Dynamics of a-synuclein aggregation and inhibition
of pore-like oligomer development by b-synuclein
Igor F. Tsigelny
1,2
, Pazit Bar-On
3
, Yuriy Sharikov
2
, Leslie Crews
4
, Makoto Hashimoto
3
,
Mark A. Miller
2
, Steve H. Keller
5
, Oleksandr Platoshyn
5
, Jason X J. Yuan
5
and Eliezer Masliah
3,4
1 Departments of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA
2 San Diego Super Computer Center, University of California San Diego, La Jolla, CA, USA
3 Department of Neurosciences, University of California San Diego, La Jolla, CA, USA
4 Department of Pathology, University of California San Diego, La Jolla, CA, USA
5 Department of Medicine, University of California San Diego, La Jolla, CA, USA
In recent years, new hope for understanding the patho-
genesis of Parkinson’s disease (PD) and Lewy body
dementia (LBD) has emerged with the discovery of

information is available about the conformation of a-syn at the initial and
end stages of fibrillation, less is known about the dynamic process of a-syn
conversion to oligomers and how interactions with antiaggregation chaper-
ones such as b-synuclein might occur. Molecular modeling and molecular
dynamics simulations based on the micelle-derived structure of a-syn
showed that a-syn homodimers can adopt nonpropagating (head-to-tail)
and propagating (head-to-head) conformations. Propagating a-syn dimers
on the membrane incorporate additional a-syn molecules, leading to the
formation of pentamers and hexamers forming a ring-like structure. In con-
trast, b-syn dimers do not propagate and block the aggregation of a-syn
into ring-like oligomers. Under in vitro cell-free conditions, a-syn aggre-
gates formed ring-like structures that were disrupted by b-syn. Similarly,
cells expressing a-syn displayed increased ion current activity consistent
with the formation of Zn
2+
-sensitive nonselective cation channels. These
results support the contention that in Parkinson’s disease and Lewy body
dementia, a-syn oligomers on the membrane might form pore-like struc-
tures, and that the beneficial effects of b-synuclein might be related to its
ability to block the formation of pore-like structures.
Abbreviations
aa, amino acid; a-syn, a-synuclein; b-syn, b-synuclein; GFP, green fluorescent protein; LBD, Lewy body disease; PD, Parkinson’s disease;
POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; tg, transgenic.
1862 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS
synaptic terminals and axons plays an important role
[11–14]. These studies suggest that a-syn oligomers and
protofibrils rather than fibrils might be the neurotoxic
species [15].
a-syn is an abundant presynaptic molecule [16] that
probably plays a role in modulating vesicular synaptic

intermediate precursor that contains hydrophobic pat-
ches. It has been proposed that the intermediate a-syn
oligomers form annular protofibrils and pore-like
structures [26–29]. The mechanism through which
monomeric a-syn converts into a toxic oligomer and
later into fibrils is currently under intense investiga-
tion. Recent reviews indicate that the kinetics of a-syn
fibrillation are consistent with a nucleation-dependent
mechanism for which a partially folded intermediate is
needed in the early stages of aggregation [30]. Factors
leading to the formation of the folded intermediates
include oxidation, phosphorylation, mutations, and
lipids in the membrane [30–34]. a-syn oligomerization
might occur on the membrane and involves interac-
tions between hydrophobic residues of the amphipathic
a-helices of a-syn [35]. These studies indicate that the
hydrophobic lipid binding domains in the N-terminal
region might be important in modulating a-syn aggre-
gation [13,36–38]. There are several studies describing
the effects of membranes and membrane-like structure
on aggregation [21,39,40], however, less is known
about the effects of membrane lipids on b-syn struc-
ture. In this context, a recent study has analyzed
by NMR the micelle-bound structure and dynamics of
b- and c-syn [41].
Thus, better understanding of the steps involved in
the process of a-syn aggregation is important in order
to develop intervention strategies that might prevent
or reverse a-syn oligomerization and toxic conversion.
The conformational state of a-syn at the initial and

inhibitory effect of b-syn may result from its interac-
tion with a-syn, which prevents formation of func-
tional a-syn channels.
Results
Conformational diversity of a-syn and b-syn
molecules during molecular dynamics simulations
To better understand the conformational changes that
a- and b -syn undergo over time and to model the
homo and heterodimeric interactions preventing or
I. F. Tsigelny et al. Modeling of a-syn oligomer formation
FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1863
leading to aggregation, molecular dynamics simula-
tions in water were performed based on the micelle-
bound structure of a-syn as resolved by NMR. This
approach allows the investigation of the dynamic
structural changes of the folded a-syn (micelle-derived)
under simplified conditions. The curved N-terminal
domain of this structure is divided into two regions
(termed helix-N and helix-C) [21] connected by a short
linker (Fig. 1A,B). In our baseline models, the two
curved helical N-terminal domains of the micelle-
derived a-syn molecular structure form an angle
around 55 ± 3° that decreases to around 42–44° dur-
ing the first 2.0 ns of the simulation, and then increa-
ses to 64–70° after 3.0–5.0 ns of simulation. During
simulation (Fig. 1A,B), the initial two curved helical
N-terminal domains (helices N and C) of a-syn trans-
form into three uncurved N-terminal helical structures.
The third helical region appears when the second
curved helix (aa 46–84) converts into two uncurved

Further analysis consisted of determining changes in
secondary structure of a-syn and b-syn over time.
After 500 ps of simulation for a-syn a coiled region
appeared, interrupting the a-helix around residue 68
(Fig. S1A). Beginning at 750 ps, turns appeared in the
a-helical structure around residue 47, then after 1.0 ns
this region was transformed into a p-helix (Fig. S1A).
The length of this p-helix increased with time, and
from 3.0 ns covered the region from residues 45–55. In
another part of the sequence, a second p-helix
appeared from 2.0 ns that includes residues 74–83
(Fig. S1A).
Changes in b-syn secondary structure over time con-
sisted of transformations from a bended a-helical
structure to the structure with two straight helices with
further conversion to p-helical structure around residue
30 and the N-terminus region (Fig. S1B). The C-ter-
minal region beyond residue 70 showed limited chan-
ges in secondary structure (Fig. S1B). Overall, b-syn
underwent significantly fewer changes in secondary
structure than a-syn during molecular dynamics simu-
lations.
Interactions of a-syn propagating dimers predict
the formation of pore-like structures
The first studies of the interactions of a-syn were per-
formed by docking the initial structures of two a-syn
monomers on a flat surface without specific limita-
tions. Under these conditions, some low energy com-
plexes of two molecules formed a ‘head-to-tail’
position. This configuration is not favorable for further

as expected along the N-terminal helices of the a-syn
conformers (Fig. 2B). To further verify the conforma-
tional changes of a-syn dimers upon interactions with
the membrane, we conducted docking of two a-syn
4 ps conformers onto a 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphocholine (POPC) membrane with a grid cell of
1A
˚
, including the membrane in calculations (Fig. 3C).
The electrostatic energy of interaction is around 30–50
kcalÆmol
)1
for docking of two a-syn molecules. Only
minimal differences (< 10% in docking energy values)
were detected between molecules docked on the flat
surface and molecules docked on the POPC membrane.
In general, two possible initial docking configurations
for a-syn molecules on the membrane were observed.
In the first one, the dimer is arranged in a head-to-tail
position and additional monomers cannot easily add to
this complex to propagate toward higher order aggre-
gates, as low-energy binding sites do not appear to exist
for consecutive docking (Fig. 2A and 3A). It is possible
for weakly propagating multimers to form over time up
to 4.0 ns (Fig. 3D), however, the binding energies of
the growing complexes (Fig. 3F) are significantly less
favorable than for propagating configurations (Fig. 3E,
Fig. 3. Modeling of docking of nonpropagating and propagating a-syn dimers and multimers on the membrane. Membrane-contacting N-ter-
minal (n-term) regions are designated by boxes and C-terminal (c-term) regions by lines, as viewed perpendicular to the membrane surface.
For docking, the second a-syn molecule (a-syn 2) docks to the first (a-syn 1), followed by docking of the third a-syn molecule (a-syn 3) to

involved in intermolecular interactions (Fig. S3).
Because the tail of a-syn carries the majority of this
protein’s positive charge, this might help to explain
why there was a significant enhancement of a-syn dimer
docking energies (and accordingly the stability of the
multimers) after 4.0 ns of simulation (Table 1). More-
over, Fig. S2 shows that the most stable conformation
of a-syn occurs after 3.8 ns of molecular dynamics
simulation time. For b-syn, the most stable conforma-
tions arise between 2.2 and 3.5 ns of simulation
(Fig. S2). The most probable a-syn multimers were
selected based on the conformers with the most favora-
ble energies of intermolecular interaction between two
monomers and the most stable conformers. Six distinct
possible multimers were generated as the result of ‘pro-
pagating docking’ (Fig. 2G). These multimers formed
low energy pentamers and hexamers with different con-
figurations that generated ring-like structures with a
central lumen (Fig. 2G). The most stable multimers of
a-syn were generated with a-syn conformers from
4.0 ns simulation and later. The theoretical pentameric
and hexameric conformations of the a-syn multimers
on the membrane are reminiscent of the pore-like
appearance of cell-free a-syn aggregates that have been
reported by atomic force microscopy (AFM) [26].
a-syn propagating dimers form pore-like
structures that are embedded in the membrane
To further investigate how closely the simulation-
derived model resembles a-syn aggregates generated
in vitro, recombinant a-syn was incubated for various

purpose, we modeled b- and b-syn, and b- and a-syn
heterodimeric interactions. Firstly, theoretical docking
of various molecular dynamics conformers of a-syn to
conformers of
b-syn was performed. All of the docked
a-syn–b-syn complexes displayed a significant level of
negative electrostatic energy of formation (Table 2). In
these simulations, b-syn was able to bind a-syn, cre-
ating stable nonpropagating heterodimers, similar to
nonpropagating a-syn homodimers (Fig. 6A). Strong
Table 1. Intermolecular interaction energies of propagating
a-syn ⁄ a-syn dimers docked on the flat membrane. MD, molecular
dynamics.
MD time (ns) Electrostatic energy (kcalÆmol
)1
)
1.50 )10.6
2.00 )10.6
2.50 )13.4
3.50 )15.1
4.00 )19.7
4.50 )32.9
I. F. Tsigelny et al. Modeling of a-syn oligomer formation
FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1867
electrostatic interactions contributed to the formation
of these a- and b-syn heterodimers. For example, com-
plexes between a-syn (2.5 ns) and b-syn (2.2 ns) dis-
played a minimum intermolecular electrostatic energy
of )31.6 kcalÆmol
)1

)1
,
Table 1). This supports the possibility that b-syn might
be able to interrupt the assembly of propagating a-syn
homodimers at various stages of the oligomerization
process.
b-syn blocks the formation of a-syn ring-like
structures and attenuates ion conductance
alterations
Previous studies have shown that when b- and a-syn
are incubated simultaneously, b-syn reduces a-syn
aggregation over time [23,24,45]. However, it is unclear
Fig. 4. Biochemical and ultrastructural analysis of a-syn aggregation, interactions with b-syn, and modeling of ring-like structures. (A) In vitro
cell-free aggregation of a-syn monomers into dimers, trimers, tetramers, pentamers, and hexamers over time without (left panel) and with
(right panel) the addition of b-syn. (B) Semiquantitative analysis of levels of a-syn multimers over time. (C–F) Electron microscopy analysis of
a-syn aggregation over time into ring-like structures and fibrils. (G–J) Electron microscopy analysis demonstrating reduction in a-syn aggrega-
tion over time in the presence of b-syn. Scale bar ¼ 20 nm. (K) Superimposition of a-syn pentamer (4.5 ns) onto the ring-like structure detec-
ted by electron microscopy. Scale bar ¼ 10 nm.
Modeling of a-syn oligomer formation I. F. Tsigelny et al.
1868 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS
whether b-syn might decrease a-syn aggregation when
added after the process of a-syn oligomerization has
started. The theoretical model presented in the previ-
ous section predicts that under experimental in vitro
conditions, addition of b-syn might prevent further
aggregation of a-syn (Fig. 4). To investigate this possi-
bility, a-syn was allowed to aggregate and then b-syn
was added for various lengths of time. When b-syn
was added 1 h after a-syn aggregation started, there
was a significant decrease in the subsequent formation

Note the penetration of the pentamer into
the membrane and the exposed membrane
in the center of the a-syn ring-like structure.
(B–E) The steps of penetration of the a-syn
pentamer into the POPC membrane during
0.8 ns molecular dynamics simulation (B, ini-
tial; C, 0.2 ns; D, 0.5 ns; E, 0.8 ns). The
depth of protein insertion into the mem-
brane was measured between the upper-
most membrane-associated atom and the
atom that is embedded deepest into the
membrane.
Table 2. Intermolecular interaction energies of the b-syn conform-
ers with 1.5 ns molecular dynamics a-syn conformer docked on the
flat membrane. MD, molecular dynamics.
b-syn conformer MD time (ns)
Electrostatic energy
(kcalÆmol
)1
)
Initial )27.4
0.25 )22.0
0.50 )29.8
0.75 )45.9
1.00 )37.8
1.50 )26.7
I. F. Tsigelny et al. Modeling of a-syn oligomer formation
FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1869
showed a significant increase in whole-cell currents eli-
cited by depolarizing the cells from a holding potential

tional a-syn molecules, leading to the formation of
pore-like structures. In contrast, b-syn dimers do not
propagate, and when interacting with a-syn aggregates
block the propagation of a-syn into multimeric struc-
tures. Recent studies have suggested that the transfor-
mation of a-syn into a neurotoxic molecule might
involve the sequential conversion of a-syn monomers
into globular oligomers and then protofibrils [46]. In
contrast, a-syn fibrils, which are present in the LBs [6],
might represent a mechanism for isolating toxic oligo-
mers [15]. Previous studies have investigated the con-
formation of a-syn either at the very initial stages of
aggregation [21] or during the process of fibril forma-
tion [42]. In micelles, a-syn monomers consist of two
curved a-helices connected by a short linker in an anti-
parallel arrangement, followed by a short extended
region and a predominantly unstructured mobile tail
[21,48]. The molecular dynamics studies described
here showed that this structure of a-syn displayed
significant changes in the organization of the N-ter-
Fig. 6. Molecular modeling of the interactions of b-syn with a-syn monomers and dimers. (A) a-syn and b-syn minimal energy nonpropagat-
ing heterodimers. (B) Primary electrostatic interactions in the minimal energy a-syn and b-syn dimer. (C) b-syn minimal energy complex with
the a-syn dimer (4.5 ns simulation for a-syn and 2.2 ns simulation for b-syn). This complex does not support further propagation.
Modeling of a-syn oligomer formation I. F. Tsigelny et al.
1870 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS
minal helices from 2 to 3 helices over time, which
might lead to more complex membrane interactions.
Computerized analysis predicted that these changes
were accompanied over time by alterations in the secon-
dary structure showing that a p-helical conformation

and corresponding current–voltage relationship (E; means ± SE) in tranduced cells. (F) Representative currents at +80mv (left panel) before
(Cont), during (Zn
2+
) and after (Wash) application of 500 l M Zn
2+
. Time course (right panel) of the change in current density before, during,
and after extracellular application of Zn
2+
. The arrows correspond to the currents shown in the left panel (Cont, a; Zn
2+
, b; and Washout, c).
I. F. Tsigelny et al. Modeling of a-syn oligomer formation
FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1871
conformations of a-syn multimers have been difficult to
study due to the inability to crystallize the oligomeric
form of this protein. Our molecular dynamics studies
support the contention that oligomers of a-syn associ-
ate with membranes and suggest that propagating
dimers might b e thermodynamically stable on membranes
in association with lipids. Moreover, the simulations
and modeling suggest that anchoring of propagating
a-syn dimers to the membrane facilitates the incorpor-
ation of additional a-syn monomers, leading to the for-
mation of trimers, tetramers, pentamers, and hexamers,
the latter oligomers forming ring-like structures.
Recent Raman and AFM studies showed that in vitro
early stage oligomers have a globular structure that
elongates over time to form protofibrils [42,51]. High-
resolution ultrastructural and AFM have suggested that
these aggregates might form pore-like structures that

ible candidate for interaction with Zn
2+
ions because it
is situated near the putative pore region of the a-syn
pentamer. The increased ion channel activity observed
in the present study is in agreement with recent results
showing that human neuronal cells expressing mutant
a-syn have high plasma membrane ion permeability
that was sensitive to calcium chelators [47]. Taken
together, these results support the contention that a-syn
aggregates might form functional ion-permeable chan-
nels that in turn might play a role in the mechanisms of
neurodegeneration in LBD.
Therefore, developing strategies that might prevent
a-syn aggregation and subsequent oligomerization into
pore-like structures, or compounds that might block
such potential ion channels could represent a viable
approach to treating disorders characterized by a-syn
aggregation. As in other neurodegenerative disorders,
such as AD similar pore-like structures are formed
[58,59], it is possible that generic antioligomer antibod-
ies that can recognize these assemblies might be useful
[60,61]. The presence of the oligomers in the membrane
might also facilitate recognition by antibodies that pro-
mote clearance of aggregated a-syn [62]. Chaperone
molecules such as heat sock proteins and b-syn might
also be useful. Remarkably, in support of this possibil-
ity, we found that, via interactions with a-syn mono-
mers and multimers, b-syn is capable of preventing
further oligomerization. Moreover, we found that

˚
. The simulation system contained 237826 atoms, 41
Na
+
and 32 Cl
–
counter ions. The namd molecular
dynamics program version 2.5 [65] was used with the
CHARMM27 force-field parameters [66] to simulate the
behavior of a-syn and b-syn molecules in water under nor-
mal conditions and the interaction of the POPC membrane
with the a-syn aggregates. The temperature was maintained
at 310 K by means of Langevin dynamics using a collision
frequency of 1 ps
)1
. A fully flexible cell at constant pressure
(1 atm) was employed using the Nose
´
–Hoover Langevin
Modeling of a-syn oligomer formation I. F. Tsigelny et al.
1872 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS
Piston algorithm [67,68], as used in the namd software
package. Initial coordinates were taken from a previously
equilibrated 500 ps system. The van der Waals’ interactions
were switched smoothly to zero over the region 10 A
˚
and
electrostatic interactions were included via the smooth
particle-mesh Ewald summation [69]. The impulse-based
Verlet-I ⁄ r-RESPA method [70,71] was used for multiple

scoring predicted membrane attractive surfaces were used
to dock the protein molecules and to subsequently calculate
possible a-syn–a-syn docking configurations on the mem-
brane. Low energy a-syn ⁄ a-syn propagating complexes
were used in simulations of consecutive docking of the next
a-syn molecules. We also simulated the behavior of a-syn
complexes on the POPC membrane. The explicit (all-atom)
membrane models were utilized for simulation.
Immunoblot and electron microscopy studies
In previous studies, we have shown that a-syn can prevent
b-syn aggregation in a cell-free system when both synuc-
leins are incubated together at the same time [23]. In this
study, we wanted to determine whether b-syn reduces a-syn
aggregation after a-syn aggregation has already started.
For this purpose, recombinant a-syn (1 lm; Calbiochem,
San Diego, CA, USA) was incubated at 65 °C for time
periods from 1 to 48 h [31]. Incubation at this temperature
allows the study of a-syn aggregation over short periods of
time [31]. After 1 h of incubation recombinant b-syn
(16 lm, purified as previously described [23,63]) was added
to the mix. Samples were subjected to immunoblot analysis
with the mouse monoclonal antibody against a-syn (LB509,
1 : 1000; Zymed Laboratories, San Francisco, CA, USA) as
previously described [31] and analyzed in the versadoc
imaging system using the quantity one software (Bio-Rad,
Hercules, CA, USA).
To investigate the ultrastructural characteristics of the
synuclein aggregates, 1-lL aliquots of a-syn either alone or
in combination with b-syn prepared under identical condi-
tions as for immunoblotting were pipetted onto formvar

2
1,
CaCl
2
2, glucose 10, and Hepes 10 (pH ¼ 7.4). During
patch-clamp recording, tetrodotoxin (0.1 lm) and CdCl
2
(0.1 mm) were added to the external solution to block volt-
age-dependent Na
+
and Ca
2+
channels. The ionic compo-
sition of the pipette solution was (in mm): CsCl 150 and
Hepes 10 (pH ¼ 7.2). The current–voltage (I–V) relation-
ship was determined by a step voltage protocol of 50 ms
duration. The membrane potential was held at )50 mV
and stepped to levels between )80 mV and +80 mV in
20-mV increments.
For verification of synuclein expression after lentivirus
infection, transduced cells were harvested in lysis buffer
and analyzed by immunoblot with antibodies against a-syn
(1 : 1000, Chemicon, Temecula, CA, USA), b-syn (prepared
as previously described [75]) and GFP (1 : 1000, Chem-
icon). For immunocytochemistry, cells were cultured on
coverslips until 50% confluence and treated as described
I. F. Tsigelny et al. Modeling of a-syn oligomer formation
FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1873
above, fixed in 4% paraformaldehyde for 20 min, and
blocked overnight at 4 °C in 10% fetal calf serum and 5%

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Modeling of a-syn oligomer formation I. F. Tsigelny et al.
1876 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS
Fig. S2. Stability of molecular dynamics (MD) simula-
tions of a-syn and b-syn monomers in water. Neigh-
boring MD conformers at 100 ps intervals were
structurally superimposed with the combinatorial
extension program [76] and rmsd between them was
calculated. The most stable MD conformations (boxed
regions of the graph) begin at 3.8 ns for a-syn (red)
and occur between 2.2 and 3.5 ns for b-syn (blue).
Fig. S3. Specific intermolecular interactions between
two head-to-head a-syn monomers over time. Sites of
interaction (arrows) between amino acids in each
of the monomers (a-syn 1 and a-syn 2) at 2.0 ns (A),
3.5 ns (B), 4.0 ns (C), 4.5 ns (D).
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