From the Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064
Received for publication, December 4, 2000, and in revised form, January 4, 2001
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ABSTRACT |
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Intracellular proteinaceous aggregates (Lewy
bodies and Lewy neurites) of Sporadic Parkinson's disease is the second most common
neurodegenerative disease and the most common age-related movement disorder. Parkinson's disease arises from the loss of dopaminergic neurons in the substantia nigra and is accompanied by the presence of
intracellular inclusions, Lewy bodies and Lewy neurites (1). Abundant
Lewy bodies and Lewy neurites in the cerebral cortex are also
pathological hallmarks of dementia with Lewy bodies, a common late-life
dementia that is clinically similar to Alzheimer's disease (2).
Furthermore, Lewy bodies have been detected in the Lewy body variant of
Alzheimer's disease (3). Structurally, Lewy bodies and Lewy neurites
are composed of filamentous and granular material (4), the major
component of which is The aggregation behavior of recombinant wild-type Unfortunately, little is currently known about the structural basis for
Materials--
Thioflavin T
(ThT)1 and
1-anilinonaphthalene-8-sulfonic acid (ANS) were obtained from Sigma.
All other chemicals were of analytical grade and were from Fisher.
Fibril Formation--
Solutions of 0.5 ml of Analysis of Fibrillation Kinetics--
The kinetics of
Circular Dichroism Measurements--
CD spectra were obtained
with an Aviv 60DS spectrophotometer using an Fluorescence Measurements--
Fluorescence measurements were
performed in semimicro-quartz cuvettes (Hellma) with a 1-cm excitation
light path using a FluoroMax-2 spectrofluorometer (Instruments S. A.,
Inc.). ThT fluorescence was recorded immediately after addition of the
aliquots to the ThT mixture from 470 to 560 nm with excitation at 450 nm, an increment of 1 nm, an integration time of 1 s, and slits of
5 nm for both excitation and emission. For each sample, the signal was
obtained as the ThT intensity at 482 nm from which was subtracted a
blank measurement recorded prior to addition of FTIR Spectra--
Attenuated total reflectance data were
collected on a Nicolet 800SX FTIR spectrometer equipped with an
MCT detector. The internal reflectance element (72 × 10 × 6 mm, 45° germanium trapezoid) was held in a modified
SPECAC out-of-compartment attenuated total reflectance apparatus. The
hydrated thin films were prepared as described previously (31, 32).
Typically, 1024 interferograms were co-added at
4-cm Small Angle X-ray Scattering Experiments--
Small angle x-ray
scattering (SAXS) measurements were made using Beam Line 4-2 at the
Stanford Synchrotron Radiation Laboratory (33). X-ray energy was
selected at 8980 eV (copper edge) by a pair of Mo/B4C
multilayer monochromator crystals (34). Scattering patterns were
recorded by a linear position-sensitive proportional counter, which was
filled with an 80% xenon and 20% CO2 gas mixture. Scattering patterns were normalized by incident x-ray fluctuations, which were measured with a short length ion chamber before the sample.
The sample-to-detector distance was calibrated to be 230 cm using a
cholesterol myristate sample. The measurements were performed in a
1.3-mm path length observation static cell with 25-µm mica windows.
To avoid radiation damage of the sample in SAXS measurements, the
protein solution was continuously passed through a 1.3-mm path length
observation flow cell with 25-µm mica windows. Background
measurements were performed before and after each protein measurement
and then averaged before being used for background subtraction. All
SAXS measurements were performed at 23 ± 1 °C.
The radius of gyration (Rg) was calculated according
to the Guinier approximation (35), ln I(Q) = ln(0) On the one hand, as noted, A fundamental question is what forces or factors will cause a natively
unfolded protein to fold. It has been shown that natively unfolded
proteins are characterized by a unique combination of low overall
hydrophobicity and large net charge (28). Based on this observation, it
is reasonable to suggest that any alterations in the protein
environment leading to an increase in its hydrophobicity and/or a
decrease in its net charge should be accompanied by at least partial
folding of the intrinsically disordered protein. The overall
hydrophobicity of a protein will increase with increasing temperature
(the hydrophobic interaction has the unusual property of increasing in
magnitude at higher temperatures due to the large change in heat
capacity with temperature and the complex thermodynamic properties
associated with changes in water structure as the temperature increases) (37), and the excess negative charge of The advantages of using high temperatures or lower pH are that these
conditions are anticipated to dramatically increase the population of
putative partially folded intermediates, thus making it easier to
characterize them. Such intermediates will be in equilibrium with the
natively unfolded conformation and presumably are not populated
significantly under normal physiological conditions.
Effect of pH on Far-UV Circular Dichroism--
Fig.
1A represents the far-UV CD
spectra of human recombinant
The question arises as to whether these spectral changes correspond to
a small fraction of the total protein being in the intermediate
conformation, in which case the intermediate has a large negative
ellipticity in the vicinity of 220 nm, or whether essentially all the
molecules are present as the intermediate, in which case it has a small
ellipticity at 220 nm. Based on the small angle x-ray scattering
results (see below), we can conclude that the majority of the molecules
are in the same conformation, and thus, the intermediate possesses
limited secondary structure.
ANS Fluorescence--
Changes in ANS fluorescence are frequently
used to detect non-native partially folded conformations of globular
proteins (38-40). This is because such intermediates are characterized
by the presence of solvent-exposed hydrophobic clusters to which ANS
binds, resulting in a considerable increase in the ANS fluorescence
intensity and in a pronounced blue shift of the fluorescence emission
maximum. Fig. 1 shows that, in the case of
Fig. 1B shows that the pH-induced structural transitions
observed by ANS fluorescence and CD ellipticity changed simultaneously in a rather cooperative manner. This means that protonation of Secondary Structure Analysis by FTIR--
FTIR represents a
powerful method for the investigation of protein secondary structure.
The main advantage of this approach in comparison with CD is that FTIR
is much more sensitive to Small Angle x-ray Scattering Studies--
SAXS is a very useful
method for the investigation of conformation, shape, and dimensions of
biopolymers in solution. Analysis of the scattering curves using the
Guinier approximation gives information about Rg.
Presentation of the scattering data in the form of Kratky plots
provides information about the globularity (packing density) and
conformation of a protein (35). For a native globular protein, this
plot has a characteristic maximum, whereas unfolded and partially
folded polypeptides have significantly different-shaped Kratky plots.
Guinier analysis of scattering data shows that, at neutral pH,
Analysis of the x-ray scattering in the form of a Kratky plot shows
that
The SAXS forward scattering intensity values give information on the
degree of protein association. The results of this analysis for
Effect of pH on
Fig. 4 represents
time-dependent changes in the ThT fluorescence during the
process of
Fig. 4 shows that decreasing the pH resulted in a very
substantial acceleration of the kinetics of Elevated Temperature Causes a Conformational Transition in
Effect of Temperature on the Secondary Structure of
Effect of Temperature on -synuclein are hallmarks of
neurodegenerative diseases such as Parkinson's disease, dementia with
Lewy bodies, and multiple systemic atrophy. However, the molecular
mechanisms underlying
-synuclein aggregation into such filamentous
inclusions remain unknown. An intriguing aspect of this problem is that
-synuclein is a natively unfolded protein, with little or no ordered
structure under physiological conditions. This raises the question of
how an essentially disordered protein is transformed into highly
organized fibrils. In the search for an answer to this question, we
have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant
-synuclein. Either a decrease in pH or an increase in temperature
transformed
-synuclein into a partially folded conformation. The
presence of this intermediate is strongly correlated with the enhanced formation of
-synuclein fibrils. We propose a model for the
fibrillation of
-synuclein in which the first step is the
conformational transformation of the natively unfolded protein into the
aggregation-competent partially folded intermediate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-synuclein (5, 6). Two different missense
mutations in the
-synuclein gene, corresponding to A53T and A30P
substitutions, have been identified in two kindreds with autosomal
dominantly inherited, early-onset Parkinson's disease (7, 8).
-Synuclein appears to be the major component of neuronal and glial
inclusions in multiple system atrophy and Hallervorden-Spatz disease
(9-12). Thus, the accumulation of
-synuclein-derived fibrillar
material represents a critical biomedical problem.
-Synuclein is a 140-amino acid protein of unknown function that was
first isolated from synaptic vesicles of Torpedo californica and rat brain (13, 14). Purified human
-synuclein has been shown to
be unstructured in aqueous solution and hence extremely heat-stable
(15). The amino acid sequence of
-synuclein is characterized by six
imperfect repeats (consensus KTKEGV) within the N-terminal half of the
polypeptide as well as by a highly acidic C-terminal region (14,
17). Although the function of
-synuclein is unclear, it is
predominantly concentrated in the cytosol and presynaptic nerve
terminals of neurons, with some fractions associated with synaptic
vesicle membranes (14, 18-20). The repeat sequences in
-synuclein
are predicted to form amphipathic helixes that can associate with lipid
vesicles (18), and it has been shown to bind to small synthetic
unilamellar vesicles (21), acidic and neutral phospholipid vesicles
(22), and rat brain vesicles (23). It has recently been reported that
-synuclein may regulate the size of presynaptic vesicular pools
(24). These studies suggest that
-synuclein may play a role in
neurotransmission or in the organization and regulation of synaptic
vesicles. The potential role of
-synuclein deposition in several
neurodegenerative diseases has focused attention on this protein.
-synuclein and its
A30P and A53T mutants has been studied under in vitro physiological conditions. It has been established that all three proteins, as well as the 1-87 and 1-120 truncated forms of
recombinant
-synuclein, are able to assemble readily into filaments
with morphologies and staining characteristics similar to those
extracted from disease-affected brain (25-27).
-synuclein fibrillation. A major fundamental question concerns how
the essentially disordered ("natively unfolded") protein is
transformed into the highly organized fibrils with characteristic
crossed
-conformation. As a start to unravel the "mystery" of
-synuclein fibrillation, we describe the effects of pH and
temperature on the structural and fibrillation properties of human
-synuclein. Since most aggregating protein systems probably involve
a transient partially folded intermediate as the key precursor to
fibrillation, we sought conditions that would be expected to favor such
a conformation for
-synuclein. The natively unfolded character of
-synuclein arises from its low intrinsic hydrophobicity and high net
charge at neutral pH (pI 4.7) (28). Thus, conditions that decrease the
net charge and that increase the hydrophobicity would be expected to
result in partial folding. Acidic pH and elevated temperatures provide
such conditions, respectively. The strong correlation observed between
the degree of protein folding and the efficiency of its fibril
formation suggests that the intermediate can be a precursor of fibrils.
Although low pH and high temperature are unphysiological, they provide
a useful model by increasing the concentration of the critical intermediate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Synuclein Expression and Purification--
A new procedure
for producing and purifying
-synuclein was developed by fusing its
gene to a chitin-binding domain/intein system (IMPACT, New England
Biolabs Inc.) and expressing the fusion protein in Escherichia
coli. This system allowed simple purification of
-synuclein by
binding of the chitin-binding domain fusion protein to a chitin column,
followed by addition of a thiol agent (dithiothreitol or cysteine) to
induce the cleavage reaction of the intein and to release the
-synuclein. The resultant protein, which was highly homogeneous upon
polyacrylamide gel electrophoresis, was indistinguishable from
authentic
-synuclein based on its electrophoretic mobility and
molecular mass determined by electrospray mass spectrometry.
-synuclein at pH
7.5 in 20 mM Tris buffer or at pH 3.0 in 20 mM
acetate buffer were stirred at 37 °C in glass vials with micro-stir
bars. Protein concentration was 0.5 mg/ml. Fibril formation was
monitored with thioflavin T fluorescence (29, 30). Aliquots of 10 µl
were removed from the incubated sample and added to 1.0 ml of 25 µM ThT in 50 mM Tris buffer (pH 8.0) (see
below). The presence of fibrils was confirmed by electron microscopy
(negative staining with uranyl acetate) and atomic force microscopy.
-synuclein fibril formation could be described as sigmoidal curves
defined by an initial lag phase, in which a negligible change in ThT
fluorescence intensity was observed; a subsequent exponential growth
phase, in which ThT fluorescence increased; and a final equilibrium
phase, in which ThT fluorescence reached a plateau, indicating the end
of fibril formation. ThT fluorescence measurements were plotted as a
function of time and fitted to a curve described by Equation 1,
where Y is the fluorescence intensity and
xo is the time to 50% of maximal fluorescence. The
initial base line during the lag phase is described by
yi + mix. The final
base line after the growth phase had ended is described by
yf + mfx. The apparent first-order rate
constant (kapp) for the growth of fibrils is
calculated as 1/
(Eq. 1)
, and the lag time is calculated as
xo
2
. This expression
is unrelated to the underlying molecular events, but provides a
convenient method for comparison of the kinetics of fibrillation.
-synuclein
concentration of 0.5 mg/ml. Spectra were recorded in a 0.01-cm cell
from 250 to 190 nm with a step size of 0.5 nm, a bandwidth of 1.5 nm,
and an averaging time of 10 s. For all spectra, an average of five
scans was obtained. CD spectra of the appropriate buffers were recorded
and subtracted from the protein spectra.
-synuclein to the
ThT solution. ANS emission spectra were recorded from 460 to 600 nm with excitation at 350 nm, an increment of 1 nm, an integration time of
1 s, and slits of 5 nm for both excitation and emission. All data
were processed using DataMax/GRAMS software.
1 resolution. Data analysis was performed
with GRAMS32 (Galactic Industries Corp.). Secondary structure content
was determined from curve fitting to spectra deconvoluted using second
derivatives and Fourier self-deconvolution to identify component band
positions. Hydrated thin film samples were prepared by drying 50 µl
of 1 mg/ml
-synuclein solution on a ZnSe crystal with dry
N2. The IR spectra were collected, followed by Fourier
transformation using the spectrum of the clean crystal as a background.
Water (liquid and vapor) components were subtracted from the protein spectrum.
Rg2/Q2/3, where
Q is the scattering vector given by Q = (4
sin
)/
, where 2
is the scattering angle and
is the
wavelength of x-ray. I(0), the forward scattering amplitude,
is proportional to
n·
c2·V2,
where n is the number of scatterers (protein molecules) in
solution,
c is the electron density difference between the
scatterer and the solvent, and V is the volume of the
scatter. This means that the value of forward scattered intensity,
I(0), is proportional to the square of the molecular mass of
the molecule (35). Thus, I(0) for a pure n-mer
sample will therefore be n-fold that for a sample with the
same number of monomers since each n-mer will scatter
n2 times as strongly as monomer; but in this
case, the number of scattered particles (n-mers) will be
n times less than that in the pure monomer sample.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-synuclein belongs to the class of
natively unfolded proteins, which have little or no ordered structure
in the purified state under physiological conditions (15); and on the
other, it forms fibrils of highly organized secondary structure. For
example, x-ray diffraction analysis of
-synuclein fibrils shows the
characteristic pattern of a crossed
-sheet structure, in which the
-strands lie perpendicular to the long fiber axis, typical of all
amyloid fibrils (27). Electron microscopy analysis indicates that human
-synuclein filaments are typically 6-9 nm in width and several
microns long (27). Clearly, within the fibril,
-synuclein cannot be
present as an extended linear polymer since, in a fully extended
conformation, the maximal linear dimension of a polypeptide with
n residues is n × 3.63 Å (36). This gives
~51 nm for
-synuclein, which is at least five times greater than
the diameter of the filament.
-synuclein at
neutral pH (pI 4.7) would be neutralized at lower pH values. Thus, we
can expect partial folding of
-synuclein under conditions of high
temperature and/or low pH. In contrast to an unfolded protein, a
partially folded intermediate is anticipated to have contiguous
hydrophobic patches on its surface, which are likely to foster
self-association and hence potentially fibrillation.
-Synuclein Structure and Fibrillation
-synuclein measured at different pH
values at 20 °C. At neutral pH, the protein possesses a far-UV CD
spectrum typical of an essentially unfolded polypeptide chain. The
spectrum has an intense minimum in the vicinity of 196 nm, with the
absence of characteristic bands in the 210-230 nm region. However, as
the pH was decreased, changes were observed in the shape of the
spectrum. Fig. 1 shows that the minimum at 196 nm became less intense,
whereas the negative intensity of the spectrum around 222 nm increased,
reflecting pH-induced formation of secondary structure. The pH
dependence of [
]222 is shown in Fig. 1B.
There was little change in the far-UV CD spectrum between pH ~9.0 and
~5.5. However, a decrease in pH from 5.5 to 3.0 resulted in a
~2-fold increase in negative intensity in the vicinity of 220 nm, and
a further decrease in pH was accompanied by a reversal in the spectral
intensity. Fig. 1B shows that the pH-induced changes in the
far-UV CD spectrum of
-synuclein were completely reversible (compare
open and closed symbols) and were independent of
protein concentration (at least in the range of 0.1-1.5 mg/ml)
(compare circles and squares). These observations
are consistent with the assumption that the pH-induced increase in the
structure of
-synuclein represents an intramolecular process and not
self-association. Based on additional structural probes, described
below, we interpret these far-UV CD changes to reflect the formation of
a partially folded intermediate with significant
-structure. The
fact that the far-UV CD spectra of
-synuclein as a function of pH
show an isosbestic point (Fig. 1A) indicates that the
transition is a two-state one, presumably between the natively unfolded
state and the partially folded intermediate conformation.
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Fig. 1.
Effect of pH on the structural properties
of -synuclein. A, far-UV CD
spectra as a function of pH. pH values were 8.9 (solid
line), 7.3, 6.4, 5.3, 4.3, 3.5, 2.7, 1.7, and 0.9 in order of the
increase in negative [
]222 value. The inset
represents ANS fluorescence spectra measured at pH 8.2, 7.5, 6.6, 5.4, 4.6, 4.0, 3.7, 3.1, 2.8, and 2.5 (in order of increasing intensity).
B, comparison of the effect of pH on far-UV circular
dichroism (circles and squares) and ANS
fluorescence (triangles) spectra. The results of the initial
titration (decrease in pH) and reverse (increase in pH) experiments are
presented as open and closed symbols,
respectively. The cell path length was 0.1 and 10 mm for far-UV CD and
fluorescence measurements, respectively. Measurements were carried out
at 20 °C. Protein concentration was 0.1 (circles), 1.0 (squares), and 0.01 (triangles) mg/ml. Data on
the pH effect on the lag time of
-synuclein fibrillation
(
) are also shown for comparison. deg,
degrees.
-synuclein, a decrease in
pH led to a large blue shift of the ANS fluorescence maximum (from ~515 to ~475 nm) (Fig. 1B, open triangles),
reflecting the pH-induced transformation from the natively unfolded
state to the partially folded compact conformation. The transition from
unfolded to partially folded conformation took place between pH 5.5 and
3.0 and was completely reversible (open and closed
symbols).
-synuclein results in transformation of the natively unfolded protein into a conformation with a significant amount of ordered secondary structure and with affinity for ANS. The position of the
transition indicates that protonation of one or more carboxylates was
responsible for the structural change. Further investigations were
devoted to the structural characterization of this conformation.
-structure. To work with low protein
concentrations, we used attenuated total reflectance FTIR and hydrated
thin films of the sample (31, 32). Fig. 2
shows the FTIR (amide I region) spectra of
-synuclein measured at pH
7.5 and 3.0. The spectrum of
-synuclein fibrils is also presented
for comparison. The FTIR spectrum of
-synuclein at pH 7.5 is typical
of a substantially unfolded polypeptide chain, whereas a decrease in pH
led to significant spectral changes, indicative of increased ordered
structure. The most evident change was the appearance of a new band in
the vicinity of 1626 cm
1, which corresponds
to
-sheet. This means that, at acidic pH, natively unfolded
-synuclein is transformed into a partially folded conformation with
a significant amount of
-structure. As expected, the FTIR spectrum
of
-synuclein fibrils shows the major contribution from
-sheet.
Fourier self-deconvolution (and second derivative) of the FTIR
spectra followed by curve fitting revealed the differences in the
secondary structure in the different conformations. These results are
summarized in Table I. Based on
the SAXS experiments (below), the protein is monomeric at pH 3, and
thus, the increased
-structure is not due to association-induced
-sheet.
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Fig. 2.
Secondary structure analysis of
-synuclein by FTIR. A, FTIR spectra
of the amide I region were measured at pH 7.5 (solid line)
and pH 3.0 (dashed line). The spectrum of
-synuclein
fibrils is shown for comparison (dashed and dotted line).
B, shown are second derivatives of the corresponding
spectra. The major
-structure bands are in the 1620-1640
cm
1 region.
Secondary structure content of human -synuclein determined by FTIR
under different experimental conditions
-synuclein is characterized by an Rg of 40 ± 1 Å (Fig. 3A). At low pH,
this parameter decreases to 30 ± 1 Å at pH 3.0, reflecting
considerable compaction of the protein, corresponding to a volume
decrease of 2.4-fold. The linear Guinier plots indicate that the
solutions are homogeneous, suggesting that, at pH 3, essentially all
the molecules of
-synuclein have the intermediate conformation,
rather than a small fraction. It is interesting to compare measured
Rg values with those calculated for the fully
globular and random-coil polypeptide chains of 140 amino acid residues.
The Rg for a native globular protein composed of
n amino acid residues is given by
RgN = 2.9·n1/3
(41). Thus, if
-synuclein were folded into a globular structure, it
would have RgN = 2.9·1401/3 = 15.1 Å. The radius of gyration of a completely unfolded (random-coil) polypeptide, RgU, may be estimated from the
corresponding Stokes radius, RSU, using the
known relation
RgU/RSU = 1.51 (42). The hydrodynamic radius of a completely unfolded globular
protein, RSU, with known molecular mass may
be calculated from the following empirical equation (43):
log(RSU) = 0.533·(M)
0.682. This gives 34.3 Å for RSU and
52.1 Å for RgU. The observed
Rg for
-synuclein at neutral pH is smaller (40 Å) than that estimated for a random-coil conformation (52 Å),
indicating that the molecule is more compact than a random coil. On the
other hand, the Rg for the partially folded intermediate (30 Å) is much larger than that of a folded globular protein of the size of
-synuclein (15 Å).
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Fig. 3.
Guinier (A) and Kratky
(B) plot representation of the results of small angle
x-ray scattering analysis of -synuclein under
different experimental conditions: pH 7.5 (line 1) and
pH 3.0 (line 2). Measurements were carried out at
23 °C. Protein concentration was 1.0 mg/ml.
-synuclein lacks a well developed globular structure at both pH
7.5 and 3.0 (Fig. 3B). The profile of the Kratky plot at
neutral pH is typical for a random-coil conformation, whereas that at
pH 3 shows changes consistent with the development of the beginnings of
a tightly packed core.
-synuclein indicate that the decrease in pH was not accompanied by a
significant change in this parameter: I(0) was 0.0124 ± 0.0004 at pH 7.5 and 0.0119 ± 0.0003 at pH 3.0. This directly
confirms that the pH-induced folding of
-synuclein is an
intramolecular process and does not result from self-association.
-Synuclein Fibrillation--
The histological
dye thioflavin T is widely used for the detection of amyloid fibrils
(29, 44, 45). In the presence of fibrils, ThT gives rise to a new
excitation maximum at 450 nm and enhanced emission at 482 nm, whereas
unbound ThT is essentially non-fluorescent at these wavelengths. ThT is
believed to interact specifically and rapidly with the crossed
-sheet structure common to amyloid fibrils, and the binding is
independent of the primary structure of the protein. Only the
multimeric fibrillar forms, not multiple
-sheet domains in native
proteins, give significant fluorescence with ThT. The binding of ThT to
-synuclein fibrils is thus a very convenient method for studying the
kinetics of fibril formation.
-synuclein fibril formation at 37 °C as a function of
pH. The kinetics of the ThT fluorescence intensity at 482 nm exhibit
characteristic sigmoidal curves, which have of an initial lag phase, a
subsequent growth phase, and a final equilibrium phase. Such curves are
consistent with a nucleation-dependent polymerization
model, in which the lag corresponds to the nucleation phase, and the
exponential part to fibril growth (elongation) (26, 46-51)
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Fig. 4.
Effect of pH on fibril formation of
human -synuclein. A, kinetics
of fibrillation monitored by the enhancement of thioflavin T
fluorescence intensity. Measurements were performed at 37 °C and pH
8.52 (circles), 7.23 (inverted triangles), 5.82 (squares), 4.08 (diamonds), 2.79 (triangles), and 1.92 (hexagons). Protein
concentration was 1 mg/ml. ThT fluorescence was excited at 450 nm, and
the emission wavelength was 482 nm. B, pH dependence of
kinetic parameters of
-synuclein fibrillation: lag time
(circles) and apparent rate constant of elongation
(squares). The lag times at rates of elongation were
determined as described under "Experimental Procedures."
-synuclein fibrillation: the lag time was ~12 times shorter (2.4 ± 0.06 versus 28.1 ± 0.6 h), and the apparent rate of fibril formation was
~10-fold larger (1.03 ± 0.003 versus 0.11 ± 0.007 h
1) at pH 3.0 compared with pH 7.5. Fig. 4B shows that pH had similar effects on both the lag
time and the elongation rate. Interestingly, the pH dependence of the
kinetic parameters of fibril formation shows transitions similar to the
pH dependences of the
-synuclein structural parameters monitored by
circular dichroism and ANS binding (cf. Figs. 1B
and 4B). Moreover, Fig. 1B shows that the pH-induced changes in the
-synuclein fibrillation kinetics were coincident with the pH-driven structural transformations. In other words, there was an excellent correlation between intramolecular conformational change and fibril formation. This is a very important observation and is consistent with the conclusion that the process of
-synuclein fibrillation is dramatically accelerated by the partial
folding of the natively unfolded protein.
-Synuclein and Facilitates Fibrillation
-Synuclein--
Fig. 5 represents the
far-UV CD spectra of
-synuclein measured at different temperatures.
At low temperatures, the protein shows a far-UV spectrum typical of an
unfolded polypeptide chain. As the temperature was increased, the
spectrum changed, consistent with temperature-induced formation of
secondary structure. The temperature dependence of
[
]222 (Fig. 5, inset) shows that the major
spectral changes occurred over the range of 3 to 50 °C. Further
heating led to a less pronounced effect. Interestingly, Fig. 5 shows
that the structural changes induced in
-synuclein by heating were
completely reversible (cf. open and closed
circles). These data indicate that high temperatures induce a
reversible transition in
-synuclein leading to a partially folded
intermediate. This intermediate has a circular dichroism spectrum
similar to that induced by low pH. Since hydrophobic interactions
increase with increasing temperature, the simplest explanation for the temperature-induced structural formation is that it is driven by
hydrophobic interactions. The enhanced rates of fibrillation at higher
temperatures probably reflect a combination of both faster rates at
higher temperatures and increased concentration of the
aggregation-competent intermediate due to the increased hydrophobic
interactions.
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Fig. 5.
Effect of temperature on the far-UV CD
spectra of -synuclein at pH 7.5. Far-UV
CD spectra were measured at increasing temperatures: 3.0, 9.0, 19.0, 31.0, 39.0, 45.0, 53.0, 62.0, 72.0, 82.0, and 92.0 °C (in order of
the increase in negative [
]222 value). The
inset shows the dependence of [
]222 on
temperature. Data for increasing temperature and cooling are shown as
open and closed circles, respectively.
-Synuclein Fibril Formation--
Fig.
6A shows the effect of
temperature on the kinetics of
-synuclein fibrillation at pH 7.5. The kinetics were strongly affected by temperature, with similar
effects on both the lag time and the elongation rate. The activation
energy values calculated from the slopes of corresponding Arrhenius
plots (Fig. 6B) were 17.9 ± 0.8 and 20.1 ± 0.8 kcal/mol for the nucleation and elongation processes, respectively.
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Fig. 6.
Effect of temperature on
-synuclein fibrillation. A,
kinetics of fibril formation monitored by the enhancement of ThT
fluorescence. Measurements were performed at 27 °C
(circles), 37 °C (inverted triangles),
47 °C (squares), and 57 °C (diamonds).
Protein concentration was 1 mg/ml. ThT fluorescence was excited at 450 nm, and the emission wavelength was 482 nm. The lag times at rates of
elongation were determined as described under "Experimental
Procedures." B, the corresponding Arrhenius plots for the
lag time (circles) and the elongation rate constant
(triangles).
Fibril Morphology from Electron Microscopy
Fig. 7 shows that the fibril
morphology was sensitive to the conditions under which the fibrils were
formed. Fibrils formed at neutral pH and 37 °C were longer and much
less clumped than those formed at higher temperature or low pH. The
latter two conditions led to fibrils that were much shorter in length
and of much higher fibril density. Since it has been frequently
observed that fibrils made in vitro from a variety of
proteins may have morphologies that are very sensitive to the
conditions under which they were grown, we do not attach much
significance to the fact that -synuclein fibril morphology varied
with the choice of incubation conditions.
|
Model for Fibrillation
The data reported herein demonstrate that low pH or elevated
temperature leads to formation of a partially folded intermediate conformation of -synuclein and that these conditions correlate with
enhanced kinetics of fibrillation, suggesting that the intermediate is
an important species on the fibril-forming pathway. The effects of low
pH are attributed to minimization of the large net negative charge
present at neutral pH, thereby decreasing charge-charge intramolecular
repulsion and permitting hydrophobic interaction-driven collapse
to the partially folded intermediate. The effects of elevated
temperatures are attributed to increased strength of the hydrophobic
interaction at higher temperatures, leading to a stronger hydrophobic
driving force for folding. Calculations indicate that the combination
of net charge and intrinsic hydrophobicity of
-synuclein is such
that it is only marginally destabilized at neutral pH; thus, either
relatively small increases in hydrophobicity or decreased charge is
sufficient to promote its partial folding (28).
The results are consistent with the following scheme for -synuclein
fibrillation: UN
I
Nucl
F, where UN, I, Nucl, and
F correspond to the natively unfolded conformation, partially folded intermediate, fibril nucleus, and fibril,
respectively. From this model, we anticipate two key kinetic steps: the
structural transformation leading to the intermediate, and formation of
the nucleus (Fig. 8). Thus, factors that
shift the equilibrium in favor of the intermediate will facilitate
fibril formation, as observed. Fibril elongation could involve the
addition of either the intermediate or the natively unfolded molecule
to existing fibrils or seeds. Fibrillation of
-synuclein, leading to
Lewy body formation and Parkinson's and related Lewy body diseases, thus may arise from various factors that would significantly
populate or increase the concentration of the
aggregation-competent intermediate. Possibilities include nonpolar
molecules (such as some pesticides) that might preferentially bind to
the partially folded intermediate and cations that might mimic the
effect of low pH (high proton concentration) as well as factors that
result in an increase in the concentration of
-synuclein itself.
|
There are two interesting features of the -synuclein partially
folded intermediate: it has some
-structure, which is the major type
of secondary structure in
-synuclein fibrils, and it is relatively
unfolded (i.e. more similar to a random-coil conformation
than a native tightly folded globular conformation). We have also
recently observed that a partially folded intermediate of IgG light
chains that form amyloid fibrils is relatively unfolded (16). Thus, it
remains to be seen if it is a common feature in amyloid fibril
formation that the critical monomeric partially folded intermediate is
relatively unfolded.
The question arises as to the physiological significance of the
observation of a partially folded intermediate of -synuclein formed
under conditions of low pH or high temperature. Clearly, these
particular environmental conditions will not be found in the
dopaminergic cells of potential Parkinson's disease patients. However,
the existence of such an intermediate, on the pathway to fibrils, means
that in vivo conditions that lead to population of the
intermediate will lead to increased risk of the disease. Thus, any
intracellular factors that lead to a shift in the equilibrium position
between the natively unfolded state and the partially folded
intermediate will increase the likelihood of
-synuclein fibril
formation. Such factors could include relatively nonpolar molecules
that would preferentially bind to the intermediate, for example.
Epidemiological studies have implicated insecticides and herbicides as
increased risk factors for Parkinson's disease. In support of such a
connection, we found in preliminary
experiments2 that several
relatively hydrophobic pesticides as well as ANS both induce
conformational changes in
-synuclein and accelerate the rate of
fibril formation, apparently by preferentially binding to the partially
folded intermediate.
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ACKNOWLEDGEMENTS |
---|
We thank Jon Krupp for help with the electron microscopy experiments and Ian Millet for assistance with the small angle x-ray scattering experiments.
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FOOTNOTES |
---|
* This work was supported in part by a grant from the National Institutes of Health.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.
Supported by fellowships from the Parkinson's Institute and the
National Parkinson's Foundation.
§ To whom correspondence should be addressed. Tel.: 831-459-2744; Fax: 831-459-2935; E-mail: enzyme@cats.ucsc.edu.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M010907200
2 V. N. Uversky, J. Li, and A. L. Fink, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ThT, thioflavin T; ANS, 1-anilinonaphthalene-8-sulfonic acid; FTIR, Fourier transform infrared; SAXS, small angle x-ray scattering.
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