimension of the initial phase was small
( 1.8). TEM analysis showed that the particles had a rounded morphology, and only a
diffusive ring was observed by selected area electron diffraction (SAED), indicating
that the initial particles were ACP. The initial state was maintained for 90 min.
After additional time had elapsed, the apparent molecular mass, gyration radius, and
fractal dimension increased simultaneously, and HAP crystal formation occurred. In
the presence of protein #64 (1
g/mL), the initial particles were ACP with a gyration
radius of 350 nm, similar to that of those obtained in the absence of the protein. In
contrast to a situation on a control experiment, the apparent molecular mass and
fractal dimension increased after 80 min, and HAP crystal formation was achieved.
It is important to note the differences in the transformation mechanisms. While
the apparent molecular mass, gyration radius, and fractal dimension increased
simultaneously in the absence of the protein, only the apparent molecular mass
and fractal dimension increased and the gyration radius remained unchanged in the
presence of the protein (Fig.
4.3
). This indicates that, in the presence of the protein,
ACP particles are transformed into crystals without a change in their size. In other
words, the rearrangement of atoms or clusters takes place within the ACP particles
during their transformation to HAP crystals. This transformation mechanism is
known as direct transformation [
67
]. During the transformation process of ACP
to HAP, artificial proteins containing peptide sequences derived from DMP1, as
designed by the motif programming method, accelerate HAP crystal formation by
altering the mode of the transformation to direct transformation [
66
].
Is direct transformation actually taking place inside the body? Beniash et
al. studied enamel formation in mouse incisor [
11
]. They found that different
developmental stages exist in different areas simultaneously, so it is suitable to
identify the mineral phase at each developmental stage. When enamel was stained
with toluidine blue dye, the staining pattern of the immature outer enamel differed
