Effect of Electrostatic Interaction on Fibril Formation of Human
Calcitonin as Studied by High Resolution Solid State 13C
NMR*
Miya
Kamihira
§,
Yuki
Oshiro
,
Satoru
Tuzi
,
Atsuko Y.
Nosaka¶,
Hazime
Saitô
, and
Akira
Naito
**
From the
Department of Life Science, Graduate School
of Science, Himeji Institute of Technology, Harima Science Garden
City, Kamigori, Hyogo 678-1297, Japan, ¶ Niigata University of
Management, Kibogaoka 2909-2, Kamo, Niigata 959-1321, Japan, and
Graduate School of Engineering, Yokohama National
University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
Received for publication, May 29, 2002, and in revised form, October 4, 2002
 |
ABSTRACT |
Fibrillation of a human calcitonin mutant (hCT)
at the position of Asp15 (D15N-hCT) was examined to
reveal the effect of the electrostatic interaction of Asp15
with charged side chains. The secondary structures of fibrils and
soluble monomers in the site-specific 13C-labeled D15N-hCTs
were determined using 13C cross-polarization magic angle
spinning and dipolar decoupled magic angle spinning NMR approaches,
sensitive to detect 13C signals from the fibril and the
soluble monomer, respectively. The local conformations and structures
of D15N-hCT fibrils at pH 7.5 and 3.2 were found to be similar to each
other and those of hCT at pH 3.3 and were interpreted as a mixture of
antiparallel and parallel
-sheets, whereas they were different from
the hCT fibril at pH 7.5 whose structure is proposed to be antiparallel
-sheets. Thus the negatively charged Asp15 in the
hCT molecule turned out to play an essential role in determining the
structures and orientations of the hCT molecules. Fibrillation kinetics
of D15N-hCT was analyzed using a two-step autocatalytic reaction
mechanism. The results indicated that the replacement of
Asp15 with Asn15 did not reduce the rate
constants of the fibril formation but rather increased the rate
constants at neutral pH.
 |
INTRODUCTION |
Calcitonin (CT)1 is a
peptide hormone consisting of 32 amino acid residues, which contains an
intrachain disulfide bridge between Cys1 and
Cys7 and a proline amide at the C terminus. In mammals CT
plays a central role in calcium-phosphorus metabolism as a thyroid
hormone (1-3). In concentrated aqueous solution, human calcitonin
(hCT), however, has a tendency to form a fibril precipitate known as a
disease-associated amyloidosis (Alzheimer's disease, type II diabetes,
Creutzfeldt-Jakob disease, etc.). Resulting fibers of 8 nm in diameter
(4) as revealed by electron microscopic studies on hCT fibrils gave
rise to a common ultrastructure with clinically and biochemically
diverse amyloid fibrils (5-8). Analogous to
-amyloid, calcitonin is
another model peptide associated with medullary carcinoma of the
thyroid (9, 10). However, the detailed molecular mechanism of fibril
formation has not yet been well understood. It is therefore important
to clarify the fibrillation process and mechanism in hCT not only to
contribute to further improvement of aqueous therapeutic formations
with a long term stability2
but also to gain insight into a general picture as to molecular mechanism of amyloid formation.
It has been shown that an amphiphilic
-helical structure is formed
in the central region of hCT in aqueous acidic solution (12).
Subsequently, local conformational transitions from an
-helix to a
-sheet structure at the central region and from a random coil to a
-sheet at the C terminus region were induced simultaneously during
the fibril formation in the acetic acid solution (pH 3.3) (13). We
further noticed that a two-step reaction model can be used to analyze
the fibrillation kinetics by the observation of a certain delay time
followed by a simultaneous decrease and increase from the DD- and
CP-MAS NMR signals, sensitive to detection of 13C NMR
signals from the soluble monomer and fibril component, respectively. The first step is a homogeneous association to form the nucleus of
fibril, and the second step is an autocatalytic heterogeneous fibrillation to mature the fibril; the kinetics parameters for the
first (k1) and second
(k2) steps were determined individually (13). As
a mechanism of the molecular association in the first nucleation
process, it was proposed that the
-helices are bundled together by
hydrophobic interaction among the side chains of the amphiphilic
-helices in the central region of hCT (14).
It was reported that the rate of fibril formation at neutral pH is much
faster than that at acidic pH, because hCT monomer is more stable in an
acidic medium in the absence of salts and buffer than in physiological
saline solution. Local molecular structures and macroscopic features of
the hCT fibrils formed in the sodium acetate solution (pH 7.5) were
different from those at pH 3.3 (13). We proposed that the fibril at pH
7.5 is comprised of antiparallel
-sheets because of the favorable
electrostatic interaction between side chains of Asp15(
)
and Lys18(+) at pH 7.5. On the contrary, the mixture of
antiparallel and parallel
-sheet structures is formed at pH 3.3, because the side chains of Lys18 and His20 and
the amino group in the N terminus were positively charged and there is
no favorable direction among the molecules to associate. Therefore, it
is worthwhile to perform further examination using mutant hCT having
different local charges whether fibril formation of hCT is affected not
only by the hydrophobic interaction but also by the electrostatic
interaction among charged side chains (13).
13C chemical shifts of carbonyl and C
and
C
carbons in amino acid residues obtained from high
resolution solid state 13C NMR spectroscopy have been
established to correlate with local secondary structures of
polypeptides (15, 16). These conformation-dependent 13C chemical shifts were proven to provide reliable
secondary structures for fibrous and membrane proteins (17, 18). Use of
site-specific 13C-labeled carbonyl groups for
Gly10 and Phe22 or methyl carbons at
Ala26 and Ala31 in hCT provided a clue as to
the local conformations at the specific sites over the entire molecule,
together with simultaneous observation of local conformational changes
of hCT from solution to fibril state by the DD-MAS and CP-MAS NMR
spectra, respectively, during the fibril formation (13).
In the present study, we focused on the effect of the electrostatic
interaction between the charged side chains in the fibril formation of
hCT. In the hCT molecule, there is only one amino acid that charges
negatively, Asp15, and there are two amino acids with
positive charges (Lys18 and His20 (see Scheme
I) besides the amino group of N terminus). Therefore an
Asn15-hCT mutant, D15N-hCT, was chemically synthesized to
examine the effect of electrostatic interaction in the structure and
kinetics of the fibril formation. The hydrophilic residue at position
15 of hCT is not required for biological activity (19), with the expectation of a slow fibrillation rate as a prerequisite for a
therapeutic agent. In contrast to this expectation, there is anticipation that the most common type of aging-related damage arising
from the neutralization of aspartate leads to an increased
-sheet
content to cause an increased propensity for fibril formation (20).
Thus the artificial mutation of hCT from Asp15 to
Asn15 can serve as a suitable model of the aging-related
damage in the
-amyloid formation, too.
 |
EXPERIMENTAL PROCEDURES |
Sample Preparation--
We prepared three types of
isotopically labeled D15N-hCT preparations,
I-III (Scheme I)
by chemical synthesis (13). Note that the Pro residue was not contained
for I and II as manifested by matrix-associated
laser desorption time-of-flight mass spectroscopy (MALDI MASS)
analysis. For the 13C solid state NMR experiments, the
lyophilized preparations were dissolved in 15 mM aqueous
acetic acid solution and 5 mM phosphate buffer. Immediately
after D15N-hCTs were dissolved in the solution, a portion of the
solution (80 µl) was placed in a 5-mm outer diameter zirconia rotor
and sealed with Araldite® (Vantico) to prevent evaporation
of the mother liquor throughout the NMR measurements. The same solution
stood in the test tubes at the same temperature as used in the NMR
measurements to check the turbidity or viscosity of the solution by
visual observation.
NMR Measurements--
13C NMR spectra were recorded
on a Chemagnetics CMX 400 NMR spectrometer at the resonance frequency
of 100.6 MHz. The 13C NMR spectra were recorded
alternatively by means of 13C CP-MAS and DD-MAS techniques
because solid fibril and soluble monomer were preferentially recorded
by the former and latter methods, respectively, in view of their
cross-polarization times (
CH) and spin-lattice
relaxation times (T1). The 13C
chemical shifts were calibrated by using the external carboxyl peak of
crystalline glycine at 176.03 ppm from tetramethylsilane. The
lengths of
/2 pulse for the carbon and proton nuclei were 5.0 µs,
and the recycle delay times were 4 and 5 s for the CP- and DD-MAS
studies, respectively. All NMR measurements were performed at 20 °C.
Acquisition of the 13C DD- and CP-MAS NMR spectra was
started at 20 °C after 6 h from dissolution, because a time
delay was necessary to achieve tight enough sealing of the sample rotor
by glue. The number of accumulations for the DD- and CP-MAS signals
were 1000 and 2000, respectively, in the time course studies.
Electron Microscope Observation--
For electron microscopic
analyses, D15N-hCT fibrils grown from the solution at pH 3.2 and 7.5 (80 mg/ml), respectively, at room temperature were diluted with each
buffer solution after 4 h and 3 days, respectively, from the
dissolution and vortexed to reduce their viscosity. 5 µl of the
suspension was placed on a Formvar-covered grid and stained with 5 µl
of uranyl acetate. The negative stained samples were viewed in a JEOL
1200 EX II electron microscope at 80 kV.
Circular Dichroism Measurements--
CD measurements were
performed on an AVIV model 62DS using quartz cuvettes with the path
length of 0.2 cm. CD spectra were recorded in the wavelength range of
250-200 nm. The concentration of D15N-hCT was 0.2 mg/ml (58.5 µM), and 20 mM phosphate buffer (pH 7.2) and
15 mM acetic acid solution (pH 3.3) were used. The temperature was controlled to 25 °C using a thermostatted cell holder throughout the CD measurements.
 |
RESULTS |
Fig. 1 illustrates the electron
micrographs for the two preparations, which clearly indicate the
presence of fibrils from D15N-hCT (II) in pH 7.5 and 3.2 solutions (80 mg/ml). The D15N-hCT fibrils at pH 7.5 (Fig.
1A) were much shorter than those at pH 3.2 (Fig.
1B). Although the distinct differences in the length of the
fibrils were observed between the two samples, the diameters of the
fibrils at pH 7.5 were about 15 nm, which was almost the same as those
at pH 3.2. The fact that fibrils at the neutral condition are shorter
than those at acidic ones is the same as the case of hCT (13).

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Fig. 1.
Electron micrographs of D15N-hCT fibrils (II)
grown in 5 mM phosphate buffer (pH 7.5, 80 mg/ml,
A) and in 15 mM acetic acid solution (pH
3.2, 80 mg/ml, B). The samples were negatively
stained with 2% (w/v) uranyl acetate. The respective scale
bars represent 200 nm.
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The local secondary structures were determined by observing DD-MAS and
CP-MAS 13C NMR spectra in
[1-13C]Phe16,[3-13C]Ala31-labeled
D15N-hCT (I) and
[1-13C]Gly10,[3-13C]Ala26-labeled
D15N-hCT (II) for soluble monomer and fibril, respectively.
In the 13C DD-MAS spectra of I at pH 7.5, the
carbonyl signal of Phe16 at 173.4 ppm and the methyl signal
of Ala31 at 17.2 ppm (Fig.
2A) of liquid-like component
were ascribed to species taking a random coil, with reference to the
conformation-dependent 13C chemical shifts;
-helix, random coil, and
-sheet are 171.6, 170.9, and 168.5 ppm
for [1-13C]Gly, 175.2, 173.2, and 169.0 ppm for
[1-13C]Phe, and 14.9, 16.9, and 20.0 ppm for
[3-13C]Ala residues (15-18). The
[1-13C]Gly10,[3-13C]Ala26,Ala31
signals of I and III at pH 7.5 and 2.6, respectively, observed in DD-MAS spectra are shown in Fig. 2
(A and D) and Fig. 3A, and the 13C
chemical shifts are also summarized in Table
I with their assignments. In the case of
Ala31 of III, we took into account the
additional upfield displacement of the peaks by 1.0 ppm arising from
the carbonyl carbon directly bonded to the amide nitrogen of a Pro
residue (21, 22). Thus, the secondary structures of D15N-hCT monomers
were exactly the same among the preparations at different pH values.
The 13C CP-MAS NMR signals of the fibrils (Fig. 2,
B, C, E, and F, and Fig. 3,
B and C) were relatively broader than the
corresponding 13C DD-MAS NMR signals of the soluble
monomers (Fig. 2, A and D, and Fig.
3A) because of the shorter spin-spin relaxation times and
the conformational heterogeneity. The peak positions of CP-MAS spectra
of the most intense one as the most populated conformation are
summarized in Table I for D15N fibrils (I-III) at pH 7.5 and 3.2 or 2.6 (for III) together with their assignments. Obviously, the conformational transition from the random
coil to
-sheet around Phe16 occurred during the fibril
formation, whereas a part of the
-helix and the random coil changed
to the
-sheet forms around Gly10, Ala26, and
Ala31 residues, respectively. We found that the presence
and absence of Pro32 at D15N-hCT does not affect the change
of secondary structures of monomer and fibril as for the central
region.

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Fig. 2.
13C DD-MAS and CP-MAS
NMR spectra of
[1-13C]Phe16,[3-13C]Ala31-labeled
D15N-hCT (I) (top six traces)
and
[1-13C]Gly10,[3-13C]Ala26-labeled
D15N-hCT (II) (bottom six
traces). A and D, DD-MAS
spectra at pH 7.5; B and E, CP-MAS spectra of
fibril at pH 7.5; C and F, CP-MAS spectra of
fibril at pH 3.3. The peptide concentration was 80 mg/ml.
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Fig. 3.
13C DD-MAS and CP-MAS
NMR spectra of
[1-13C]Gly10,[3-13C]Ala31-labeled
D15N-hCT (III). A, DD-MAS spectra at pH 2.6 (80 mg/ml);
B, CP-MAS spectra of fibril at pH 7.5 (30 mg/ml);
C, CP-MAS spectra of fibril at pH 2.6 (80 mg/ml).
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Table I
13C chemical shifts (ppm from tetramethylsilane) and the
assignment of D15N-hCT monomers (observed in DD-MAS spectra) and
fibrils (observed in CP-MAS spectra)
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The secondary structure of D15N-hCT (III) monomer was also
examined using CD measurements (0.2 mg/ml) at pH 7.2 and 3.3 (Fig.
4). They showed similar spectra as those
of hCT in the wavelength between 200 and 250 nm (23, 24). This means
that the D15N-hCT monomer takes mainly the random coil form as
confirmed by the CD data of the hCT solution (23, 24).

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Fig. 4.
Far-UV circular dichroism spectra of D15N-hCT
(III) in 20 mM phosphate buffer (pH 7.2, A) and 15 mM acetic acid solution (pH 3.3, B). The concentration of D15N-hCT
(III) was 0.2 mg/ml (58.5 µM). The spectra
were measured in the first 5 min from dissolution.
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Fig. 5 shows the time courses of the
variation of the 13C NMR signal intensities at 20 °C for
D15N-hCT (III) with the concentration of 25 mg/ml at pH 2.9, because the fibrils were formed in a few minutes under the higher
concentration (80 mg/ml) at both pH 7.5 and 3.2. The aqueous solution
of III at pH 2.9 became a gel after 8 h from the
dissolution. The Gly10 C=O signal in the 13C
DD-MAS spectra decreases gradually during the first 40 h (Fig. 5A). On the contrary, the Gly10 C=O signal
(170.1 ppm) in the CP-MAS NMR spectra was visible after 20 h from
the dissolution and increased rapidly during the next 20 h. It
turned out that the fibrillation mechanism for D15N-hCT was interpreted
as a two-step reaction model that is the same as that of wild type hCT
(13) on account of the rate constants, k1 and
k2, obtained from the fitting to the data points
(Fig. 5B, Table II).

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Fig. 5.
A, plots of peak intensities for
[1-13C]Gly10 in 13C DD-MAS and
CP-MAS spectra of D15N-hCT (III) at pH 2.9 (25 mg/ml)
against the elapsed time. Acquisition was started after 4.8 h from
dissolution. Closed diamond, DD-MAS signal;
open circle, CP-MAS signal. The time of
dissolution was regarded as 0 h. Acquisition was started after
6 h from dissolution by alternatively accumulating 1000 scans for
DD-MAS and 2000 scans for CP-MAS experiments. B, the same
plot as the intensity of CP-MAS signal (A) presenting the
vertical line with the fraction of fibril after normalizing the
intensity observed 60 h after dissolution as unity.
Dotted line is the best fit to the equation
representing the two-step reaction mechanism (13).
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The time course of the fibril formation of D15N-hCT (III) at
pH 7.5 was also examined by CD measurements. The monomeric component
assigned to random coil (205 nm) was decreased gradually as a result of
fibril formation after 2 h from dissolution (Fig. 6A). The fitting plot is shown
in Fig. 6B, and the rate constants obtained by fitting to
the data points are summarized in Table II. The characters of
k1 and k2 will be
discussed later.

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Fig. 6.
A, time course of CD spectra of D15N-hCT
(III) at pH 7.2 (0.2 mg/ml). B, plot of peak
heights at 205 nm against the elapsed times. Vertical line presents the
normalized fraction of the peak height with that observed after 5 h from dissolution.
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 |
DISCUSSION |
Conformation of D15N-hCT Monomer and Fibril--
It was found that
the D15N-hCT (I, II) monomer forms the local
-helical structure in the vicinity of Gly10 and random
coil around Phe16, Ala26, and Ala31
residues in the neutral and acidic solutions, showing the same structures as those of hCT in view of their
conformation-dependent 13C chemical shift data
(13). The CD spectra of the D15N-hCT (III) solution (Fig. 4)
also showed similarities to that of hCT, which indicated disordered
secondary structure in solution (24, 25). Although the chemical shift
values indicate that the vicinity of Phe16 forms a random
coil, the central region of D15N-hCT monomer might be in equilibrium
between the
-helix structure and random coil in solution as
suggested for hCT that could form partially an
-helix in the central
region (Thr13-Phe19) and undergo exchange with
another structure to show the random coil form (12). The methyl signal
from Ala31 (I) was observed at 17.2 ppm, which
is shifted from the standard value of random coil (16.9 ppm). This
discrepancy indicates that the C terminus may form a turn structure as
suggested by a solution NMR study (12), because the chemical shift is
similar to that of loop structure of bacteriorhodopsin (18).
During fibril formation, it was shown that the local random coil formed
around the Phe16 residue in D15N-hCT was converted into the
-sheet structure (Figs. 2 and 3). Although 13C chemical
shifts of [1-13C]Phe16 can be assigned to
-sheet structure, the deviation from the standard value is 2.7 ppm.
This large discrepancy may be because of the formation of
-turn/
-sheet structure in the Asn17-Thr21
region (26). Because this structure is crucial for bioactivity, it is
expected that the bioactivity of D15N-hCT will be the same as that of
hCT. A part of the
-helical component around Gly10
residue is also changed to the
-sheet structure, although the local
-helical structure remains as the major component around Gly10 in the D15N-hCT fibril. The chemical shifts of
169.7-170.0 ppm and 18.9-19.9 ppm for [1-13C]Gly and
[3-13C]Ala, respectively, in D15N-hCT fibrils are quite
similar to those of silk fibroin (type II), distinguished from the
polymorphic form, silk I (27, 28), and of the native dragline silk
fibroin (29).
Interestingly, two conformations exist also in the C terminus region as
viewed from the 13C NMR peaks of
[3-13C]Ala26 and
[3-13C]Ala31 in the D15N-hCT fibril except
for those of D15N-hCT fibril (III) at pH 7.5. These
conformations are similar to the case of hCT fibrils at pH 3.3 (13).
These results led to a model of the fibril formation of D15N-hCT that
indicates the fibrils form the mixture of antiparallel and parallel
-sheet structures at pH 7.5 and 3.3 as depicted in Fig.
7. It is emphasized that the conformation of D15N-hCT fibrils is almost the same as those grown at pH 3.2 and 7.5 and those of hCT at pH 3.0 except for the existence of the
-helix
component around Gly10 in the D15N-hCT fibrils. These
structures, however, were quite different in the case of hCT at pH 7.5 (13).
Influence of Local Charges to Molecular Structure of the hCT
Fibrils--
Artificially mutated
-amyloid peptides changed from an
ionic side chain to a neutral one have been investigated by means of
electron microscopy, Fourier transform infrared spectroscopy, fiber x-ray diffraction, and NMR spectroscopy (30-32). The previous studies in the formation of amyloid fibrils from the fragments of
amyloid
peptides showed that the charge to charge interactions stabilize the
-sheet conformation and promote the assembly of protofibrils into longer amyloid fibers (30). In contrast to the
-amyloid peptides, hCT has a limited number of ionic charged amino
acids, such as Asp15, Lys18, and
His20, and the present study of the hCT fibril formation
could be served as a more straightforward means to elucidate the effect
of the electrostatic interaction on the formation of amyloid fibril.
The local charges in the hCT and D15N-hCT molecules are as follows: hCT
at pH 7.5: Asp15(
), Lys18(+),
NH3(+);hCT at pH 3.3: Lys18(+),
His20(+), NH3(+); D15N-hCT at pH 7.5:
Lys18(+), NH3(+); D15N-hCT at pH 3.3:
Lys18(+), His20(+), NH3(+). As for
the molecular association, we suggested that hCT fibril at pH 7.5 forms
the antiparallel
-sheet by a favorable electrostatic interaction
between Asp15 and Lys18, in addition to the
hydrophobic interaction among the amphiphilic helices of the core
region of hCT during the nucleation process (13). This kind of
electrostatic interaction is also important for the stabilization of
the coiled-coil structure (11). The hCT fibril formed the mixtures of
antiparallel and parallel
-sheets up to the most C-terminal region
at pH 3.3 because of the absence of an attractive ionic interaction to
determine the direction for molecular association (13). On the other
hand, the molecular structure of D15N-hCT fibril at pH 7.5 was similar
to that of D15N-hCT fibril at pH 3.3. They are also similar to
that of the hCT fibril at pH 3.3 and are quite different from that of
the hCT fibril at pH 7.5. This result clearly shows that the presence of a negative charge at Asp15 determines the
direction to lead the antiparallel
-sheet during molecular
association (Fig. 7).
Effect of Local Charges on Kinetics of hCT
Fibrillation--
Fibril formation of D15N-hCT also showed the same
two-step autocatalytic reaction mechanism as hCT. In the reaction, we
can compare the rates for samples of one concentration with those of
different concentrations, because the rate constant
k1 (s
1) is independent of sample
concentration and ak2 (s
1) is the
comparative value where a is the initial sample
concentration (13). The rate constant, k1,
obtained for D15N-hCT in the acidic condition showed a smaller value by
5-fold as compared with that of hCT at pH 3.3, although the
k2 values are comparable
(k1 = 3.28 × 10
6
s
1; ak2 = 4.10 × 10
5 s
1 for hCT) (13). Interestingly, both
k1 and k2 values of
D15N-hCT at neutral condition are larger than those of hCT at pH 7.5 (k1 = 2.79 × 10
6
s
1, ak2 = 1.34 × 10
4 s
1) (13).
Fig. 8A summarizes the local
and the net charges in the central core region of hCT and D15N-hCT at
various pH values as compared with their rate constants (Table II)
(13). The fraction of charged groups is obtained from the
Henderson-Hasselbalch equation for pH using the pKa
values of free amino acids and summarized in Fig. 8A.
Apparently, smaller net charges of Lys18 and
His20 side chains (Net*) lead to relatively
larger k2 values. This observation can be
rationalized by considering that the side chains of Lys18
and His20 are standing in the neighborhood of the
-strand structure. It is emphasized from this analysis that this
arrangement of two positive charges in one side prevent monomers from
associating with the
-sheet (Fig. 8B) because of
repulsive electrostatic interaction. On the other hand, the order of
k1 values is not correlated to the net charge of
the side chains but rather related to the hydrophobic nature of the
side chains. It is interesting to note that the absence of the negative
charge at the Asn15 does not reduce both the
k1 and k2 values. It is
therefore concluded that the rate of fibril formation does not depend
on the favorable electrostatic interaction among the charged side
chains. It is stressed that the mutation from Asp15 to
Asn15 causes even faster fibril formation at neutral pH.
The result supports the evidence that aging-related damage accompanied
by such mutation to Asn leads to faster fibril formation (20). It is
suggested that the propensity and stability for
-sheet formation
increase by the mutation from Asp to Asn.

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Fig. 8.
A, local charges in the central core
region of hCT and D15N-hCT at various pH values with the respective and
net charges (Net). Net charges for Lys and His
(Net*) are also shown. The sign of inequalities indicates
the order of rate constants summarized in Table II. B,
-strands in the central core region of hCT and D15N-hCT
(Leu9-Phe22).
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 |
CONCLUSION |
We demonstrate by using D15N-hCT that absence of the negatively
charged group in hCT leads to the fibril structure as a mixture of
parallel and antiparallel
-sheets. This result demonstrates that the favorable electrostatic interaction among the charged side chains of Asp15, Lys18, and
His20 residues or amide nitrogen in hCT plays an important
role to determine the direction of the molecular association to form
the antiparallel
-sheet structure. It was found that the absence of
a negative charge at Asp15 does not reduce the ability of
fibril formation, as D15N-hCTs form the amyloid fibrils even faster
than hCT does. On the contrary, positively charged side chains of
Lys18 and His20 residues standing near the
-strand structure delay the maturation of the fibril formation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. S. Sonobe, T. Shimmen, S. Kimura, and T. Iyanagi of Himeji Institute of Technology for help and
advice in the electron microscopy and the CD measurements.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-aid for
Scientific Research 11694096 and 12034217 from the Ministry of
Education, Science, Culture and Sports of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a research fellowship for young scientists from the
Japan Society for the Promotion of Science.
**
To whom correspondence should be addressed: Graduate School of
Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku,
Yokohama 240-8501, Japan. Tel.: 81-45-339-4232; Fax: 81-45-339-4251;
E-mail: naito@ynu.ac.jp.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M205285200
2
T. Arvinte and K. Ryman, European patent
application, publication number 0490549A (1992).
 |
ABBREVIATIONS |
The abbreviations used are:
CT, calcitonin;
hCT, human calcitonin;
D15N-hCT, hCT mutant substituted from
Asp15 to Asn15;
CP-MAS, cross-polarization
magic angle spinning;
DD-MAS, dipolar decoupled magic angle
spinning.
 |
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