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INTRODUCTION |
Amyloidoses are protein misfolding disorders in which soluble
proteins aggregate to form insoluble fibrils, accumulation of which has
been implicated in several diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases, and systemic
amyloidoses (1, 2). Numerous in vitro studies have
supported the thesis that partially folded protein molecules are the
precursors for the nucleation and growth of amyloid fibrils (1, 3),
even though the detailed molecular mechanism underlying fibril
formation is not well defined. However, it is well established that
thermodynamic and conformational stability of native proteins, which
are determined by intrinsic or extrinsic factors, are inversely related
to their propensity to form amyloid fibrils (4-7). Therefore
substantial efforts have been made to discover ligands (which could be
used to treat or prevent amyloidoses) that can strongly bind to and stabilize the fully folded, native state of proteins, resulting in
inhibition of fibril formation (e.g. 8).
Congo red (CR)1 has been the
subject of several studies investigating its effects on the formation
of fibrils (e.g. 9-13) and is often used diagnostically as
a stain for amyloid fibrils (2). CR is a symmetrical sulfonated azodye
with a hydrophobic center consisting of a biphenyl group spaced between
the negatively charged sulfate groups. CR binds to amyloid fibrils to
induce a characteristic shift in maximal absorbance from ~490 to
~540 nm and an enhanced apple-green birefringence and dichroism under
polarized light (14-16). The binding mechanism has been proposed to be
intercalation of CR molecules between the antiparallel
-pleated
sheets of amyloid fibrils by aromatic stacking (14, 15, 17, 18) or
alignment of CR molecules along the fibril axes by electrostatic
interactions between the negatively charged sulfate groups of CR and
the positively charged amino acid residues of proteins (16, 19).
CR has been reported to stabilize A
monomer and to inhibit its
oligomerization (20), to inhibit the structural conversion of normal
prion protein into its aggregation-competent pathogenic form (9, 10),
and to reduce A
-amyloid neurotoxicity by binding to preformed
fibrils (21). Because of these findings, CR and its analogs have been
screened as potential therapeutic inhibitors of amyloid fibril
formation (11, 12). However, although CR at high concentrations
inhibited amyloid fibril formation by A
-peptides (13) and prion
proteins (12), at lower levels CR accelerated fibril formation. Also,
the binding of CR to some proteins induces formation of soluble
oligomers (22, 23), which could be precursors of fibrils. Thus, even
the phenomenological effects of CR on protein aggregation and
fibrillogenesis appear to be highly variable, and the mechanism(s)
underlying these differences have not been determined.
There have been studies that document CR binding to native or partially
folded states of several different proteins, regardless of their
secondary structures (9, 14, 22-28). However, there have not been any
detailed reports on the effects of CR binding on protein conformation
nor on the potential roles that binding-induced structural changes
might play in modulation of protein fibrillization by CR. In
addition, the driving forces of CR binding to proteins remain
controversial, as to whether the binding is caused by hydrophobic interactions, electrostatic interactions, or both (18, 23, 27, 28).
Recently we showed that CR enhanced amyloid fibril formation by an
amyloidogenic immunoglobulin light chain variable domain (VL), SMA, under high pressure (29). In the present study,
to gain further insight into the mechanism(s) for CR effects on amyloid fibril formation, we characterized (at atmospheric pressure) the aggregation kinetics, conformational changes, and thermal stability of
SMA with a range of molar ratios of CR. In addition, we used isothermal
titration calorimetry to study the thermodynamics of CR binding to SMA.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant VL SMA was expressed in
Escherichia coli and purified by the methods of
Wilkins-Stevens et al. (30) and Raffen et al. (5)
with the following modifications. Instead of 5-ml prepacked Econo-Pac Q
and S cartridges, High Q and S resins (Bio-Rad) were packed in 1 × 50-cm glass columns (Bio-Rad). Fractions were eluted with an 8×
volume (400 ml), 0-900 mM NaCl gradient, collected, and
assayed by SDS-PAGE. The fractions containing the protein were pooled
and concentrated to 15-20 mg/ml with a stirred ultracentrifuge cell
(Amicon) and a YM3 membrane (Amicon). All purification procedures were
performed at 4 °C. The purity of the protein exceeded 99% based on
SDS-PAGE analysis. The purified protein was stored at 4 °C in 10 mM potassium phosphate, pH 7.4, plus 100 mM
NaCl buffer (5, 7, 30). The SMA concentration was estimated using an
extinction coefficient of 1.71 mg ml
1 cm
1
at 280 nm (30). Recombinant SMA has the same sequence as the VL that originated from lymph node-derived amyloid fibrils
of a patient with immunoglobulin light chain-related amyloidosis (30).
SMA is a homodimer with a dimerization constant of
7 × 105 M
1 (0.054 mg/ml) (30).
The SMA molar concentration was calculated using a dimer molecular
mass of 25.5 kDa because the protein dominantly exists as a
dimer under the experimental concentrations used in the present study.
Throughout the experiments, 10 mM potassium phosphate, pH
7.4, plus 100 mM NaCl buffer ("buffer") was used. CR
(Sigma C6767) solutions were prepared daily in buffer and filtered
three times using a 0.45-µm pore size polysulfone filter (Whatman)
(29). The CR concentration was determined using an extinction
coefficient of 5.93 × 104 M
cm
1 at 505 nm after dilution into 40% (v/v) ethanol
water (16). Other chemicals were of reagent or higher grade.
Aggregation of SMA with Various Molar Ratios of CR with
Agitation--
Solutions of 40 µM (1 mg/ml) SMA with
various molar ratios of CR (r = [CR]/[SMA dimer]),
i.e. r = 0, 0.3, 1.3, 4.8, and 8.8, were
incubated in buffer, containing 0.05% NaN3, at 37 °C
with agitation at 250 rpm in an orbital shaker (6, 7). After centrifugation of the samples (14,000 rpm for 5 min), the supernatants were analyzed by size-exclusion high performance liquid chromatography (SEC). The supernatants of the samples were also analyzed on reducing SDS-PAGE with a 12% Tris-HCl ready gel (Bio-Rad).
Some samples were analyzed with SEC coupled with a triple detector
(model 300TDA Detectors, Viscotek Co.), which monitors light
scattering, refractive index, and intrinsic viscosity of proteins. Ovalbumin (Sigma A2512A, grade VI) was used for the calibration of the refractive index and light-scattering detectors and
has a molecular mass of 42.7 kDa and refractive index increment with
protein concentration (dn/dc) of 0.185 ml/g (31). Chromatograms were
analyzed by the TriSEC 3.0 GPC software (Viscotek Co.) to determine
molecular mass, radius of gyration (Rg),
hydrodynamic radius (Rh), and intrinsic
viscosity of the samples.
Circular Dichroism (CD) Spectroscopy--
CD spectroscopy was
performed with an Aviv 62DS spectrometer (Aviv) equipped with a Peltier
temperature control unit. Far-UV CD spectra (190-260 nm) for 117 µM (2.97 mg/ml) SMA with various molar ratios of CR
(r = 0, 1.2, 2.3, 4.8, and 8.8) were collected every
0.5 nm with an averaging time of 5 s at 25 °C in a 0.01-cm path
length quartz cuvette. Near-UV CD spectra (260-340 nm) for 40 µM (1 mg/ml) SMA with various molar ratios of CR
(r = 0, 1.3, 4.8, and 8.8) were collected every 0.5 nm
with an averaging time of 5 s at 25 °C in a 0.1-cm path length
quartz cuvette. The far- and near-UV CD spectra were collected
immediately after mixing the samples.
To monitor protein aggregation directly in the sample cell, far-UV CD
spectra of SMA (117 µM = 2.97 mg/ml) with various molar ratios of CR (r = 0, 1.3, 4.8, and 8.8) were acquired
as described above during isothermal incubations at 37 °C (up to 140 min at 20-min intervals) and during heating from 25 to 60 °C at
5 °C intervals, with a dwell time of
25 min at each temperature.
For the samples incubated isothermally at 37 °C for 140 min in the CD cell, the induced-CR CD spectrum (400-650 nm) was acquired at
37 °C every 1 nm with an averaging time of 5 s (14, 24, 25).
The induced-CR CD spectrum (400-650 nm) was also collected at 37 °C
for 40 µM native SMA and preformed amyloid fibrils of SMA
in the presence of 160 µM CR.
For far- and near-UV CD spectra, raw spectra were corrected for the
appropriate buffer blank and converted to mean residue ellipticity,
[
] (deg cm2/dmol) (32),
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(Eq. 1)
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where
obs is observed ellipticity in
millidegrees, Mo is the mean residue molecular
mass calculated from a molecular mass of 12.7 kDa and 108 residues
(30), c is the protein concentration in mg/ml, and
l is the path length of the quartz cell in cm.
Thermal unfolding of SMA (4 µM = 0.1 mg/ml) with various
molar ratios of CR (r = 0, 1.3, 4.8, and 8.8) was
studied by measuring ellipticity at 217 nm during heating from 25 to
80 °C at 1 °C intervals in a 0.1-cm path length cell with an
averaging time of 5 s. Values for the midpoint of the transition
region, Tm, for the unfolding curve for each
sample were obtained as described before (6, 7).
Fourier Transform Infrared (FT-IR) Spectroscopy--
IR spectra
were acquired with a Bomem MB-104 FT-IR spectrometer equipped with a
dTGS detector (6, 7). The spectra for 0.7 mM (16 mg/ml) SMA
with various molar ratios of CR (r = 0, 1.3, and 2.8)
were recorded at 25 °C in a liquid IR cell (P/N 20500, Specac Inc.) with CaF2 windows separated by a 6-µm Mylar spacer (Chemplex Industries) (6, 7). Reference spectra for the
appropriate buffer blank were recorded under identical scan conditions
for the subtraction from the protein solution spectrum. Protein spectra
were processed and analyzed as described previously (6, 7).
To monitor protein aggregation, IR spectra for 0.7 mM (16 mg/ml) SMA with various molar ratios of CR (r = 0, 1.3, and 2.8) were recorded at 37 °C as a function of time. The
temperature was controlled by circulating ethylene glycol around a
custom made IR cell holder and was measured by a thermal couple probe, inserted into a depression in the cell. Protein aggregation was monitored by following band intensity at 1627.8 cm
1 in
the second derivative amide I spectra, which is indicative of
intermolecular
-sheet (33, 34).
Hydrogen-deuterium exchange (HX) was initiated by mixing 1 volume of
protein stock solution (1.6 mM = 40 mg/ml in
H2O buffer) with 3 volumes of 100% D2O buffer
containing an appropriate CR concentration to give a final protein
concentration of 0.4 mM (10 mg/ml) SMA with various molar
ratios of CR (r = 0, 0.02, 1.3, and 1.9) in 75%
D2O buffer. Samples were immediately placed in a liquid IR
cell (P/N20500, Specac) with CaF2 windows separated by a
12-µm polypropylene spacer (Chemplex Industries). A
time-dependent series of spectra was acquired at 25 °C.
The time from the sample preparation to acquisition of the first
spectrum was about 90 s. HX was monitored by the shift in the
frequency of the second derivative amide I band, which was at 1,639 cm
1 in spectra for the proteins in H2O, as a
function of the length of time of exposure to 75% D2O
buffer (6).
ThT Fluorescence Assay and Characterization of SMA
Aggregates--
The ThT fluorescence assay for the samples without CR
was performed as described previously (6, 7). Samples with CR could not
be analyzed by this method because of the absorbance of CR in the
spectral region used for excitation of ThT. IR spectra were obtained to
determine the secondary structure of the insoluble protein aggregates
formed in the various conditions (6, 7). CR birefringence under
crossed-polarization microscopy and transmission electron microscopy
(TEM) of precipitated proteins were performed as described previously
(6, 7). Precipitates formed without CR were stained with CR as
described previously (29).
Isothermal Titration Calorimetry (ITC)--
The thermodynamics
of CR binding to SMA were measured using an isothermal titration
microcalorimeter (VP-ITC, MicroCal Inc.) (35). The samples were
thoroughly degassed before each titration. The experiments were
performed at 25 °C with 20.1 µM (0.5 mg/ml) SMA in the
sample cell and 760 µM CR in the titrant syringe, at 25 °C with 32.3 µM (0.8 mg/ml) SMA and 1,036 µM CR, and at 30 °C with 20.1 µM (0.5 mg/ml) SMA and 760 µM CR. The sample in the cell was
stirred at 300 rpm by the syringe, and 10 µl of titrant was delivered
over 20 s with 180-s intervals between injections to allow
complete equilibration. The data were collected automatically and
subsequently analyzed by Origin software from MicroCal, Inc. Before the
curve-fitting process, a background titration, consisting of the
identical CR solution but only the buffer solution in the sample cell,
was subtracted from each experimental titration to account for the heat
of dilution (35). From the nonconstrained fitting to the plot of heat
evolved/mol of CR injected versus the molar ratio of CR to
SMA dimer, the binding stoichiometry (n), the binding
enthalpy (
H), and the dissociation constant (Kd) were determined (35). The Gibbs free energy
of binding (
G) was calculated by
G =
RTln(1/Kd). The entropy of
binding (T
S) was calculated by
T
S =
H
G.
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RESULTS |
Effects of CR on Aggregation of SMA during Incubation at 37 °C
with Agitation--
Solutions of 40 µM (1 mg/ml) SMA
with various molar ratios of CR (r = 0, 0.3, 1.3, 4.8, and 8.8) were incubated at 37 °C with agitation. The amount of
soluble SMA remaining in solution was measured with SEC, as a function
of incubation time (Fig. 1). Without CR,
there was a lag phase during which soluble SMA levels did not decrease.
After 3 days of incubation, there was a progressive loss of SMA from
solution, with a concomitant increase of ThT fluorescence (Fig. 1).
These results are characteristic of nucleation-dependent amyloid fibril formation kinetics (7). In the presence of CR, there was
no apparent lag phase, and loss of soluble SMA was much faster, with
the rate of loss increasing with increasing molar ratios (r)
(Fig. 1). The SEC chromatograms showed no soluble oligomers for the
samples with r = 0, 0.3, and 1.3 during the
incubations.

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Fig. 1.
Effects of CR on the levels of soluble native
dimer (A) and non-native dimer species (B) of SMA
during incubation at 37 °C with agitation in the presence of CR at
r = 0 ( , ), 0.3 ( , ), 1.3 ( , ), 4.8 ( ,
), and 8.8 ( , ). Data are shown as the percentage of the
peak area in the SEC chromatogram relative to that for an unincubated
control sample of 40 µM SMA dimer without CR. Also shown
in A is the ThT fluorescence ( ) for the SMA sample
incubated without CR. The inset in B represents a
light scattering signal for SMA samples incubated with CR at
r = 8.8 for 0, 1, 3.5, and 10 h. C,
reducing SDS-PAGE for supernatants of the samples incubated for 10 h (lanes 1-4) and 20 h (lanes 5-8).
Lanes 1 and 5, CR at r = 0;
lanes 2 and 6, CR at r = 1.3;
lanes 3 and 7, CR at r = 4.8;
lanes 4 and 8, CR at r = 8.8. Errors bars indicate the S.D. for triplicate samples.
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Interestingly, in the chromatograms for samples with CR at
r = 4.8 and 8.8, a new peak appeared before the elution
of native dimer in samples incubated for
30 min (Fig. 1B,
inset). To characterize further the protein molecules
eluting at this time point, the SEC eluate was analyzed with Viscotek
triple detectors that have light-scattering, refractive index, and
viscosity instrumentation (31). The molecular mass values of the
molecules in the early eluting and the native dimer peaks both were the
same as that calculated for the native dimer (25.5 kDa), suggesting
that the earlier peak may be the result of a non-native dimer species
(Table I). Rg and
Rh values for the putative non-native
dimer species were about 20% greater than those for the native dimer,
consistent with the degree of structural expansion characteristic of
molten globules (36). Consistent with these observations, the
non-native dimer species also had a greater intrinsic viscosity than
the native dimer. However, the
Rg/Rh ratio of both
native and non-native dimer species was ~0.89, which is much closer
to ~0.78 for compact spherical molecules than to ~1.5 for random
coil structure (36, 37). Thus, the structurally expanded non-native
dimer species maintains a spherical shape.
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Table I
Physical and hydrodynamic parameters of native dimer and non-native
dimer species of SMA determined by the SEC triple detector
Highly purified ovalbumin (Sigma, grade VI) was used for the
calibration of light-scattering and refractive index detectors. The
theoretical molecular mass of the SMA dimer is 25.5 kDa (30).
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The level of non-native dimer species initially increased during
incubation, concomitant with the loss of native SMA dimer. Then there
was a gradual loss of the non-native dimer species, coinciding with the
further reduction in the level of native SMA dimer (Fig.
1B). These results suggest that the non-native dimer species
may be an intermediate on the aggregation pathway.
For samples with CR at r = 0, 0.3, 1.3 and 4.8, the
loss of soluble SMA coincided with an increase of visible precipitated aggregates. In contrast, for samples with r = 8.8, there were no detectable precipitates until 20 h of incubation,
even though soluble native SMA dimer was completely depleted after
5 h of incubation, and the non-native dimer species was populated
after 30 min of incubation (Fig. 1). After 20 h, a small amount of
precipitate (sufficient to account for about 10-20% of the total
protein) was observed visually, but the precipitate amount did not
increase during further incubation up to 4 days. To reconcile the
inability to account for the loss of native protein caused by formation of non-native aggregates or precipitates, samples incubated for 10 and
20 h were centrifuged, and the supernatants were analyzed on
reducing SDS-PAGE. As shown in Fig. 1C, the supernatants for samples with CR at r = 1.3 and 4.8 showed significant
decreases in the amount of soluble SMA, consistent with SEC data.
However, the supernatant for the sample with r = 8.8 showed an amount of protein similar to that of the sample for
r = 0, which remained soluble without forming any
aggregates for up to 3 days of incubation. So the majority of the
protein in the incubated samples with CR at r = 8.8 remained soluble but appeared to interact with the SEC column,
preventing detection in SEC analysis. Thus, the apparent loss of
soluble protein indicated by the SEC analysis was caused by the
formation of a soluble species that would not elute from the SEC column.
Real Time Measurements of SMA Aggregation by CD and IR
Spectroscopies--
To monitor structural changes of SMA during
aggregation and determine the kinetics of aggregation, real time
aggregation measurements were made with CD and IR spectroscopies. In
studies with far-UV CD spectroscopy, solutions of 117 µM
(2.97 mg/ml) SMA with various molar ratios of CR (r = 0. 1.3, 4.8, and 8.8) were incubated isothermally at 37 °C or heated
from 25 to 60 °C (Fig. 2). Without CR,
the native secondary structure of SMA was not significantly changed by
incubations up to 140 min at 37 °C (Fig. 2A) or by
heating up to 45 °C (Fig. 2E). Secondary structure was
rapidly lost during heating above 50 °C with large increases in
negative ellipticity at 217 nm, which were most likely the result of
non-native intermolecular
-sheets in protein aggregates (38). The
short path length cell (0.01 cm) inhibited settling of insoluble
aggregates on the time scale of the spectroscopic measurements (38).
Aggregation of SMA at temperatures above 50 °C was expected because
the onset temperature and Tm of thermal
unfolding of SMA were 50 and 54 °C (see Fig. 9B),
respectively.

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Fig. 2.
Effects of CR on far-UV CD spectra of SMA
(117 µM) in the presence
of CR at r = 0 (A and
E), 1.3 (B and F),
4.8 (C and G), and 8.8 (D
and H). A-D,
time-dependent spectra collected every 20 min during
isothermal incubations at 37 °C for 140 min. E-H,
temperaturedependent spectra collected every 5 °C during
heating from 25 to 60 °C. Arrows indicate the spectral
shifts with time or temperature increases.
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Compared with results in the absence of CR, at r = 1.3 and 4.8 (Fig. 2, B, C, F, and
G), the aggregation was much faster during isothermal
incubation at 37 °C and occurred at lower temperatures during
heating from 25 to 60 °C. The most rapid aggregation and the
earliest onset temperature for aggregation were noted at
r = 4.8. During isothermal incubation at 37 °C, the
spectra indicated the presence of aggregates after 20 min. During
heating, aggregates were noted at 30 °C. With CR at
r = 8.8 in both isothermal and heating incubations, as
time or temperature increased, the spectra gradually had increased
negative ellipticity. The position of the minimum shifted to lower
wavelengths, asymptotically approaching 205 nm (Fig. 2, D
and H), which is close to the wavelength expected for
unordered conformations (32).
Also, during far-UV CD experiments (Fig. 2), a negative CD band around
233 nm decreased in intensity as aggregation progressed in solutions
with CR at r = 1.6 and 4.8, and unfolding progressed at
r = 8.8. The negative band at 233 nm is contributed by
aromatic residues, i.e. 2 Trp, 7 Tyr, and 2 Phe per monomer
(30, 32, 39-41), consistent with studies on other proteins (42, 43). The spectra for the sample with CR at r = 8.8 showed a
clear isosdichrotic point at 228 nm (Fig. 2, D and
H), indicating a two-state unfolding process of SMA.
For the sample without CR, precipitates were visible after the
completion of the thermal scan but not after isothermal incubation at
37 °C. In contrast, with CR at r = 1.3 and 4.8, precipitates were visible after incubations and thermal scans.
Interestingly, with CR at r = 8.8, neither isothermal
incubation nor thermal scans resulted in visible precipitates. Thus, in
a solution of SMA with CR at r = 8.8, the combination
of the dramatic structural changes evident in far-UV CD spectra, the
absence of a intermolecular
-sheet signal at 217 nm, and the lack of
visible precipitates suggest that SMA unfolded without aggregating.
Similar results were observed by FT-IR spectroscopy during isothermal
incubations at 37 °C for 0.7 mM (16 mg/ml) SMA with CR
at r = 0, 1.3, and 2.8 (Fig.
3). The second derivative IR spectra of
native SMA without CR had a dominant band at 1639 cm
1,
indicating a predominance of
-sheet (29). During incubation at
37 °C in the absence of CR, there were no changes in the secondary structure of SMA during the 14 h of the experiment. With CR at r = 1.3 and 2.8, however, the native
-sheet
structure of SMA gradually decreased, and a new band around 1,628 cm
1 grew in intensity, indicative of formation
intermolecular
-sheet associated with protein aggregation (33, 34).
The spectra showed an isosbestic point at 1,632.7 cm
1,
indicating two state-structural transitions of native SMA into the
aggregates. The aggregation rate was faster for CR at r = 2.8 than at r = 1.3.

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Fig. 3.
Effects of CR on IR spectra of SMA (0.7 mM) with CR at r = 0, 1.3, and 2.8 during
incubation at 37 °C. A, time course second
derivative IR spectra of SMA with CR at r = 2.8, recorded at 0.015, 0.15, 0.5, 1, 2, 3, 4, 5, 6, 10, and 14 h.
Arrows indicate the spectral shift with duration of
incubation. B, time course intensity change at 1,627.8 cm 1 in second derivative IR spectra for SMA in the
presence of CR at r = 0 ( ), 1.3 ( ), and 2.8 ( ).
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Characterization of the Aggregates--
Precipitates formed during
incubation at 37 °C with agitation (Fig. 1) were characterized by
TEM, CR green birefringence, and FT-IR spectroscopy (Figs.
4-6). TEM images of the SMA aggregates formed with CR at r = 0 and 0.3 showed clumps of
fibrils that were unbranched and ~8-12 nm in diameter (Fig. 4,
A and B), which are typical of amyloid fibril
morphology (2, 7). Further, CR staining of these samples showed strong
green birefringence under polarized light (Fig.
5, A-D), which is also
diagnostic of amyloid fibrils. TEM images of the SMA aggregates with CR
at r = 1.3 and 4.8 showed mixed morphology with both
fibrils and amorphous aggregates (Fig. 4, C and
D), consistent with the reduced birefringence of CR-stained
samples (Fig. 5, E and F; cf. Ref. 29). TEM images of the aggregates generated during thermal scans of SMA
alone to 60 °C (Fig. 2A) also showed a mixed morphology of fibrils and amorphous aggregates (Fig. 4G). The TEM
images of the precipitate formed in samples agitated at 37 °C with
CR at r = 8.8 showed spherical structures with
diameters of 20-34 nm (Fig. 4E). Similar and much larger
(>150 nm) spheres were also observed for samples with CR only (Fig.
4F), which were generated under the same conditions used for
incubating the SMA sample with CR at r = 8.8, (i.e. a 352 µM CR solution was incubated at
37 °C with agitation for 48 h). Thus the spheres most likely
were CR micelles.

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Fig. 4.
Representative TEM images for
insoluble SMA aggregates. A-E, images of SMA
aggregates formed during incubation at 37 °C with agitation (Fig. 1)
in the presence of CR at r = 0 for 103 h
(A), r = 0.3 for 40 h (B),
r = 1.3 for 40 h (C), r = 4.8 for 20 h (D), and r = 8.8 for
48 h (E). F, image of CR only (355 µM) after incubating at 37 °C with agitation for
48 h. G and H, images for insoluble
aggregates formed during heating from 25 to 60 °C with CR at
r = 0 (G) and r = 1.3 (H), under conditions identical to those used in the
experiments for which results are given in Fig. 2, A and
B, respectively. Scale bars represent 100 nm.
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Fig. 5.
Representative images of CR staining
and green birefringence of insoluble SMA aggregates formed during
incubation at 37 °C with agitation in the presence of CR at
r = 0 for 103 h (A and
B), 0.3 for 40 h (C and
D), 4.8 for 20 h (E and
F) in the bright field (A,
C, and E) and under cross-polarized
light (B, D, and
F). Magnification, ×200.
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IR spectra of the precipitated SMA samples had dominant bands around
1,626-1,628 cm
1 and 1,689-1,693 cm
1 (Fig.
6), which are characteristic of
non-native intermolecular antiparallel
-sheet (6, 29, 33, 34). Minor
differences between the spectra for precipitates formed in different
levels of CR may be caused by differences in morphologies of the
aggregates.

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Fig. 6.
Second derivative IR spectra of the insoluble
SMA aggregates formed during incubation at 37 °C with agitation
(Fig. 1) in the presence of CR at r = 0 ( ),
0.3 ( ), 1.3 ( ), 4.8 ( ), and 8.8 ( ). Also shown
is the second derivative IR spectrum for the native SMA (solid
line).
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For the samples incubated isothermally at 37 °C for 140 min in the
CD cell (Fig. 2, A-D), binding-induced CR CD spectra
(400-650 nm) were collected to determine whether the aggregates
contained amyloid fibrils (Fig. 7). The
induced CD spectrum is caused by conformational changes of CR resulting
from its binding to native proteins or amyloid fibrils (23-25). CR
(Fig. 7) or protein alone (data not shown) did not show any optical
activity in the 400-650 nm spectral region. Native SMA was mixed with
CR and spectra acquired immediately. The spectrum showed a maximum peak
around 530 nm, indicative of CR binding to SMA with distorted,
nonsymmetric conformations (23-25). The spectrum of CR in the presence
of SMA amyloid fibrils (generated by agitation at 37 °C for 4 days)
had a maximum peak around 548 nm (Fig. 7), which is characteristic of
the induced CD spectrum of CR upon binding to amyloid fibrils (14, 23). The aggregates formed during isothermal incubation with CR at r = 1.3 and 4.8 showed spectra with peak maxima close
to 548 nm, indicating that the aggregates contained amyloid fibrils,
consistent with the TEM results. However, in the spectrum for the SMA
sample with CR at r = 8.8, the peak maximum was near
530 nm because SMA in this sample unfolded instead of forming fibrils
(Fig. 7).

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Fig. 7.
CD spectra of CR induced by binding to SMA
aggregates formed during the incubations for which results are shown in
Fig. 2, B-D. Spectra are shown for samples with CR at
r = 1.3 ( ), 4.8 ( ), and 8.8 ( ) after
incubation at 37 °C for 140 min. Also shown are the induced CD
spectra of CR in the presence of native SMA (with CR at
r = 4.8, time = 0) ( ), amyloid fibrils of SMA
( ), and CR only (280 µM) ( ).
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Effects of CR on the Secondary and Tertiary Structures of
SMA--
The stimulatory effects of CR on SMA amyloid fibril formation
and aggregation prompted us to examine CR effects on secondary and
tertiary structures of SMA at 25 °C, using far- and near-UV CD and
IR spectroscopies (Fig. 8). The far-UV CD
spectrum of native SMA without CR showed a negative band around 217 nm
and a positive band around 200 nm, which are contributed by native
-sheet structure, as well as a negative band at 233 nm which is the
result of aromatic residues, as discussed above. CR alone did not show
any optical activity in the far-UV CD region (Fig. 8). The spectrum for
denatured SMA in 4 M urea (7) had a minimum band around 204 nm, as expected for unordered conformations (32). In the spectra for
native SMA in the presence of CR, the negative band around 233 nm
gradually increased in intensity with increasing levels of CR, with an
isosdichrotic point at 224 nm which is indicative of a two-state
structural perturbation of the microenvironments of the aromatic
residues. However, the
-sheet band at 217 nm was not altered in the
presence of CR, indicating that CR does not greatly perturb the
secondary structure of SMA. Similar results were obtained from second
derivative IR spectra of SMA, which were minimally affected by CR at
the ratios tested (Fig. 8B).

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Fig. 8.
Effects of CR on the secondary and tertiary
structures of SMA. A, far-UV CD spectra of SMA in the
presence of CR at r = 0 ( ), 1.2 ( ), 2.3 ( ),
4.8 ( ), and 8.8 ( ) at 25 °C. Also shown is the spectrum for
SMA incubated overnight in 4 M urea ( ) and for CR (280 µM) buffer only (solid line). B,
second derivative IR spectra of SMA (0.7 mM) with
r = 0 (solid line), r = 1.3 (dotted line), and r = 2.8 (dashed
line) at 25 °C. C, near-UV CD spectra of SMA with
r = 0 ( ), 1.3 ( ), 4.8 ( ), and 8.8 ( ) at
25 °C. Also shown is the spectrum for CR only (280 µM)
(solid line).
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The near-UV CD spectrum of native SMA without CR showed positive maxima
around 296 and 272 nm, a shoulder around 287 nm, and a minimum around
283 nm (Fig. 8C), consistent with previously reported
results for SMA (41). A positive band at 296 nm has been ascribed to
Trp and a negative band around 283 nm to Tyr (39-41). CR alone did not
show any optical activity (Fig. 8C). In the spectra for SMA
in the presence of CR, the peaks around 296, 283, and 272 nm were
gradually blue shifted with increasing levels of CR, indicating
CR-induced solvent exposure of aromatic groups in the protein (39,
40).
Taken together, the far-UV CD, near-UV CD, and IR spectroscopic results
documented that CR perturbed the tertiary structure of native SMA
without significantly affecting the secondary structure of the protein.
It is important to note that these spectroscopic studies were conducted
at 25 °C, and the spectra do not show any signal (e.g.
because of non-native
-sheet or light scattering) indicative of
protein aggregates.
Effects of CR on the Thermal Stability and HX of SMA--
Thermal
unfolding of SMA (4 µM = 0.1 mg/ml) was studied with
far-UV CD spectroscopy at various molar ratios of CR (r = 0, 1.3, 4.8, and 8.8) (Fig. 9,
A and B). Far-UV CD spectra of all samples at
80 °C showed a negative maximum centered at about 205 nm (Fig.
9A), indicating that thermal unfolding rather than
aggregation occurred at the relatively low protein concentration used
in these experiments. The thermal unfolding was irreversible. The
Tm values were 53.9 ± 0.3 °C for SMA
alone, 47.0 ± 0.5 °C for r = 1.3, 43.9 ± 0.4 °C for r = 4.8, and 39.5 ± 1.6 °C for
r = 8.8, showing that CR lowered the thermal stability
of SMA (Fig. 9B).

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Fig. 9.
Effects of CR on thermal stability and HX of
SMA. A, far-UV CD spectra of SMA (4 µM)
collected at 80 °C in the presence of CR at r = 0 ( ), 1.3 ( ), 4.8 ( ), and 8.8 ( ). Also shown is the spectrum
for SMA (4 µM) in buffer only at 25 °C before heating
(solid line). B, fraction of unfolded SMA as a
function of temperature in the presence of CR at r = 0 ( ), 1.3 ( ), 4.8 ( ), and 8.8 ( ). C, HX rates of
SMA in the presence of CR at r = 0 ( ), 0.02 ( ),
1.3 ( ), and 1.9 ( ). HX was monitored by the shift in band
frequency around 1,638 cm 1 as a function of the length of
time of exposure to 75% D2O buffer. Inset,
changes in second derivative amide I IR spectrum of SMA in the presence
of CR at r = 1.9 as a function of the length of time of
exposure to 75% D2O buffer. The arrow indicates
the direction of time-dependent spectral shifts.
Errors bars indicate the S.D. for duplicate samples.
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The HX of SMA (400 µM = 10 mg/ml) was measured in real
time with FT-IR spectroscopy at 25 °C, in solutions with various
molar ratios of CR (r = 0, 0.02, 1.3, and 1.9) (Fig.
9C). During the 6-h HX experiment, all SMA samples
maintained native secondary structure (Fig. 9C,
inset). During HX, there was a time-dependent shift in the position of the major
-sheet band at about 1,638 cm
1. The rate at which HX occurred was faster in the
presence than in the absence of CR (Fig. 9C).
Thermodynamics of CR Binding to SMA Measured by ITC--
Fig.
10 shows representative data from ITC
for a titration of CR to SMA at 25 °C. The CR titration to buffer
only was endothermic with greater heat absorption in the initial
injections (Fig. 10A). CR readily forms self-assembled
micelle-like structures in water at high ionic strength or above 5 µM at physiological pH (24, 25), but monomeric CR may
also exist transiently (44). The initial larger endothermic peaks
presumably were caused by the dissociation of CR from micelles during
dilution in the reaction cell, an effect that decreased with further
injections because the dilution of CR was decreased (45).

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Fig. 10.
Representative ITC data analysis for
titration of CR (760 µM titrant
concentration) into buffer (A) and into a solution of
20.1 µM SMA dimer (B
and C) at 25 °C. Titrations consisted of
25 injections (10 µl each) of CR. A and B show
the raw heat data as a function of injection number, and C
shows enthalpy changes as a function of molar ratios of CR to SMA
dimer.
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CR titrations to SMA were exothermic (Fig. 10B). The
enthalpy change as a function of the molar ratios of CR to SMA dimer
increased without a clear inflection point (Fig. 10C). The
results were fit best with a model in which binding to each site has
equal energy, using Origin software supplied by MicroCal, Inc. The
binding stoichiometry (n) was about 3.4 at 25 °C and 5.1 at 30 °C. Kd values were in the
µM range, showing that the binding between CR and SMA is
relatively strong (35, 46). The enthalpic contribution to the free
energy of binding was dominant (Table
II). After the titration experiments, samples were removed from the cell and analyzed by SEC. Aggregates were
not detected in samples titrated at 25 or 30 °C (data not shown). In
contrast, when titrations were conducted at 37 °C, soluble oligomers
were formed (data not shown), precluding the use of these data to
calculate binding parameters.
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Table II
Thermodynamic parameters for the interactions between SMA and CR
measured by ITC
Values are the mean ± S.D. from two or three experiments.
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DISCUSSION |
Interactions of CR with SMA--
The binding molar ratios of CR
molecules/SMA dimer increased from ~3.4 at 25 °C to ~5 at
30 °C. The increased binding at higher temperature is likely the
result of exposure of new binding sites on the protein as temperature
increases. A previous study also showed that the binding molar ratios
of CR molecules to a
-type VL dimer were ~8 at
40-45 °C and ~16 at 55 °C, measured by gel extraction analysis
(22). The dissociation constant (~19 µM) of CR binding
to SMA is similar to that (~9 µM) of CR binding to
A
-amyloid fibrils determined by filtration method using radiolabeled CR (47).
Even though CR has been shown to bind to several proteins (9, 14,
22-28), the main binding interactions still remain controversial. The
structural features of CR suggest that the binding most likely occurs
through hydrophobic interactions, electrostatic interactions, or both.
Some earlier studies proposed that binding occurs through hydrophobic
interactions between the biphenyl rings of CR and hydrophobic pockets
and clefts on proteins (14, 17, 22, 48). However, our ITC data showed
that the formation of a CR·SMA complex was dominantly enthalpically
driven, excluding hydrophobic interactions (35, 46). Hydrophobic
interactions are characterized mainly by small enthalpy changes and
large positive entropy changes, resulting from the release into the
bulk solution of relatively highly ordered water molecules that
surround the hydrophobic surfaces of two interacting molecules (35,
46). Further, the observed decrease in the magnitude of negative
enthalpy with increased temperature (Table II) is opposite the
phenomenon associated with the burial of solvent-accessible hydrophobic
surfaces (35, 46). Thus it seems that the electrostatic interactions
between the sulfate groups of CR and the positively charged residues of
SMA are responsible for the formation of a CR·SMA complex. Previous studies also showed that electrostatic interactions were responsible for the binding of CR to A
-peptides (27), prion proteins (9, 10),
and
2-microglobulin, which is a structural homolog of VL (28), indicating that the sulfate groups of CR are
important for its binding to proteins.
Effects of CR on Aggregation and Conformation of SMA--
With CR
at r = 0.3, 1.3, 4.8, aggregation of SMA was greatly
accelerated (Figs. 1-3), and the resulting aggregates and fibrils had
a non-native intermolecular
-sheet structure that is typical of
amyloid fibrils and protein precipitates. Accelerated aggregation was
associated with binding of CR to the protein, which reduced the thermal
stability of SMA and greatly favored SMA species with perturbed
tertiary structure and native secondary structure (i.e. molten globules). Our HX results also indicated that in the presence of
CR, the molecular population of SMA was shifted toward partially unfolded species, which are highly reactive to aggregation (1, 3, 6,
49). Previous studies showed that CR stabilized the molten globule-like
states of IgG molecules and a
-light chain dimer (26, 50). A similar
amphipathic dye, 8-anilino-1-naphthalenesulfonate (ANS), also favored a
partially folded conformation of pectate lyase C (51).
As can be shown through application of the Wyman linkage function (52),
preferential binding of CR and ANS to partially unfolded protein
molecules shifts the equilibrium between protein substates
toward these species. Because the partially unfolded molecules are
aggregation-competent, ligand binding greatly accelerates protein
aggregation. This effect of ligands contrasts with the inhibition of
protein aggregation which can be realized with ligands that bind
strongly with the fully native protein conformation (8). In this case,
the same Wyman linkage function describes how ligand binding shifts the
equilibrium toward the native state and away from
aggregation-competent, partially unfolded species.
CR binds to partially folded SMA with a stoichiometry of about 4 mol of
CR/mol of SMA dimer. At higher CR concentrations (e.g. r = 8.8), CR binds to denatured SMA molecules, favoring
their population. Protein aggregation occurs through partially unfolded molecules and not from the fully unfolded state (1, 3, 49). High
concentrations of CR reduce aggregation (Figs. 1 and 2) by populating
the denatured state of SMA.
Thus, the overall effect of CR binding on protein aggregation depends
on the concentrations of CR and protein, which dictate the amount of CR
bound and hence the equilibrium between protein species. At lower
concentrations CR binds to and populates partially unfolded forms of
SMA, which accelerates aggregation. In contrast, higher concentrations
of CR favor unfolding of SMA, and, hence, result in reduced
aggregation. These concentration-dependent effects of CR on
protein aggregation resemble those of chaotropes such as urea and
guanidine hydrochloride. Previous studies have shown that at relatively
low concentrations, these denaturants populated partially unfolded
protein molecules and foster aggregation, whereas at higher
concentration they greatly favor the unfolded state and thus prevent
aggregation (5, 53).
These insights can be used to reconcile the apparently conflicting
results from earlier reports on the effects of CR on protein aggregation and fibril formation. At high molar ratios of CR to the
protein, e.g. r
60 for prion protein (9,
10, 12) and r
300 for A
-peptides (13, 20),
amyloid fibril formation by these proteins was inhibited. In contrast,
at lower molar ratios CR accelerated fibril formation from
A
-peptides (13) and prion proteins (12). Although the detailed
mechanism by which CR operates in a dose-dependent manner
remains to be established for these and other proteins, we speculate
that at relatively low concentrations, CR binding populates partially
folded, aggregation-competent species of the proteins. At higher
concentrations, CR inhibits fibril formation because it favors the
denatured state, which is much less prone to aggregate.
It has been suggested previously that CR inhibits fibril formation by
prion proteins by stabilizing the so-called "scrapie" conformation
to such a high degree that further unfolding, which is thought to be
required for this conformation to assemble into fibrils, is inhibited
(54). It also has been proposed that CR inhibits fibril formation by
blocking binding sites for other molecules that are promoters of
protein aggregation (48). However, neither of these mechanisms can
account for the concentration-dependent effects of CR on
protein aggregation and fibrillogenesis.
The dose-dependent effects of CR binding on protein
conformational equilibria have implications for practical therapeutic use of CR or its analogs. During dosing to obtain the high level of CR
potentially needed to inhibit amyloid fibril formation, there would be
periods of time during which lower concentrations of CR were present.
The lower concentration of CR could induce fibril formation. Thus CR or
its analogs could cause increased rather than decreased pathology.
Biological Implications--
CR has two anionic sulfate groups and
in that respect resembles the sulfated glycosaminoglycans (GAGs), which
are the main components of basement membrane glycoproteins
(e.g. heparan sulfate, chondroitin sulfate, and demartan
sulfate) and in vivo frequently deposit coincidentally with
amyloid fibrils of a variety of proteins including A
-peptides,
prions, serum amyloid A, and immunoglobulin light chains (55).
Interestingly, sulfated GAGs stimulate amyloid fibril formation by
A
-peptides and
-synuclein in vitro (56-58), suggesting that they could enhance amyloid fibril formation in vivo. CR competes with sulfated GAGs for binding with prion
proteins, suggesting that they bind to same sites, most likely through
sulfates (9). A previous study showed that several VL's,
including SMA, interact with GAGs by electrostatic interactions between
the sulfate groups of GAGs and the positively charged residues of the
proteins (59). These interactions may be responsible for the enhanced fibrillogenesis of proteins in the presence of GAGs which, analogously to CR, may favor population of partially unfolded, aggregation competent protein species.
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CONCLUSIONS |
The concentration-dependent effects of CR on SMA
aggregation and fibril formation depend on which protein species is
favored upon binding of this ligand. For any given ligand, its effect on amyloid fibril formation will similarly be dictated by how its
binding affects the equilibrium between protein species. Thus, in
efforts to discover potential therapeutic compounds for inhibition of
amyloidosis, it is important that in vitro screening studies take into account not only the binding affinity of the ligands for the
target protein, but also the effect of binding on the molecular
population of the protein. In addition,
concentration-dependent effects of compounds on both
amyloid fibril formation and protein structure should be studied in
these screening efforts.