Amyloid-like Fibril Formation in an All
-Barrel Protein
PARTIALLY STRUCTURED INTERMEDIATE STATE(S) IS A PRECURSOR FOR
FIBRIL FORMATION*
Sampath
Srisailam
,
Thallampuranam Krishnaswamy
S.
Kumar
,
Dakshinamurthy
Rajalingam
,
Karuppanan Muthusamy
Kathir
,
Hwo-Shuenn
Sheu§,
Fuh-Jyh
Jan¶,
Pei-Chi
Chao¶, and
Chin
Yu
From the
Department of Chemistry, National Tsing Hua
University, Hsinchu 30043, Taiwan, § Synchrotron Radiation
Research Center, Science-Based Industrial Park, Hsinchu 300, Taiwan, and ¶ Regional Instrumentation Center, National
Chung Hsing University, Taichung 40277, Taiwan
Received for publication, January 13, 2003
 |
ABSTRACT |
Acidic fibroblast growth factor from newt
(Notopthalmus viridescens) is a ~15-kDa, all
-sheet
protein devoid of disulfide bonds. In the present study, we investigate
the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt
acidic fibroblast growth factor (nFGF-1). The protein aggregates
maximally in 10% (v/v) TFE. Congo red and thioflavin T binding
experiments suggest that the aggregates induced by TFE have properties
resembling the amyloid fibrils. Transmission electron microscopy and
x-ray fiber diffraction data show that the fibrils (induced by TFE) are
straight, unbranched, and have a cross-
structure with an average
diameter of 10-15 Å. Preformed fibrils (induced by TFE) of nFGF-1 are
observed to seed amyloid-like fibril formation in solutions containing
the protein (nFGF-1) in the native
-barrel conformation.
Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding,
multidimensional NMR, and Fourier transformed infrared spectroscopy
data reveal that formation of a partially structured intermediate
state(s) precedes the onset of the fibrillation process. The native
-barrel structure of nFGF-1 appears to be disrupted in the partially
structured intermediate state(s). The protein in the partially
structured intermediate state(s) is found to be "sticky" with a
solvent-exposed non-polar surface(s). Amyloid fibril formation appears
to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and
intermolecular
-sheet formation among the extended, linear
-strands in the protein.
 |
INTRODUCTION |
Protein aggregation is a problem of importance not only in
biotechnology but also in health-related industries (1, 2). Globular
proteins in aqueous solution often tend to aggregate under a variety of
conditions of concentration, temperature, pH, and ionic strength
(3-6). The morphology of aggregates formed varies considerably and
ranges from amorphous forms to highly structured fibrils (7-9).
Structured fibrils formed in vitro closely resemble the
highly organized amyloid fibrils found in association with a variety of
human disorders, including Alzheimer's disease, Creutzfeldt-Jakob
disease, Huntington's disease, and type II diabetes (10-19). Several
studies show proteins that are apparently unrelated in sequence and, in
their native conformation, aggregate into fibrils that have
characteristic amyloid-like structural and histological features (16,
20-26). Recently, Bucciantini et al. (27) demonstrated that
amyloid-like fibrils of SH3 domain (from bovine phosphatidylinositol
3-kinase) induced in vitro in 25% (v/v) 2,2,2-trifluoro
ethanol (TFE)1 (under
appropriate conditions) are cytotoxic to fibroblast NIH3T3 cells. The
amyloid fibrils generated ex vivo are observed to seed fibrillate in cultured NIH3T3 cells (23). Therefore, it is now increasingly believed that amyloid represents a generic form of polypeptide conformation, and all peptides/proteins have the potential to form amyloid-like fibrils under appropriate conditions (21-27).
The molecular mechanism underlying the amyloid fibril formation is
poorly understood. Recent studies on proteins such as transthyretin (16, 25), lysozyme (26),
-synuclein (28),
-microglobulin (29),
immunoglobulin light chain variable domain (30) suggest that amyloid
fibril formation from the native state occurs via conformational
changes leading to the formation of sticky amyloid-prone, partially
structured intermediate(s). The partially structured intermediate
state(s) is postulated to associate into oligomers and subsequently
undergo structural rearrangement to form amyloid-like fibrils (8, 16).
However, very little information exists on the conformational features
of the "amyloid-prone" partially structured intermediate state(s).
In the present study, we investigate the TFE-induced conformational
transitions in a
-barrel protein such as the newt acidic fibroblast
growth factor (nFGF-1, Refs. 31 and 32). TFE is observed to induce
amyloid-like fibrils in nFGF-1. The formation of amyloid-like fibrils
is triggered by the accumulation of a partially structured intermediate
state(s) in the TFE-induced unfolding pathway of nFGF-1.
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MATERIALS AND METHODS |
Heparin-Sepharose was purchased from Amersham Biosciences.
Labeled 15NH4Cl, D2O, and
TFE-d3 were purchased from Cambridge Isotope Laboratories. 1-Anilino-8-naphthalene sulfonate (magnesium salt), thioflavin T (ThT),
and Congo red were purchased from Sigma. All other chemicals used were
of high quality analytical grade. Unless otherwise mentioned, all
solutions were made in 100 mM phosphate buffer (pH 7.0)
containing 100 mM sodium chloride. All experiments were
performed at 20 °C.
Protein Expression and Purification--
Recombinant nFGF-1 was
prepared from transformed Escherichia coli BL21(DE3)pLysS
cells. The nFGF-1 DNA construct consisting of 486 base pairs was
inserted between the NdeI and BamH1 restriction sites. The expressed protein was purified on a heparin-Sepharose affinity column over a NaCl gradient (0-1.5 M). Desalting
of the purified protein was achieved by ultra filtration using an
Amicon set-up. The purity of the protein was assessed using SDS-PAGE. The first 22 residues of the full form of nFGF-1 were digested by
subjecting the expressed full form of nFGF-1 to the action of
chymotrypsin. Chymotrypsin digestion was carried out by incubating the
column material (heparin-Sepharose containing the bound protein) with
the enzyme (at an enzyme to protein ratio of 20:1) in 10 mM
phosphate buffer (pH 7.2) containing 0.85 M NaCl. The
incubated mixture was stirred mildly at room temperature for 3 h.
The incubated material was repacked into a column and washed with 10 mM phosphate containing 0.85 M NaCl until the
absorbance of the eluate plateaued to a steady base line. Truncated
nFGF-1 was later eluted with 10 mM phosphate buffer (pH
7.2) containing 1.5 M NaCl. The homogeneity of the
truncated nFGF-1 sample was checked by SDS-PAGE. The authenticity of
the truncated sample was verified by electrospray ionization-mass spectrometry analysis. Protein concentration was estimated based on the extinction co-efficient value of the protein at 280 nm. It
should be stated that the truncated newt FGF-1, which we label as
nFGF-1, is used in all the experiments described ahead in the present work.
Preparation of Isotope Enriched nFGF-1--
15N
isotope labeling was achieved using M9 minimal medium containing
15NH4Cl. To maximize expression yields, the
composition of the M9 medium was modified by the addition of a mixture
of vitamins. The expression host strain, E. coli
BL21(DE3)pLysS, is a vitamin B1-deficient host, and hence,
the medium was supplemented with thiamine (vitamin B1).
Protein expression yields were in the range of 25-30 mg/liter of the
isotope-enriched medium. Purification and chymotrypsin digestion
methods to obtain truncated nFGF-1 were the same as described in the
previous section. The extent of 15N labeling was verified
by electrospray ionization-mass spectrometry analysis.
Turbidity Measurements--
Turbidity measurements were
performed on a Hitachi U-3310 spectrophotometer. All measurements were
made after 3 h of incubation (at 20 °C) of the protein (nFGF-1)
in appropriate concentrations of TFE. The concentration of the protein
used in the turbidity experiments was 100 µg/ml. The turbidity of the
solutions was measured by absorbance at 350 nm. The path length of the
sample cell used was 10 mm.
Circular Dichroism--
All CD measurements were made on a Jasco
J-720 spectropolarimeter. CD experiments were performed using a 0.2-cm
quartz cell for far (195-250 nm) and 10-cm quartz cell for the near UV
CD (250-320) regions. A step size of 0.1 nm, an average time of 3 s, and an average of 10 scans were recorded to generate the data. The
concentration of the protein inside was 0.1 mg/ml. The far and near UV
CD spectra were smoothed using the noise-reducing option in the
software supplied by the vendor (Jasco).
Congo Red and Thioflavin T Binding--
The concentration of
protein used in these experiments was 100 µg/ml. Protein samples were
incubated in various concentrations of TFE for 3 h before the dye
(Congo red or thioflavin T) was added. The solutions were stirred upon
the addition of the dye. Fluorescence spectra of ThT were measured on a
Hitachi F2500 spectrofluorimeter using a quartz cell with a light path
of 10 mm. The excitation wavelength was set at 440 nm, and bandwidths
for excitation and emission lights were 2.5 and 10 nm, respectively.
For the Congo red binding assay, absorption spectra of the samples
containing 10 µl of a 1 mM solution of the dye (Congo
red) were recorded on a Hitachi U-3310 spectrophotometer in the range
of 400-700 nm.
Transmission Electron Microscopy--
Electron micrographs of
amyloid fibrils (generated in TFE) of nFGF-1 were acquired on a Hitachi
H-7500 transmission electron microscope. A 2-µl sample of the protein
at a concentration of about 100 µg was applied to Formvar- and
carbon-coated copper grids. The fibrils were washed by the successive
addition of 3 wash steps (10-µl aliquots of water) followed by drying
with filter paper. The fibrils were stained by the addition of 10 µl
of uranyl acetate (1% w/v) and immediately dried with filter paper.
X-ray Diffraction--
The fibers induced in 15% (v/v) TFE were
suspended between the ends of 2 wax-filled glass capillaries and
allowed to dry. The x-ray scattered image of TFE-induced fibrils were
collected using a wavelength 1.11 Å on an off-line image plate
detector at BL17B2 of the synchrotron radiation. The specimen was
mounted on a 1-mm loop for free standing to avoid the x-ray beam
touching any material other than fibrils. The image was processed by
using the XPRESS software.
Seeding Experiment--
Aggregation of nFGF-1 (5 mg/ml) in 15%
(v/v) TFE were incubated for about 2 weeks at room temperature to
obtain dense fibers. The TFE-induced fibers were repeatedly washed in
10 mM phosphate buffer (pH 7.2) containing 100 mM NaCl, filtered, and dried. For the seeding experiments,
various aliquots 100-500 µl of seeding solution (TFE-induced
preformed fibrils) of the protein was added to freshly prepared
solution (TFE induced preformed fibrils) of the protein. The seeding
experiments were carried out with appropriate controls. The first
control involved the incubation of an identical solution of nFGF-1 in
the absence of the aliquot of the seeding solution, and the second
control involved the dilution of an approximate aliquot of the seeding
solution into phosphate buffer. The seeding (aggregation) was monitored
by the increase in the absorbance at 350 nm. In addition the seeding
solution was analyzed by transmission electron microscopy to obtain
definite evidence for the existence of fibrils and analyzing their morphology.
Steady State Fluorescence--
Fluorescence experiments were
performed on a Hitachi F2500 spectrofluorimeter at 2.5- or 10-nm
resolution. Intrinsic fluorescence measurements were made at a protein
concentration of 100 µg/ml using an excitation wavelength of 280 nm.
1-Anilino-8-napthalene sulfonate (ANS) binding experiments were carried
out on nFGF-1 at various concentrations of TFE using an excitation
wavelength of 390 nm. The emission was monitored between 420 and 600 nm. The concentrations of the dye (ANS) and the protein (nFGF-1) used were 200 µM and 100 µg/ml, respectively.
NMR Experiments--
All NMR experiments were performed on
Bruker Avance-600 NMR spectrometer at 20 °C. A 5-mm inverse probe
with a self-shielded z-gradient was used to obtain all
gradient-enhanced 1H,15N heteronuclear single
quantum coherence (HSQC) spectra. 15N decoupling during
acquisition was accomplished using the globally optimized
alternating-phase rectangular pulse sequence. 2048 complex data
points were collected in the 1H,15N HSQC
experiments. 512 complex data points were collected in the
15N dimension. The HSQC spectra were recorded at 32 scans
at all concentrations of TFE. The concentration of the protein sample was 0.5 mM in 95% H2O and 5% D2O
(containing 100 mM phosphate and 100 mM sodium
chloride). 15N chemical shifts were referenced using the
consensus ratio of 0.01013291189. All spectra were processed on a
Silicon Graphics work station using XWINNMR and AURELIA software.
Fourier Transform Infrared Spectroscopy (FT-IR)--
The samples
for the FT-IR spectral measurements were prepared as follows. Protein
aggregates formed in 15% (v/v) TFE (at a protein concentration of 1 mg/ml) were centrifuged at 10,000 rpm using a desktop centrifuge for 20 min, and the supernatant was removed carefully. The precipitant was
dried overnight in a vacuum desiccator. The dry powder of the
aggregates was enclosed in a KBr tablet by a conventional high pressure
method. FT-IR spectra of nFGF-1 at 0% (v/v) and 70% (v/v) TFE were
acquired by dissolving the protein in 99% D2O containing
appropriate concentrations of TFE-d3. All spectra were
recorded with a wave number resolution of 2 cm
1. For each
spectrum, 64-200 interferograms were collected and averaged, and a
Happ-Genzel apodization function was applied before Fourier
transformation. All processing procedures were carried out so as to
optimize the quality of the spectrum in the amide I region, between
1600 and 1700 cm
1.
 |
RESULTS AND DISCUSSION |
nFGF-1 is a ~15-kDa, all
-sheet protein with no disulfide
bonds (31). The protein lacks helical segments, and the secondary structural elements include 12
-strands arranged into a
-barrel architecture (Fig. 1).

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Fig. 1.
MOLSCRIPT representation of the structure of
nFGF-1. The secondary structural elements in nFGF-1 include 12 -strands arranged into a -barrel motif. The -strands are
numbered from 1 to 12.
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Effect(s) of TFE on the Backbone Conformation--
The original
objective of this study was to investigate the
specificity/non-specificity of the helix-inducing effect(s) of 2,2,2-trifluoroethanol. In this context, we monitored the secondary structural changes in nFGF-1 at various concentrations of TFE using
far-UV circular dichroism (CD) spectroscopy. The far-UV CD spectrum of
nFGF-1 (200-250 nm) shows two prominent ellipticity bands (a positive
ellipticity band at 228 nm and an intense minimum at around 205 nm)
characteristic of type II
-barrel proteins (Fig.
2, inset). The 228-nm CD band
is believed to primarily represent the arrangement of the
-strands
in the
-barrel architecture of the protein. No significant change(s)
occurs in the intensity of the 228-nm CD band at a TFE concentration
lower than 5% (v/v) (Fig. 2). However, at a TFE concentration of 8%
(v/v), the far-UV CD spectra of the protein show dramatic changes (Fig.
2). The positive CD band centered at 228 nm is lost, implying the
disruption of the native
-barrel architecture. The loss of the
positive CD band at 228 nm is paralleled by the appearance of a
negative CD band at around 218 nm in 20% TFE (v/v) (Fig. 2),
suggesting the formation of extended
-sheet conformation. The
intensity of the negative ellipticity band at 218 nm is observed to
steadily increase with the increase in the TFE concentration from 10 (v/v) to 50% (v/v) (Fig. 2). At higher concentrations of TFE (>50%
(v/v)), the far-UV CD spectra of the protein show two negative
ellipticity bands at 208 and 222 nm, indicating the induction of
helical conformation (Fig. 2). Because nFGF-1 in its native
conformation lacks helical segments, the induced helix conformation (at
higher concentrations of TFE (>50% v/v)) appears to be essentially
non-native in origin. The percentage of non-native helix estimated from
the far-UV CD spectrum of the protein 70% v/v is about 20%. The
majority (>60%) of the backbone of the protein (33, 34) (in 70%
(v/v) TFE) is estimated to be unstructured. The results discussed above
clearly suggest that the conformational transitions induced by TFE in nFGF-1 occur in two stages. In the first stage, the
-barrel
conformation is disorganized, resulting in the formation of extended
-sheet conformation. In the second stage, portions of the backbone
of the protein form non-native helix conformation.

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Fig. 2.
Far-UV CD spectra of nFGF-1 in various
concentrations of TFE. No or insignificant change(s) occur in the
spectra of the protein in TFE concentrations lower than 8% (v/v). In
15% (v/v) TFE, the 228-nm ellipticity band (representing the native
-barrel architecture) completely disappears, giving rise to a
negative far-UV CD band at 218 nm, indicating the formation of extended
parallel/anti-parallel -sheet elements in the protein. The
inset depicts the far-UV CD spectra of nFGF-1 in the
native and denatured states.
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Influence of Backbone Conformation on Protein Aggregation--
The
protein samples treated with TFE in the concentration range of 10%
(v/v) to 45% (v/v) and incubated at room temperature (~25 °C) for
more than 3 h are observed to turn turbid. Hence, the aggregation
of the protein (nFGF-1) is examined systematically by monitoring the
changes in the 350-nm absorbance (scattering) at various concentrations
of TFE (after 3 h of incubation of the protein at 25 °C in
various concentrations of TFE). Interestingly, the aggregation profile
shows that the protein tends to aggregate maximally between 8 and 45%
(v/v) TFE, wherein the protein is observed to exist in an extended
-sheet conformation (Fig. 3). The
extent of aggregates formed appears to increase with the increase in
the formation of the extended
-sheet conformation in the protein. In
addition, the aggregates formed (at TFE concentrations between 10% to
50% (v/v) TFE) are long and thread-like. Beyond this range of TFE
concentration (>50% (v/v) TFE), the protein solution turns clear
(with low 350-nm absorbance values) even after 48 h of incubation at 25 °C (Fig. 3). These results suggest that the aggregation of
nFGF-1 is related to the nature of non-native secondary structural elements induced in the protein (by TFE). Formation of non-native extended
-sheet conformation appears to promote aggregation, and
induction of non-native helix conformation correlates with the
inhibition of protein aggregation. Similar observations were made by
Dobson and co-workers, based on exhaustive point mutations in the
muscle acylphosphatase protein (35, 36). A clear-cut kinetic
partitioning between aggregation and folding of proteins was observed.
Protein aggregation was strongly correlated to the
-sheet propensity
of the regions of the protein (muscle acylphosphatase) in which the
point mutations were located (35). In addition, mutations that
stabilize helix conformation were shown to decrease the aggregation
process, and those that destabilized the helix increased the
aggregation rate (35).

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Fig. 3.
Absorbance (350 nm), shown in closed
triangles, and ellipticity (222 nm), shown
in closed squares, changes at various percentage
concentrations of TFE. It could be deduced that the increase in
the intensity of the 222 nm CD band (signifying the formation of
non-native helical segments) is correlated with the decrease in the
turbidity of the protein solution (indicating a decrease in
protein aggregation).
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TFE-induced Aggregates Possess Amyloid Fibril-like
Properties--
The aggregates of nFGF-1 formed in TFE (in 8-40%
(v/v) TFE) have a fibrous texture, suggesting that they could possess
properties characteristic of amyloid fibrils. This possibility was
examined by characterizing the tinctorial and ultrastructural
properties of the TFE-induced aggregates.
Congo red is a hydrophobic dye routinely used to identify amyloid
fibril formation by proteins (37). The absorption maximum of the dye is
known to undergo a red shift upon binding to ordered repetitive
-sheet structures in the amyloid fibrils (38). Although the
TFE-induced aggregation is maximum at 10% (v/v), Congo red binding is
observed maximally at 15% (v/v) TFE (Fig.
4A). The dye binding is not
only accompanied by a significant increase in the absorbance intensity
at 490 nm but also by a prominent red shift in the absorbance maximum
(490 to 520 nm, Fig. 4A, inset). Beyond 15%
(v/v) TFE the intensity of the dye at 490 nm decreases steadily with
the increase in concentration of the fluoro alcohol. These results
suggest that the fibrils formed maximally at 15% (v/v) TFE have
amyloid-like characteristics.

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Fig. 4.
Panel A depicts the binding of Congo red
to nFGF-1 at various concentrations of TFE. The inset
(panel A) shows the absorption spectra of Congo red at
various concentrations of TFE (panel A). The absorption
spectrum of Congo red in 15% (v/v) TFE shows a red shift (from 490 to
520 nm) in the absorption maximum, implying the formation of
amyloid-like fibrils. Panel B depicts the changes in the
emission intensity of thioflavin T upon binding to nFGF-1 in various
concentrations of TFE. The emission intensity of the dye is maximum in
15% (v/v) TFE, suggesting maximum accumulation of the amyloid type of
fibrils under these conditions. The inset (in panel
B) shows the emission spectra of thioflavin T in various
concentrations of TFE.
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The nature of the aggregates formed were also probed using ThT. ThT is
a fluorescent dye used as a diagnostic to identify amyloid fibril
formation (39). The emission intensity of ThT is known to increase
significantly upon binding to the linear array of
-strands in the
amyloid fibrils. There is no appreciable change(s) in the fluorescence
intensity of the dye at TFE concentrations lower than 10% (v/v) (Fig.
4B). However, an 8-fold increase in the fluorescence
intensity at 485 nm is observed when the dye binds to the protein in
15% TFE (Fig. 4B). These results provide further evidence
suggesting that amyloid-like fibrils of nFGF-1 accumulate maximally in
15% (v/v) TFE.
The ultrastructure of the aggregates (of nFGF-1) formed in 15% (v/v)
TFE was examined by transmission electron microscopy and x-ray fiber
diffraction. Electron micrographs reveal that the aggregates formed are
straight and unbranched, with an average diameter of about 10-15 nm
(Fig. 5A) and is similar to
the width of the fibrils formed from other amyloidogenic proteins (39). X-ray fiber diffraction analysis of the nFGF-1 aggregates shows a
dominant reflection at 4.7 Å on the meridian and associated equatorial
reflection at 10.3 Å, typical of those expected for fibrils with a
cross-
structure (Fig. 5B). The meridional
reflection (at 4.7 Å), which primarily arises from the spacing between
the
-sheet structure, is sharp and intense (Fig. 5B). The
equatorial reflection (at 10.3 Å), which is generally attributed to
the intersheet spacing, is relatively diffused (Fig. 5B).
Thus, the results of the electron microscopy and x-ray fiber
diffraction experiments authenticate that the aggregates formed in 15%
(v/v) TFE possess features resembling that of the amyloid fibrils.

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Fig. 5.
Panel A depicts the transmission
electron micrograph of the TFE-induced amyloid fibrils in nFGF-1. The
average diameter of the induced fibrils is estimated to be about 10-15
nm. Panel B depicts the x-ray fiber diffraction of the
TFE-induced fibrils in nFGF-1. The pattern shows dominant reflections
at 4.8 Å on the meridian and 10.3 Å on the equator. These reflections
are characteristic of the amyloid fibrils.
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Seeding Fibril Formation with Preformed Fibrils--
Seeding is a
common phenomenon in aggregation and gelation processes, as it is in
crystallization (40). It has been reported that aliquots of pre-formed
fibrils formed by AB peptide when it is added to clear solutions
substantially accelerated the rates of formation of amyloid-like
fibrils (8). Recently, Bucciantini et al. (27) demonstrate
that pre-formed amyloid fibrils (formed in vitro) of
proteins including SH3 domain from phosphatidylinositol 3-kinase and
the N-terminal domain of the E. coli HypF protein, which are
not associated with amyloidoses, are highly cytotoxic and induced
amyloid-like fibrils in cultured NIH3T3 fibroblast cells (27). In this
context, we investigated the seeding potency of the amyloid-like
fibrils of nFGF-1 induced in TFE. The potency of the pre-formed fibrils
of nFGF-1 (induced in 15% TFE (v/v)) to seed fibril formation was
examined by monitoring the time-dependent changes(s) in the
350-nm absorbance upon the addition of aliquots of pre-formed fibrils
into solutions (1 mg/ml) of native nFGF-1. It could be observed from
Fig. 6A that the 350-nm
absorbance increased significantly upon the addition of the pre-formed
fibrils (induced in TFE). Seeding experiments performed by the addition
of increasing aliquots of pre-formed nFGF-1 fibrils into a fixed
concentration (1 mg/ml) of native nFGF-1 solution revealed that the
rate of aggregation increases with the increase in the amount of the
preformed fibrils added (Fig. 6A). However, the extent of
induced aggregation (as deduced from the 350-nm absorbance) appears to
be independent of the amount of the pre-formed fibrils added (Fig.
6A). It should be mentioned that control experiments, with
an aliquot of the pre-formed fibrils diluted ~20-fold into the buffer
(10 mM phosphate containing 100 mM NaCl) and a
solution of nFGF-1 diluted 20-fold with the buffer to a final protein
concentration of 1 mg/ml under the same conditions, show no signs of
increases in aggregation (as observed from the 350-nm absorbance).
These experiments demonstrate qualitatively that pre-formed fibrils of
nFGF-1 (formed in TFE) are capable of seeding fibril formation in
solutions containing the protein (nFGF-1) in the native
conformation.

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Fig. 6.
Aggregation induced by pre-formed fibrils
(formed in TFE) of nFGF-1 in a solution containing nFGF-1 in its native
conformation (panel A). 100-, 200-, and 500-µl
aliquots (drawn from the pre-formed fibrils formed at 2 mg/ml native
protein concentration) were added to solutions of nFGF-1 in its native
state. It could be observed that the rate of induction of aggregation
depends on the amount of the pre-formed fibrils added. However, the
total aggregation induced appears to be independent of the
concentration of the pre-formed fibrils used for seeding. Panel
B shows the thioflavin T binding affinity of the induced
aggregates. The control experiment (open triangles) with ThT
added to nFGF-1 (in 10 mM phosphate buffer (pH 7.2)
containing 100 mM NaCl (in the absence of the pre-formed
fibrils)) shows no time-dependent increase in the emission
intensity at 485 nm. These results suggest that the induced aggregates
possess properties of the amyloid fibrils.
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We performed ThT binding experiments to verify if the aggregates seeded
by the pre-formed fibrils have amyloid-like characteristics. A
significant time-dependent increase in the fluorescence
intensity of the dye (ThT) at 485 nm could be observed upon incubation
of the native protein with pre-formed fibrils (Fig. 6B).
These results suggest that the induced aggregates (upon seeding) indeed
possess properties resembling the amyloid fibrils. In addition,
transmission electron micrographs revealed that the induced aggregates
are fibrillar with an average diameter of about 15 nm.2 In summary, the results
of the seeding experiments clearly demonstrate that fibril growth (in
nFGF-1) is dominated by a nucleation mechanism similar to that observed
in crystal growth and gelation process. Such in vitro
seeding has also been observed in studies of human lysozyme and its
amyloidogenic variants (41). Recently, fibrils generated under in
vitro conditions in hen egg white lysozyme (which is not
associated with amyloid diseases) have been shown to seed fibril
formation of protein in the native conformation (42). Similarly,
in vitro studies with the AB peptide associated with
Alzheimer's diseases suggest that origin of the rapid onset of amyloid
diseases and infectivity of prion-related diseases occurs by a
nucleation (seeding) mechanism (8).
Formation of a Partially Structured Intermediate(s)--
Recently,
there is increased interest in understanding the molecular events
leading to the formation of amyloid fibrils. Based on experimental
evidence available in different proteins, it is now increasingly
believed that amyloid fibril formation involves the ordered
self-assembly of partially folded species that are crucial soluble
precursors of fibrils (22-26, 35-37). In this context, we monitored
the conformational changes in the protein before amyloid-like fibril formation.
The fluorescence spectrum of nFGF-1 shows an emission maximum at around
308 nm (Fig. 7, inset). The
fluorescence of the lone tryptophan residue located at position 121 is
significantly quenched in the native state of the protein (43-46).
This quenching effect is attributed to the presence of imidazole and
pyrrole groups in the vicinity of the indole ring of the tryptophan
(Trp-121) residue (31). However, the quenching effect is relieved upon unfolding of the protein, yielding a fluorescence spectrum with an
emission maxima at around 350 nm (Fig. 7, inset). Hence, the ratio of 350- to 308-nm fluorescence reliably describes the
conformational transitions occurring during the unfolding of the
protein (46). The fluorescence spectra of nFGF-1 does not appreciably
change at lower concentrations of TFE (<8% (v/v) TFE). However, in
the TFE concentration range of 8 to 15% (v/v) TFE, the ratio of the 350- to 308-nm fluorescence drastically increases, suggesting substantial loss of tertiary structural interactions in the protein (Fig. 7). The TFE-induced unfolding effects on the protein are maximum
at 15% (v/v) TFE. Thus, analyzing the intrinsic fluorescence data in
conjunction with that obtained using far-UV CD (indicating
-sheet
segments in the protein) suggests that nFGF-1 exists in a partially
structured state(s), with extended
-sheets and loosely packed side
chains. Interestingly, at higher concentrations of TFE (>50 (v/v)
TFE), the fluorescence spectra show a significant decrease in the
350-nm emission, suggesting a change in the microenvironment of the
tryptophan residue upon induction of non-native helical conformation in
the protein.

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Fig. 7.
Changes in the intrinsic fluorescence
intensity of nFGF-1 in various concentrations of TFE. The
inset shows the emission spectra of the protein in its
native and denatured states. The tertiary structural interactions in
the protein are substantially perturbed in 15% (v/v) TFE.
|
|
ANS is a popular hydrophobic dye that is known to bind to
solvent-exposed hydrophobic surfaces in a manner characteristic of the
formation of partially structured state(s) (47). The dye generally
exhibits weak binding affinity to the native and unfolded state(s) of
protein (46). The binding affinity of ANS to the protein does not
significantly change below 8% (v/v) TFE (Fig.
8). However, beyond this TFE
concentration (>8% (v/v) TFE), the fluorescence intensity of the dye
at 485 nm increases drastically, reaching a maximum value at 15% (v/v)
TFE. The intensity of the dye upon binding to the protein in 15% (v/v)
TFE is about 20 times that observed when it (ANS) is bound to the
native state of the protein (Fig. 8). In addition, the dye upon binding
to the protein in 15% (v/v) TFE shows a prominent blue shift of about
42 nm (from 520 to 475 nm) in the wavelength of maximum emission (Fig.
8). These spectral characteristics clearly indicate that a partially structured state(s) with a solvent-exposed non-polar surface(s) accumulates (maximally at 15% (v/v) TFE) before the formation of
amyloid-like fibrils. Beyond 15% (v/v) TFE, the fluorescence intensity
of ANS at 485 nm decreases progressively (with the increase in the
concentration of the fluoro alcohol (TFE)), indicating a depletion in
the population of the partially structured intermediate (exhibiting
high binding affinity to ANS) state(s) (Fig. 8). It appears that the
partially structured intermediate state(s), which is accumulated
maximally at 15% (v/v) TFE, is sticky and has a high tendency to
aggregate through solvent-exposed hydrophobic surface(s) and
intermolecular
-sheet formation (to be discussed below). The
resultant aggregates possibly rearrange to form organized amyloid-like
fibrils.

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Fig. 8.
Emission spectra of ANS binding upon binding
to nFGF-1 in various concentrations of TFE. The emission maximum
blue shifts by 47 nm from 520 to 473 nm in 15% (v/v) TFE, indicating
the presence of solvent-accessible non-polar surface(s) in the protein.
The inset represents the emission intensity changes of the
dye (ANS) at various concentrations of TFE. It could be observed that
the emission intensity is maximum in 15% TFE, suggesting maximal
accumulation of the "molten globule-like" intermediate.
|
|
NMR spectroscopy facilitates the study at the level of individual amino
acids residues during folding/unfolding of proteins (48). Heteronuclear
correlation experiments have been shown to be very sensitive because of
high magnetization transfer between directly bond nuclei (48). This
aspect enables the use of 1H,15N HSQC technique
to investigate the conformational changes induced by TFE at high
resolution. In general, 1H,15N HSQC spectrum
serves as a fingerprint of the conformational state of a protein (48).
The HSQC spectrum of nFGF-1 in its native conformation (0% TFE) is
well dispersed, and all the expected 126 1H,15N
cross-peaks in the spectrum could be unambiguously assigned (31). No
discernable changes(s) could be observed in the HSQC spectrum of the
protein acquired below a TFE concentration of 5% (v/v) (Fig.
9). We could not assess the structural
features of the partially structured intermediate state(s) accumulated at 15% (v/v) TFE because the protein (even at 0.1 mM
concentration) aggregates seriously after sample preparation (in 15%
(v/v) TFE). Under these circumstances, the structural characteristics
of the partially structured intermediate state(s) were predicted from the 1H,15N chemical shift perturbation observed
in the HSQC spectrum at 8% (v/v) TFE. 1H,15N
HSQC spectrum of the protein in 8% (v/v) TFE shows that many cross-peaks (in the spectrum) undergo significant chemical shift perturbation (Fig. 9B). These spectral features are
indicative of gross conformational changes leading to the protein. Many
cross-peaks corresponding to residues in the secondary structure
regions show prominent chemical shifts in perturbation. Interestingly,
residues (Lys-26, Lys-142, Ala-143, Leu-145, Leu-147, Leu-149, and
Asp-154) that bridge the N- and C-terminal ends of the nFGF-1 molecule through hydrogen bonds also show significant chemical shift
perturbation, (Ref. 31, Fig. 9B). This observation strongly
suggests that the native
-barrel architecture is disrupted in the
partially structured intermediate state(s) that accumulates before
fibril formation. Although the structural features of the protein in 8% (v/v) TFE and 15% (v/v) TFE (wherein the partially structured state(s) is maximally populated) cannot be directly compared, it may
not be far-fetched to infer that the conformational flexibility of the
protein in the partially structured intermediate state(s) (at 15%
(v/v) TFE) could be similar (if not higher) to that observed in 8%
(v/v) TFE. The 1H,15N HSQC spectra of the
protein in 70% (v/v) TFE reveals that the chemical shift dispersion of
the cross-peaks is vastly diminished, implying that large portions of
the protein molecules are in a disordered conformation (Fig.
9A). The far-UV CD and NMR data (obtained in 70% (v/v) TFE)
analyzed in conjunction suggests that the protein possibly exists
primarily as a random coil with only small portions of the polypeptide
backbone existing in non-native helix conformation. It should be
mentioned that in the absence of detailed triple resonance data, the
chemical shift indices of the resonances
(1H,15N) in 70% (v/v) TFE cannot be estimated.
This aspect precludes the assignment of non-native helical segment
formed in the protein in 70% (v/v) TFE.

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Fig. 9.
1H,15N HSQC spectra of
nFGF-1 in various concentrations of TFE (panel A).
Panel B depicts the 1H,15N chemical
shift perturbation in nFGF-1 in 8% (v/v) TFE. The protein undergoes
gross conformational changes in 8% (v/v) TFE. Amide protons of
residues Lys-26, Lys-142, Ala-143, Leu-145, Leu-147, Leu-149, and
Asp-154 (indicated at the top of the respective
bars in panel B) connecting the N- and C-terminal
ends of the protein (through hydrogen bonds) also show significant
chemical shift perturbation. The 1H,15N
chemical shift perturbation data shown in panel B represents
the weighted average (of 15N and 1H) (31)
chemical shift differences of residues in the protein in 0% (v/v) TFE
and 8% (v/v) TFE. The drastic decrease in the dispersion of the
cross-peaks in the HSQC spectrum of the protein in 70% (v/v) TFE
indicates that most portions of the protein exist in a disordered
state(s). Only small portions of the sequence of nFGF-1 appear to
assume non-native helical conformation in 70% (v/v) TFE.
|
|
Mechanism of Amyloid-like Fibril Formation--
We are tempted to
propose a model for the TFE-induced amyloid-like fibrils in nFGF-1. It
is well known that alcohols such as TFE exert mild denaturant effects
by partially disorganizing the tertiary and quaternary structures of
proteins (49, 50). The denaturant effects of TFE on proteins primarily
stem from their low polarity, which weakens the hydrophobic
interactions that stabilize the compact native structures of proteins
(51). In this background, the first conformational transition induced by TFE (at concentrations greater than 10% (v/v)) appears to be the
disorganization of the hydrophobic contacts stabilizing the native
-barrel structure leading to the formation of a sticky partially
structured intermediate state(s) (Fig.
10). The presence of solvent-exposed,
non-polar surface(s) in the partially structured state(s) (populated in
15% (v/v) TFE) is evident from the high binding affinity of the
protein in the intermediate state(s) to ANS (Fig. 8). Similarly, the
disorganization of the native
-barrel architecture is probably
reflected in the significant chemical shift perturbation of most of the
cross-peaks in the 1H,15N HSQC spectrum in 8%
TFE and the loss of the positive ellipticity band at 228 nm with the
concomitant appearance of the negative CD band at 218 nm (Figs. 2 and
9). The loss of the native
-barrel architecture is also evident from
the FT-IR spectrum acquired in 15% (v/v) TFE, wherein the 1618 and
1639 cm
1 amide I doublet bands characterizing the
-barrel structure disappear (Fig.
11). Aggregation of the protein
(observed upon prolonged incubation (>3 h) of the protein in the TFE
concentration range of 10 to 40% (v/v)) is probably triggered by the
coalescence of the sticky extended
-sheet elements in the partially
structured intermediate state(s) (Fig. 10). The amyloid-like fibril
formation appears to arise due to the rearrangement and annealing of
the extended
-sheet elements through intermolecular hydrogen bond formation. Formation of intermolecular
-sheets is corroborated by
the presence of the 1688-cm
1 band in the FT-IR spectrum
of the TFE-induced amyloid-like fibril (8) (Fig. 11). In addition to
the intermolecular hydrogen bonds that favor aggregation, the enhanced
solvent exposure of the non-polar side chains in the protein (in the
non-native extended
-sheet conformation) also appears to provide a
conducive environment for the condensation of the polypeptide chain to
form a higher order aggregates, such as the amyloid-like fibrils (Fig.
10). The inhibition of aggregation observed in higher concentrations of TFE (>50% (v/v) TFE) is possibly due to weakening of the hydrophobic interactions stabilizing the intermolecular
-sheet structures in the
aggregates (50, 51). The loss of non-polar contacts (at higher
concentration of TFE) appears to favor the formation of intramolecular
hydrogen bonds (over intermolecular hydrogen bonds) conducive for the
induction and formation of helix conformation. These results
clearly suggest that protein aggregation is intricately linked to the
backbone conformation (52, 53). Induction of non-native
-sheet
conformation appears to promote aggregation (subsequently to form
fibrils), and formation of non-native helical segments in the backbone
appears to inhibit aggregation. Therefore, these findings could provide
useful information for the design of new strategies to thwart protein
aggregation in vivo and in vitro. In addition,
although TFE is not a physiologically relevant solvent, the
-sheet
to
-helix structural transition induced by TFE could be used as a
useful model to understand the conformational switch of the prion
protein from the soluble
-helical form to the
-sheet conformation
leading to amyloid fibril formation.

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Fig. 11.
Resolution-enhanced second derivative FT-IR
spectra of nFGF-1 in its native (0% TFE) and the amyloid-like fibril
(in 15% TFE) forms. It could be observed that the 1618 and 1639 cm 1 amide I bands signifying the native -barrel
structure disappear in the amyloid-like fibril to yield a new band at
1625 cm 1 (indicating the formation of a extended
parallel/antiparallel -sheets). Amide I bands at 1661 and 1688 cm 1 are representative of the intermolecular -sheet
interactions in the amyloid-like fibrils.
|
|
In general, the results of the present study support the proposal of
Dobson and coworkers (21) that formation of amyloid fibrils is a
generic property of peptides and proteins and not one restricted to the
particular species observed in diseased states. Although TFE is not a
physiologically relevant solvent, understanding the mechanism of
TFE-induced amyloid formation in proteins (such as nFGF-1) provides
valuable insights into the fibrillation process.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Ing-Ming Chiu for providing
the clone for nFGF-1. We also thank for Prof. Gu-Gang Chang, Prof.
Yen-Chung Chang, and Prof. Ann Shyn Chiang for cooperation.
 |
FOOTNOTES |
*
This work was supported by the National Science Council of
Taiwan.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.
To whom correspondence should be addressed. Fax:
886-35-711082; E-mail: cyu@mx.nthu.edu.tw.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M300336200
2
S. Srisailam, T. K. S. Kumar, D. Rajalingam, and C. Yu, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
TFE, 2,2,2-trifluoroethanol;
nFGF-1, newt acidic fibroblast growth factor;
ThT, thioflavin T;
ANS, 1-anilino-8-napthalene sulfonate;
HSQC, heteronuclear single quantum coherence;
FT-IR, Fourier transform
infrared spectroscopy.
 |
REFERENCES |
1.
|
Chan, W.,
Helms, L. R.,
Brooks, I.,
Lee, G.,
Ngola, S.,
McNulty, D.,
Maleeff, B.,
Hensley, P.,
and Wetzel, R.
(1996)
Fold. Des.
1,
77-89[Medline]
[Order article via Infotrieve]
|
2.
|
Klein, J.,
and Dhurjati, P.
(1995)
Appl. Environ. Microbiol.
61,
1220-1225[Abstract]
|
3.
|
Finke, J. M.,
Gross, L. A.,
Ho, H. M.,
Sept, D.,
Zimm, B. H.,
and Jennings, P. A.
(2000)
Biochemistry
39,
15633-15642[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Nielsen, L.,
Khurana, R.,
Coats, A.,
Frokjaer, S.,
Brange, J.,
Vyas, S.,
Uversky, V. N.,
and Fink, A. L.
(2001)
Biochemistry
40,
6036-6046[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Uversky, V. N.,
Li, J.,
and Fink, A. L.
(2001)
J. Biol. Chem.
276,
44284-44296[Abstract/Free Full Text]
|
6.
|
Jiang, X.,
Buxbhaum, J. N.,
and Kelley, J. W
(2001)
Proc. Natl. Acad. Sci. U. S. A.
277,
1310-1315
|
7.
|
Seshadri, S.,
Khurana, R.,
and Fink, A. L.
(1999)
Methods Enzymol.
309,
559-576[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Harper, J. D.,
and Lansbury, P. T.
(1997)
Annu. Rev. Biochem.
66,
355-407
|
9.
|
Caughey, B.
(2001)
Trends Biochem. Sci.
26,
235-242[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Hamada, D.,
and Dobson, C. M.
(2002)
Protein Sci.
11,
2417-2426[Abstract/Free Full Text]
|
11.
|
Shtileman, M. D.,
Ding, T. T.,
and Lansbury, P. T.
(2002)
Biochemistry
41,
3855-3860[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Cohberg, J. A.,
Li, J.,
Uversky, V. N.,
and Fink, A. L.
(2002)
Biochemistry
41,
1502-1541[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Caughey, B.
(2001)
Philos. Trans. R. Soc. Lond. B. Biol. Sci.
356,
197-202[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Padrick, S. B.,
and Miranker, A. D.
(2001)
J. Mol. Biol.
78,
783-794
|
15.
|
Come, J. H.,
Fraser, P. E.,
and Lansbury, P. T.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
5959-5963[Abstract]
|
16.
|
Kelly, J. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
930-932[Free Full Text]
|
17.
|
Dobson, C. M.
(1999)
Trends Biochem. Sci.
24,
329-332[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Bouchard, M.,
Zurdo, J.,
Nettleton, E. J.,
Dobson, C. M.,
and Robinson, C. V.
(2000)
Protein Sci.
9,
1960-1967[Abstract]
|
19.
|
Tito, P.,
Nettleton, E. J.,
and Robinson, C. V.
(2000)
J. Mol. Biol.
303,
267-278[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Kad, N. M.,
Thomson, N. H.,
Smith, D. P.,
Smith, D. A.,
and Radford, S. E.
(2001)
J. Mol. Biol.
313,
559-571[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Fandrich, M.,
Fletcher, M. A.,
and Dobson, C. M.
(2001)
Nature
410,
165-166[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
MacPhee, C. E.,
and Dobson, C. M.
(2000)
J. Am. Chem. Soc.
122,
12707-12713[CrossRef]
|
23.
|
Pertinhez, T. A.,
Bouchard, M.,
Tomlinson, E. J.,
Wain, R.,
Ferguson, S. J.,
Dobson, C. M.,
and Smith, L. J.
(2001)
FEBS Lett.
495,
184-186[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Zurdo, J.,
Guijarro, J. L.,
Jimenez, J. L.,
Saibil, H. R.,
and Dobson, C. M.
(2001)
J. Mol. Biol.
311,
325-340[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Lai, Z.,
Colon, W.,
and Kelly, J. W.
(1996)
Biochemistry
35,
6470-6482[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Booth, D. R.,
Sunde, M.,
Belloti, V.,
Robinson, C. V.,
Hutchinson, W. L.,
Fraser, P. E.,
Hawkins, P. N.,
Dobson, C. M.,
Radford, S. E.,
Blake, C. C.,
and Pepys, M. B.
(1997)
Nature
385,
787-793[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Bucciantini, M.,
Giannoni, E.,
Chiti, F.,
Baroni, F.,
Formgli, L.,
Zurdo, J.,
Taddei, N.,
Ramponi, G.,
Dobson, C. M.,
and Stefani, M.
(2002)
Nature
416,
507-511[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Uversky, V. N.,
Li, J.,
and Fink, A. L.
(2001)
J. Biol. Chem.
276,
10737-10744[Abstract/Free Full Text]
|
29.
|
McParland, V. J.,
Kad, N. M.,
Kalverda, A. P.,
Brown, A.,
Kirwin Jones, P.,
Hunter, M. G.,
Sunde, M.,
and Radford, S. E.
(2000)
Biochemistry
39,
8735-8746[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Khurana, R.,
Gilleppie, J. R.,
Talapatra, A.,
Minert, L. J.,
Jonesiu-Zanetti, C.,
Millett, I.,
and Fink, A. L.
(2001)
Biochemistry
40,
3525-3535[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Arunkumar, A. I.,
Kumar, T. K. S.,
Kathir, K. M.,
Srisailam, S.,
Wang, H. M.,
Leena, P. S. T.,
Chi, Y. H.,
Chen, H. C.,
Wu, C. H.,
Wu, R. T.,
Chang, G. G.,
Chiu, I. M.,
and Yu, C.
(2002)
Protein Sci.
11,
1050-1061[Abstract/Free Full Text]
|
32.
|
Arunkumar, A. I.,
Srisailam, S.,
Kumar, T. K. S.,
Kathir, K. M.,
Peng, C. L.,
Chen, C.,
Chiu, I. M.,
and Yu, C.
(2000)
J. Biomol. NMR
17,
279-280[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Chang, C. T.,
Wu, C. S.,
and Yang, J. T.
(1978)
Anal. Biochem.
91,
13-31[Medline]
[Order article via Infotrieve]
|
34.
|
Sreerama, N.,
and Woody, R. W.
(2000)
Anal. Biolchem.
287,
252-260[CrossRef]
|
35.
|
Chiti, F.,
Taddei, N.,
Baroni, F.,
Capanni, C.,
Stefani, M.,
Ramponi, G.,
and Dobson, C. M.
(2002)
Nat. Struct. Biol.
9,
137-143[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Taddei, N.,
Capanni, C.,
Chiti, F.,
Stefani, M.,
Dobson, C. M.,
and Ramponi, G.
(2001)
J. Biol. Chem.
276,
37149-37154[Abstract/Free Full Text]
|
37.
|
Glenner, G. G.
(1980)
N. Engl. J. Med.
302,
1333-1343[Medline]
[Order article via Infotrieve]
|
38.
|
Inouye, H.,
and Kirschner, D. A.
(2000)
J. Struct. Biol.
130,
123-129[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Sunde, M.,
and Blake, C.
(1997)
Adv. Protein. Chem.
50,
123-159[Medline]
[Order article via Infotrieve]
|
40.
|
Mullins, J.
(1993)
Crystallization
, 3rd Ed.
, Butterworth-Heinemann, Oxford
|
41.
|
Morozova-Roche, L. A. J.,
Zurdo, J.,
Spencer, A.,
Noppe, W.,
Pepys, M.,
Receveru, V.,
and Dobson, C. M.
(2000)
J. Struct. Biol.
130,
339-351[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Krebs, M. R. H.,
Wilkins, D. K.,
Chung, E. W.,
Pitkeathly, M. C.,
Chamberlain, A. K.,
Zurdo, J.,
Robinson, C. V.,
and Dobson, C. M.
(2000)
J. Mol. Biol.
300,
541-549[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Srisailam, S.,
Wang, H. M.,
Kumar, T. K. S.,
Rajalingam, D.,
Sivaraja, V.,
Sheu, H. S.,
Chang, Y. C.,
and Yu, C.
(2002)
J. Biol. Chem.
277,
19027-19036[Abstract/Free Full Text]
|
44.
|
Samuel, D.,
Kumar, T. K. S.,
Srimathi, T.,
Hsieh, H.,
and Yu, C.
(2000)
J. Biol. Chem.
275,
34968-34975[Abstract/Free Full Text]
|
45.
|
Samuel, D.,
Kumar, T. K. S.,
Balamurugan, K.,
Lin, W. Y.,
Chin, D. H.,
and Yu, C.
(2001)
J. Biol. Chem.
276,
4134-4141[Abstract/Free Full Text]
|
46.
|
Dabora, J. M.,
Sanyal, G.,
and Middaugh, C. R.
(1991)
J. Biol. Chem.
256,
23637-23640
|
47.
|
Kuwajima, K.
(1989)
Proteins Struct. Funct. Genet.
6,
87-103[Medline]
[Order article via Infotrieve]
|
48.
|
Schulman, B. A.,
Kim, P. S.,
Dobson, C. M.,
and Redfield, C.
(1997)
Nat. Struct. Biol.
6,
630-634
|
49.
|
Rajan, R.,
and Balaram, P.
(1996)
Int. J. Pept. Protein Res.
48,
328-336[Medline]
[Order article via Infotrieve]
|
50.
|
Arunkumar, A. I.,
Kumar, T. K. S.,
and Yu, C.
(1997)
Biochim. Biophys. Acta
1138,
69-76
|
51.
|
Thomas, P. D.,
and Dill, K. A.
(1993)
Protein Sci.
2,
2050-2065[Abstract/Free Full Text]
|
52.
|
Srisailam, S.,
Kumar, T. K. S.,
Srimathi, T.,
and Yu, C
(2002)
J. Am. Chem. Soc.
124,
1884-1888[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Uversky, V. N.,
Gillepse, J. A.,
and Fink, A. L.
(2000)
Proteins
41,
415-427[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.