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Biochemical and Biophysical Research Communications 368 (2008) 772778

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Sequence determinants regulating brillation of human a-synuclein

Hyun-Jung Koo, Hak-Joo Lee, Hana Im *
Department of Molecular Biology, Sejong University, 98 Gunja-dong, Kwangjin-gu, Seoul 143-747, Republic of Korea
Received 24 January 2008
Available online 7 February 2008

Abstract
a-Synuclein is a neural protein that comprises the brillar core of Lewy bodies, a histologically dening lesion of Parkinsons disease.
To investigate the role of each specic residue of the a-synuclein molecule in bril formation, amino acid substitutions were introduced
throughout the molecule. Incorporation of proline, especially in the region spanning residues 3789, drastically retarded bril formation.
Substitutions with polar residues showed that the hydrophobicity of the central hydrophobic region is also important in brillation regulation. In the N-terminal repeated region, increasing the number of negative charges interfered with brillation. In contrast, single
amino acid substitutions in the C-terminal acidic region of a-synuclein had only minimal eects on brillation. More than 20 dierent
single amino acid substitutions that were sucient to prevent brillation of a-synuclein were obtained, and most of them were impaired
in both nucleation and bril elongation. Identication of sequence determinants regulating brillation of amyloidogenic proteins may
provide valuable information for designing peptide analog drugs to prevent protein amyloidosis.
2008 Elsevier Inc. All rights reserved.
Keywords: Conformational switch; Protein amyloid; Protein brils; Protein folding; a-Synuclein

Parkinsons disease (PD) is the second most common

neurodegenerative disease, with a prevalence of about 2%
of those 65 years of age and older. It results from loss of
dopaminergic neurons in the substantia nigra, which causes
subsequent movement disorders including resting tremor,
rigidity, balance impairment, and slowness of movement
[1]. The intracellular protein aggregates that dene the
intracellular lesions of PD, which are known as Lewy
bodies and Lewy neuritis, are composed primarily of a-synuclein. Involvement of a-synuclein in PD is supported by
the identication of three missense alleles (A30P, A53T,
and E46K) of the a-synuclein gene; these genes are inherited in an autosomal dominant fashion in cases of familial
early-onset PD [2]. Furthermore, overexpression of wildtype (WT) a-synuclein by gene duplication or triplication
is sucient to cause inheritable PD [3]. Overexpression of
a-synuclein in animal models, such as transgenic ies [4]
Abbreviations: PD, Parkinsons disease; ThT, thioavin T; WT, wildtype; NAC, non-amyloid b component; CD, circular dichroism.
*
Corresponding author. Fax: +82 2 3408 4336.
E-mail address: hanaim@sejong.ac.kr (H. Im).
0006-291X/$ - see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.01.140

and mice [5], also caused the neuropathological symptoms

of PD.
The 140-amino acid a-synuclein polypeptide chain
(Fig. 1) contains an N-terminal imperfect repeat (consensus
KTKEGV)-containing region, a central hydrophobic cluster, and a highly acidic C-terminal region [6]. In aqueous
solution, puried human a-synuclein is unstructured [7],
but the b-strand content can be increased, accompanying
formation of protein brils. X-ray and electron diraction
studies suggest that a-synuclein brils have an amyloid-like
cross-b conformation [8].
The N-terminal repeated region transforms into amphipathic helices upon association with lipid vesicles [9], and
the presence in this region of the three missense mutations
associated with familial PD suggest that it has a role in
a-synuclein brillation or pathogenecity of PD [2]. Truncation of the N-terminal repeats promotes formation of bsheet-rich oligomers and brils, and addition of extra
repeats suppresses b-sheet and bril formation [10]. Amino
acid residues 6195 of a-synuclein comprise the central
hydrophobic segment, which is known as the NAC
(non-amyloid b component). The NAC was isolated,

H.-J. Koo et al. / Biochemical and Biophysical Research Communications 368 (2008) 772778
1 MDVFM

KGLSK

AKEGV

VAAAE

KTKQG

*
26 V A E A A

GKTKE

GVLYV

GSKTK

*
E
GVVH

51 G V A T V

AEKTK

EQVTN

VGGAV

VTGVT

76 A V A Q K

TVEGA

GSIAA

ATGFV

KKDQL

101 G K N E E

GAPQE

GILED

MPVDP

DNEAY

126 E M P S E

EGYQD

YEPEA

Fig. 1. Primary sequence of a-synuclein [6]. The 11-residue imperfect

repeats in the N-terminal domain are boxed, and the central hydrophobic
region is shown in bold. The acidic C-terminal region is shown in italics.
The sites of three PD-related point mutations, A30P, E46K, and A53T,
are marked by asterisks.

together with amyloid b fragments, from plaques of postmortem brain tissue of Alzheimer patients [11]. Since bsynuclein, a non-brillogenic member of the synuclein family, lacks residues 7182, which fall in the middle of this
hydrophobic region, the NAC may play a central role in
a-synuclein brillation. Indeed, synthetic peptides corresponding to this sequence promote brillation of a-synuclein in vitro [12]. The C-terminal acidic tail of a-synuclein
contains 10 Glu and 5 Asp residues. This high charge density may be the reason that the C-terminal region acts as a
solubilizing domain responsible for the thermostability of
a-synuclein [13]. Deletion of the tail region accelerates bril
formation, reducing the t of brillation by more than 90%
[14].
Although aggregation of a-synuclein is associated with
pathogenesis of the synucleinopathies, the molecular mechanisms underlying a-synuclein brillation are poorly
understood. Protein aggregation has generally been considered to occur as a nonspecic coagulation of unfolded
polypeptides that is driven by hydrophobic interactions
between inappropriately exposed hydrophobic surfaces.
However, recent studies have shown that protein aggregation is due to highly specic intermolecular interactions
among partially structured folding intermediates [15]. Likewise, brillation of amyloidogenic proteins requires very
specic interactions, as shown by the observation that each
protein can be nucleated by brils formed by peptides of
the same sequence, but not by brils with closely related
sequences [16]. Site-directed spin labeling and electron
paramagnetic resonance spectroscopy of a-synuclein brils
have shown that the central region of a-synuclein is packed
in a highly ordered parallel fashion, and that identical residues from adjacent molecules are in exact register [17].
Since the above observations support the hypothesis that
a-synuclein aggregates via sequence-specic interactions,
we decided to pinpoint the sequence determinants of bril
formation. Our aim was to identify factors that can prevent
the conformational switch of a-synuclein that is associated
with the etiology of PD.
Materials and methods
Cloning, mutagenesis, expression, and purication of a-synuclein. The
cDNA encoding human a-synuclein was amplied by PCR, and restriction

773

endonuclease recognition sites for NdeI and HindIII were introduced at

the N- and C-terminal ends, respectively. The PCR products were cloned
into pAED4 [18], an Esherichia coli expression vector. Substitution
mutations were introduced using oligonucleotide-directed mutagenesis
[19]. a-Synuclein was overexpressed in E. coli BL21(DE3) and puried as
described previously [10]. Protein concentration was measured using BioRad DC protein assay kit (Bio-Rad Laboratories Inc.).
a-Synuclein bril formation. A 350 lM solution of monomeric a-synuclein protein in PBS (1.76 mM KH2PO4, 10 mM Na2HPO4, 2.7 mM KCl,
and 137 mM NaCl, pH 7.4) was incubated at 37 C with shaking at
150 rpm. A 20 ll aliquot was removed at various time points and added to
3 ml of 20 lM thioavin T solution (ThT; Sigma Co.). Fluorescence
emission was measured using a spectrophotometer (RF-5301PC, Shimadzu) with excitation and emission wavelengths of 450 and 482 nm,
respectively, and slit widths of 5 and 3 nm, respectively. The degree of
brillation was also monitored by following the amount of soluble asynuclein protein remaining in solution. Insoluble brils were removed by
centrifugation at 9800g for 30 min, and the supernatant was analyzed by
15% SDSPAGE. Proteins were visualized by staining with Coomassie
Brilliant Blue.
Electron microscopy. a-Synuclein brillation reactions were diluted to
0.2 lM protein, and placed on formava-coated 300-mesh copper grids.
The samples were negatively stained with 1% aqueous uranyl acetate, and
examined using a JEOLJEM-100B transmission electron microscope.
Circular dichroism (CD) measurements. The protein concentration was
200 lg/ml. Spectra were obtained using a Jasco-720 spectropolarimeter
with a 1-mm path-length cell at 25 C. Spectra were recorded from 190 to
250 nm at a scan speed of 20 nm/min. For each sample, ve scans were
taken and averaged.

Results
b-Packing and hydrophobicity of the central hydrophobic
region is critical in brillation of a-synuclein
To investigate the role of each specic residue of the asynuclein molecule in bril formation, we introduced single
amino acid substitutions throughout the molecule and
monitored their eects on bril formation. The brillation
process was observed by monitoring the uorescence
change of the dye ThT, which binds specically to brils
(Fig. 2A). Consistent with previous studies, the patterns
of ThT uorescence change were typical of a nucleationdependent polymerization process [20]: ThT uorescence
did not change signicantly during an initial lag phase; following this, it increased exponentially for a period; and
then it remained almost constant. For WT a-synuclein
samples, the uorescence began to increase at 1.75 days,
and it reached its maximum level at 3 days. Amyloid formation was also monitored by measuring the decrease in
the amount of soluble monomers or oligomers in the reaction solution (Fig. 2B). Consistent with the above ThT
uorescence results, the amount of soluble a-synuclein
decreased more quickly for a brillation-enhanced mutant
(E35Q) than for the WT protein. In addition, the disappearance of soluble a-synuclein molecules occurred more
slowly in a brillation-retarded mutant (A78T), and most
a-synuclein molecules remained soluble in a brillationsuppressed mutant (V63P). Indeed, there was a good correlation between the increase in ThT uorescence and the
decrease in soluble a-synuclein protein. Observation of

774

H.-J. Koo et al. / Biochemical and Biophysical Research Communications 368 (2008) 772778
1.2

Relative fluorescence

1.0
0.8

0.6
0.4
0.2
0.0
0

Time (day)

WT

Incubation -

E35Q

A78T

+ -

V 63P

+ - +

a. 1.5 day

b. 2.5 day

c. 5.5 day

WT

G93S

retarded a-synuclein brillation, as judged by an extended

lag phase and a delay in reaching maximum brillation
(Table 1). In particular, Pro substitutions in the region
spanning residues 3789 had drastic eects on the kinetics
of brillation; many of these mutants had formed only a
few, if any, brils by the end of the experimental incubation
period, which was longer than two weeks. These results
suggest that amyloid packing in the central region of a-synuclein is critical in regulating the switch to the bril
conformation.
To evaluate the importance of hydrophobicity of the
central region, substitutions of charged or polar residues
(such as Glu and Ser) for hydrophobic ones (such as Val
and Ala) were introduced (Table 2). Substitution of
charged or polar residues for Val residues in the region
spanning residues 6374 prevented brillation during
the incubation period of more than 20 days. In addition,
the T72E, T75K, and V95S substitutions, which increase
the polarity of the central region, signicantly retarded
brillation. The A78T mutation, another polarity-increasing substitution, also retarded aggregation of a-synuclein,
whereas the T72A mutation, which increases hydrophobicity of this region, was the most rapidly brillating var-

E123A
Table 1
Fibrillation kinetics of proline-scanning a-synuclein mutants

Fig. 2. Fibrillation of some representative a-synuclein mutants. (A) The

degree of bril formation was analyzed by monitoring the uorescence of
ThT binding. Symbols: d, WT a-synuclein; j, E35Q (accelerated bril
formation); N, A78T (retarded bril formation); ., V63P (no brillation).
(B) Mutational eects on bril formation were assessed by monitoring the
amount of a-synuclein protein remaining in the soluble fraction. Aliquots
of the WT and mutant a-synuclein brillation reactions were removed for
analysis at the time points indicated by arrows in (A). The amount of
soluble a-synuclein protein remaining was monitored by 15% SDS
PAGE. (C) Transmission electron microscopy of a-synuclein brils. The
a-synuclein variants with dierent maximum ThT uorescence values (250
for WT, 40 for G93S, and 420 for E123A) also diered in their number of
brils.

the laments using transmission electron microscopy

showed that the morphologies of the brils formed from
brillation-competent a-synuclein mutants and from WT
a-synuclein were similar (Fig. 2C). Those a-synuclein variants that did not exhibit a signicant increase in ThT uorescence in the above experiments also did not exhibit
brils observable by transmission electron microscopy.
Overall, the number of laments observed by transmission
electron microscopy was approximately proportional to the
maximum uorescence value (Fmax) for each variant.
To evaluate the role of packing at specic sites in a-synuclein brillation, Pro residues were introduced throughout
the N-terminal repeated region and the central hydrophobic core. All of the Pro substitution mutants exhibited

Mutant

Lag time (day)a

Max. time (day)b

Fmaxc

WT
V16P
A18P
T22P
Q24P
V26P
A27P
A29P
A30P
V37P
L38P
V40P
V48P
V49P
V52P
T59P
V63P
T64P
N65P
G67P
G68P
A69P
V70P
T72P
T75P
T81P
E83P
A89P
A90P

1.75 0.3
5 0.7
4 0.5
2.5 0.6
2 0.1
4.5 0.8
4.5 0.5
2.75 0.4
3 0.1
>20
4.5 0.5
3.75 0.6
>25
>15
3.75 0.4
12.5 0.2
>30
>50
>50
>30
>49
>25
15 0.6
>53
>46
>25
>25
>21
2.5 0.4

3 0.2
8.5 0.7
9 0.6
4.5 0.4
3.5 0.1
7.75 0.9
6 0.4
7.5 0.6
4 0.1
>20
5.5 0.4
5.5 0.8
>25
>15
5.75 0.5
14 0.3
>30
>50
>50
>30
>49
>25
19 0.9
>53
>46
>25
>25
>21
4 0.5

250 32.7
140 18.4
80 10.6
117 9.2
100 10.6
25 3.2
300 28.3
250 35.4
300 31.8
0
60 7.1
55 7
0
0
22 4.2
55 5.4
0
0
0
0
0
0
135 14.1
0
0
0
0
0
600 28.3

a
The incubation time during which no change in ThT uorescence
occurred, indicating that no brils were formed.
b
The incubation time at which the ThT uorescence signal reached its
maximum intensity and remained constant.
c
The maximum ThT uorescence value (arbitrary units) at Max. time.

H.-J. Koo et al. / Biochemical and Biophysical Research Communications 368 (2008) 772778
Table 2
Fibrillation kinetics of a-synuclein mutants which increase polarity or
change charges
Mutant

Lag time (day)a

Max. time (day)b

Fmaxc

WT
V63E
V63G
V66S
V70G
V70E
V74E
V74G
T72E
T75K
V95S
A78T
T72A
K10Q
K12E
V15E
K21Q
K23E
K23Q
V26E
V26Q
K32Q
E35Q
G36N
V37E
K45E
K45R
E46K
G47Q
A53T
K60Q
E61K
T72K
K80Q
G93S
E104A
E105A
D121A
E123A
E130A

1.75 0.3
>20
>20
>21
>26
>20
>20
>26
>20
4.5 0.3
2.5 0.1
5 0.5
0.75 0.1
5.5 0.5
5.5 0.4
5.5 0.4
3 0.25
5.5 0.6
5.5 0.3
>20
2.75 0.1
3 0.35
1.0 0.25
1.5 0.25
3.5 0.5
8.5 0.5
2.75 0.2
0.75 0.1
2.5 0.3
1.5 0
2.5 0.25
1.5 0.1
1.5 0.1
>25
1.5 0.25
2 0.1
1.75 0.1
2 0.3
1.5 0.3
1.5 0

3 0.2
>20
>20
>19
>26
>20
>20
>26
>20
7 0.5
3.75 0.1
6.75 0.7
2.75 0.3
7.5 0.7
9.5 0.5
9.5 0.5
4 0.25
9 0.4
8 0.2
>20
3.75 0.1
5.5 0.5
2.0 0.3
3 0.2
4.75 0.7
14.5 1.2
5 0.3
2.5 0.2
4 0.2
2.25 0.1
6 0.3
3 0.2
3 0.2
>25
2.75 0.5
3 0.5
4 0.1
3.5 0.4
3 0.5
3.5 0.1

250 32.7
0
0
0
0
0
0
0
0
100 7.1
150 19.8
37 5.7
150 7.1
50 12.6
60 7.1
60 7.1
260 30.2
300 47
160 21
0
150 31.1
95 5.2
250 24.3
100 10.2
50 5.4
120 10
170 21.5
220 11.6
105 7
200 20.1
370 27.2
150 13.4
140 20
0
40 12
160 28.2
100 10.6
130 23.3
420 25.5
360 60.6

a,b,c

Denition of the lag time, max. time, and Fmax are the same as in
Table 1.

iant observed in this study (shown in bold in Table 2).

Since Thr is frequently found in b-sheets [21] and is
therefore unlikely to impair b-packing of a-synuclein into
brils, this result illustrates the importance of hydrophobicity in this region.
Since the N-terminal repeated region transforms into ahelices upon binding to synthetic or membrane lipids [9],
amino acids that are frequently found in a-helical structures (Glu, Gln, and Lys) were introduced into the N-terminal region. The eects of these substitutions on
brillation were not as great as those of the substitutions
in the central region (Table 2). The CD spectra of the Nterminal mutants were typical of unstructured proteins
and indistinguishable from the spectrum of WT a-synuclein (data not shown). When an a-helical structure was
induced using 7% hexauoroisopropanol [10], the CD spec-

775

tra of all the variants tested were transformed to those

typical of proteins with predominantly a-helical secondary
structure. The CD signals of two Pro-substituted a-synuclein mutants (V26P and A30P) were slightly less intense
than that of the WT protein (data not shown). Since the
tendency to form a-helices was not changed signicantly
by single substitutions, and since a-synuclein variants did
not form a-helices in the brillation reaction buer
(PBS), dierences in a-helix-forming ability are not the primary cause of the eects of these N-terminal mutations.
Charge eects of the N-terminal region may, instead, be
important in regulation of a-synuclein brillation. All 11
mutations that increased the total negative charge (introduction of negatively charged Glu, removal of positively
charged Lys, or both) signicantly retarded or even prevented brillation of a-synuclein (Table 2). On the other
hand, both mutations that increased the total positive
charge (E35Q and E46K) facilitated brillation (shown in
bold in Table 2). Notably, E46K was one of the most rapidly brillating mutants, with a lag time of 0.75 days, consistent with a previous study [22], and is also one of the
three PD-linked a-synuclein variants. The eects of the
mutations present in the two other PD-linked a-synuclein
variants [23] were also conrmed in this study. The A53T
mutation, in which a b-sheet-favoring Thr replaces an ahelix-favoring Ala, exhibited accelerated brillation, and
A30P, in which a secondary structure-disrupting Pro is
introduced, retarded brillation. In contrast, removal of
one of the 15 acidic residues in the C-terminal region had
only marginal eects on brillation kinetics, causing small
changes in the lag time (60.25 day) (Table 2).
Most brillation-blocked a-synuclein variants are defective in
both nucleation and elongation
For a-synuclein variants that showed retarded brillation kinetics, the aected step(s) in the brillation process
were elucidated by seeding the a-synuclein brillation reaction with preformed nuclei. As previously reported [20],
WT a-synuclein nuclei seeded at 3% of total protein, accelerated brillation of the WT protein, as shown by a
decrease in lag time from 1.75 to 0.75 days (Fig. 3 and
Table 3). It has been also shown that the A53T a-synuclein
mutant can cross-seed with WT a-synuclein [20]. If a
mutant a-synuclein is competent in the bril-elongation
step but defective in nucleus-formation, addition of preformed WT nuclei should facilitate brillation of the
mutant. In the brillation-retarded mutants, brillation
was accelerated by the addition of WT nuclei, whereas
the nal ThT uorescence signal was not changed greatly.
Therefore, the major step aected in these brillationretarded a-synuclein mutants is likely to be that of
nucleus-formation. On the other hand, in most brillation-blocked mutants, brillation was not induced by
inclusion of preformed WT nuclei, suggesting that these
mutants were defective at least in bril elongation (as
shown for T72P in Fig. 3, and Table 3). Three of the bril-

H.-J. Koo et al. / Biochemical and Biophysical Research Communications 368 (2008) 772778

Relative fluorescence

776

Table 3
Seeding eects of the WT a-synuclein nuclei on mutant a-synuclein
brillation

1.0
0.8
0.6
0.4
0.2
0.0
0

Time (day)
Fig. 3. Seeding of the a-synuclein brillation reaction with WT nuclei.
WT a-synuclein was pre-incubated for 1.75 days to promote formation of
nuclei, and the pre-formed nuclei were added to other brillation reactions
to constitute 3% of the total protein. Fibrillation reactions were followed
as described in Fig. 2A. Symbols: d, WT a-synuclein; j, WT a-synuclein
seeded with WT nuclei; s, T72P mutant a-synuclein; h, T72P mutant asynuclein seeded with WT nuclei.

lation-suppressed mutants (G67P, G68P, and K80Q)

formed some brils upon addition of preformed WT nuclei,
but the lag time was delayed and the nal uorescence signal was relatively weak, compared to those of the WT asynuclein.
To evaluate the nucleus-forming abilities of the brillation-blocked mutants, the mutant brillation solutions
were pre-incubated for 20 days and then used to seed
WT a-synuclein brillation reactions. If the pre-incubated
mutants formed some nuclei, despite being defective in
bril elongation, their addition to the WT a-synuclein
should accelerate the brillation reaction. However, neither
the brillation kinetics nor the nal ThT uorescence of
WT a-synuclein was changed greatly by the addition of
the pre-incubated mutants (Table 4). The results suggest
that few, if any, nuclei were formed in the brillation reaction solutions of these mutant a-synuclein proteins. Therefore, most of the brillation-blocked a-synuclein mutants
examined in this study were impaired in both nucleation
and bril elongation.
Discussion
In the hope of identifying a structural motif in a-synuclein that regulates amyloidogenesis, we examined the
sequence determinants of a-synuclein that are critical in
its brillation. Our results show that the hydrophobicity
of the central region and b-packing, especially in the region
spanning amino acid residues 3789, are important structural determinants of a-synuclein brillation. The results
are consistent with a previous electron paramagnetic resonance study, which showed that in a-synuclein brils, residues 34101 are packed in a highly ordered, parallel
fashion, and the N- and C-terminal regions remain
unstructured [17]. A highly ordered structure of the central
region is further supported by the resistance of this region
to digestion of a-synuclein brils with proteinase K [24].

WT + WT
seed
K23Q + WT
seed
K23E + WT
seed
V26P + WT
seed
K45E + WT
seed
T59P + WT
seed
V48P + WT
seed
V49P + WT
seed
V63E + WT
seed
V63G + WT
seed
V63P + WT
seed
T64P + WT
seed
N65P + WT
seed
V66S + WT
seed
G67P + WT
seed
G68P + WT
seed
G69P + WT
seed
V70E + WT
seed
V70G + WT
seed
T72P + WT
seed
V74E + WT
seed
V74G + WT
seed
T75P + WT
seed
A78T + WT
seed
K80Q + WT
seed
T81P + WT
seed
E83P + WT
seed
A89P + WT
seed

Lag time (day)a

Max. time
(day)b

Fmaxc

0.75 0.2
(1.75 0.3)
0.75 0.2
(5.5 0.6)
1.5 0.4
(5.5 0.3)
4.5 0.5
(4.5 0.8)
4.75 1.2
(8.5 0.5)
7 0.7
(12.5 0.2)
>15 (>25)

1.5 0.4
(3 0.2)
3 0.4 (8 0.2)
4.5 0.5
(9 0.4)
6.75 0.7
(7.75 0.9)
8.5 0.7
(14.5 1.2)
10 0.7
(14 0.3)
>15 (>25)

300 14.1
(250 32.7)
165 24.7
(160 21)
280 14.1
(300 47)
55 4.7
(25 3.2)
130 7.1
(120 10)
65 3.5
(55 5.4)
0 (0)

>15 (>15)

>15 (>15)

0 (0)

>15 (>20)

>15 (>20)

0 (0)

>15 (>20)

>15 (>20)

0 (0)

>15 (>30)

>15 (>30)

0 (0)

>15 (>50)

>15 (>50)

0 (0)

>15 (>50)

>15 (>50)

0 (0)

>15 (>21)

>15 (>21)

0 (0)

2.75 0 (>30)

4 0.2 (>30)

35 3.5 (0)

5.75 0.2 (>49)

8.5 0 (>49)

30 3.5 (0)

>15 (>25)

>15 (>25)

0 (0)

>15 (>20)

>15 (>20)

0 (0)

>15 (>26)

>15 (>26)

0 (0)

>20 (>53)

>20 (>53)

0 (0)

>15 (>20)

>15 (>20)

0 (0)

>15 (>26)

>15 (>26)

0 (0)

>15 (>46)

>15 (>46)

0 (0)

3.5 0.2
(5 0.5)
6 0.4 (>25)

6.5 0.2
(6.75 0.7)
9 0.2 (>25)

240 28.3
(37 5.7)
30 3.5 (0)

>15 (>25)

>15 (>25)

0 (0)

>15 (>25)

>15 (>25)

0 (0)

>15 (>21)

>15 (>21)

0 (0)

The values shown in parenthesis are without addition of nuclei.

a,b,c
Denition of the lag time, max. time and Fmax are the same as in
Table 1.

H.-J. Koo et al. / Biochemical and Biophysical Research Communications 368 (2008) 772778
Table 4
Seeding eects of pre-incubated mutant a-synuclein proteins on WTasynuclein brillation
Seed

Lag time (day)a

Max. time (day)b

Fmaxc

WT + no seed
WT + V37P seed
WT + V48P seed
WT + V63E seed
WT + V63G seed
WT + V63P seed
WT + T64P seed
WT + N65P seed
WT + V66S seed
WT + G67P seed
WT + G68P seed
WT + A69P seed
WT + V70P seed
WT + V70E seed
WT + V70G seed
WT + T72E seed
WT + V74E seed
WT + V74G seed
WT + K80Q seed
WT + T81P seed
WT + E83P seed
WT + A89P seed

1.75 0.3
2 0.4
1.75 0
2 0.2
20
2.5 0.4
1.75 0.2
1.5 0
2 0.2
1.75 0.2
1.75 0
1.75 0
1.5 0.2
1.5 0
2 0.2
20
1.75 0.2
1.75 0
1.5 0.2
2 0.2
2.5 0.4
2 0.4

3 0.2
3.5 0.2
2.75 0.2
3.5 0
3.75 0.2
4.5 0
3 0.4
3 0.4
3.5 0
3.75 0.2
3 0.4
3.5 0.4
3 0.4
2.75 0.2
3.75 0.2
3.5 0
3 0.2
3 0.2
3 0.4
3.75 0.2
4 0.4
3 0.4

250 32.7
220 14.1
230 7.1
210 21.2
200 31.8
200 21.2
220 17.7
190 10.6
240 28.3
80 3.5
200 7.1
230 10.6
230 23.3
240 21.2
200 10.6
200 17.7
255 27.6
235 9.2
220 24
210 14.1
200 11.3
210 9.9

a,b,c

Denition of the lag time, max. time, and Fmax are the same as in
Table 1.

The net charge of the N-terminal region also seems to be

important in regulating a-synuclein brillation; increasing
the negative charge drastically retarded brillation, and
increasing the positive charge facilitated bril formation.
Since only a few substitutions promoted brillation, this
last result has signicant implications for the charge eects
of this region in brillation. In contrast, removal of single
acidic residues from the highly acidic C-terminal region
had only minor eects on a-synuclein brillation. One possible explanation for these results is that the highly acidic
C-terminal region might interact with Lys residues in the
N-terminal region to form a partially folded intermediate
conformation that is prone to aggregation. In this case, loss
of a positive charge from the N-terminal region would
weaken the interaction between two domains, retarding
brillation. Increasing the negative charge of the N-terminal region might cause electrostatic repulsion between the
two domains and also interfere with self-assembly. At the
same time, increasing positive charges would strengthen
the interaction, and facilitate brillation. Another possibility is that removal of Lys (or introduction of Glu) destabilizes the N-terminal amphipathic a-helices that are formed
upon interaction with acidic phospholipids of the membrane; this interaction is implicated in promoting brillation of a-synuclein [25]. The N-terminal domain of asynuclein is suggested to form class A2 a-helices, in which
basic residues (preferentially Lys) are characteristically
clustered at the polar/nonpolar interface and positioned
at about 100 of the center of the nonpolar face [26]. Since
this orientation would stabilize the interaction with
membranes, Lys removal or Glu incorporation would

777

destabilize the interaction between a-synuclein and

phospholipid vesicles. Although the latter possibility is less
likely to occur in our brillation assays, it may contribute
to a-synuclein brillation inside neuronal cells, in which
a-synuclein molecules are also found in association with
synaptic vesicles.
Several a-synuclein oligomeric species with various morphologies, including spherical and annular protobrils,
have been observed in vitro prior to bril formation [27].
Although the identity of the pathogenic a-synuclein species
remains the topic of vigorous debate, a toxic protobril
hypothesis has been proposed. This hypothesis posits that
the pathogenic protobrils are transiently populated during the brillation process and that the bril end products
may be inert by themselves and protective by reducing the
toxic intermediates, rather than cell-damaging [28]. The
CD spectra of protobrils puried by gel ltration are suggestive of structures with a signicant b-strand content,
rather than of random coils of monomeric protein [29].
To our surprise, more than 20 dierent single amino acid
substitutions sucient to prevent brillation of a-synuclein
were obtained (Tables 1 and 2). In our CD experiments, the
spectra of brillation-suppressed mutants did not indicate
any noticeable increase in secondary structure content,
even after incubation at 37 C for more than 20 days (data
not shown), conrming that a conformational switch to bstructures did not occur in these variants. Our results suggest that the pathogenic species, whatever its identity, was
not formed by our brillation-decient a-synuclein
mutants. The results therefore allow for the possibility of
developing peptidomimetic small molecules that can suppress brillation of amyloidogenic a-synuclein proteins.
Acknowledgments
We greatly appreciate the gift of human a-synuclein
cDNA from Dr. H. Rhim (Catholic University, Korea).
This work was supported by Grant No. R01-2006-00011154-0 from the Basic Research Program of the Korea
Science and Engineering Foundation, by Grant number
FPR05B2-211 of 21C Frontier Functional Proteomics Program from the Korea Ministry of Science and Technology,
and by the Korea Research Foundation Grant funded by
the Korean Government (C00044).
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