α-Synuclein as an intrinsically disordered monomer - fact or...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/febs.12471 This article is protected by copyright. All rights reserved.

α-Synuclein as an intrinsically disordered monomer: fact or

artifact?

Eduardo Coelho-Cerqueira1#, Phelippe do Carmo-Gonçalves1#, Anderson Sá Pinheiro2,

Juliana Cortines3, Cristian Follmer1*

1Department of Physical Chemistry, Institute of Chemistry, Federal University of Rio de

Janeiro, Rio de Janeiro 21941-909, Brazil. 2Department of Biochemistry, Institute of

Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil.

3Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro 21941-

909, Brazil.

*Corresponding author: Tel: 55 21 2562 7752. Fax: 55 21 2562 7265.

E-mail: [email protected]

# E.C.C. and P.C.G. contributed equally to this work.

Article type:Original Article

Running title: Native oligomeric state of recombinant α-syn

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This article is protected by copyright. All rights reserved.

Abbreviation list: BCIP: 5-bromo-4-chloro-3-indolyl-phosphate; BOG: n-octyl-β-

glucopyranoside; BSA: bovine serum albumin; CD: circular dichroism; ESI-MS:

electrospray ionization mass spectrometry; GHD: glutaraldehyde; GST: glutathione S-

transferase; HPLC: high-performance liquid chromatography; HSQC: heteronuclear

single quantum coherence; MALDI-TOF: matrix-assisted laser desorption/ionization –

time of flight; NAC: non-amyloid-β component; NBT: nitro blue tetrazolium; NMR:

nuclear magnetic resonance; SAXS: small-angle X-ray scattering; SDS-PAGE: sodium

dodecyl sulfate polyacrylamide gel electrophoresis; SEC: size exclusion chromatography.

Abstract

Fibrillization of the protein α-synuclein (α-syn) is a hallmark of Parkinson’s disease and

other α-synucleinopathies. The well-established idea that α-syn is a natively disordered

monomer prone to forming fibrils was recently challenged by data showing that the

protein mostly exists in vitro and in vivo as helically folded tetramers that are resistant to

fibrillization. These apparently conflicting findings may be reconciled by the idea that α-

syn exists as a disordered monomer in equilibrium with variable amounts of dynamic

oligomeric species. In this context, varying the approaches used for protein purification

such as the method used to lyse cells or the inclusion of denaturing agents could

dramatically perturb this equilibrium and hence alter the relative abundance of the

disordered monomer. Herein, we investigated how the current methods for α-syn

purification affect the structure and oligomeric state of the protein, discussing the main

pitfalls associated with the production of recombinant α-syn in Escherichia coli. We

demonstrated that α-syn was expressed in E. coli as a disordered monomer independent

of both the cell lysis method and the use of heating/acidification for protein purification.

In addition, we provide convincing evidence that the disordered monomer exists in

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equilibrium with a dynamic dimer, which is not an artifact of the cross-linking protocol as

previously suggested. Unlike the helically folded tetramer, α-syn dimer is prone to

fibrillate and thus it may be an interesting target for anti-fibrillogenic molecules.

Keywords: α-synuclein; disordered monomer; dimer; purification; Escherichia coli.

Introduction:

The pathological characteristic of Parkinson’s disease (PD) is the degeneration of

dopaminergic neurons in the substantia nigra pars compacta accompanied by the

formation of intracellular fibrillar inclusions, termed Lewy bodies and Lewy neurites,

which are mainly composed of the small protein α-synuclein (α-syn) [1,2]. Besides PD,

α-syn is also involved in the Lewy body variant of Alzheimer’s disease (AD), diffuse

Lewy body disease (LBD) and multiple system atrophy [2-4]. α-Syn is a 14 kDa

presynaptic protein that acts as a non-classical chaperone during the formation of a

soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-

complex assembly in presynaptic terminals [5]. The role of α-syn in PD

neuropathogenesis is evidenced by the fact that three missense mutations in this protein,

A53T, A30P and E46K, are linked to rare, early-onset forms of PD [6,7].

α-Syn has long been thought to exist as a natively disordered monomer [8]. For this

reason, it has been postulated that the utilization of denaturing conditions to purify

bacterially expressed α-syn does not affect the native protein structure. However, this

view was challenged by recent data showing that α-syn isolated from neuronal and non-

neuronal cell lines exists predominantly as a putative tetramer that is rich in α-helices and

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resistant to fibrillization [9]. Corroborating this hypothesis, an N-terminal GST fusion

construct of α-syn expressed in E. coli and purified under non-denaturing conditions

forms a non-fibrillogenic tetramer in addition to the unfolded monomer [10]. Moreover,

α-syn was found in intact cells mostly as tetramers that undergo destabilization and form

monomers after cell lysis [11]. In contrast with these data, Lashuel and colleagues

demonstrated that bacterially expressed recombinant α-syn behaves as a disordered

monomer when purified under denaturing or nondenaturing conditions. α-Syn has also

been found mostly as a disordered monomer in various mammalian cell lines and in

homogenates from rat, mouse and human brains, including human brain homogenates

from AD and LBD patients [12]. Unlike α-syn produced in bacteria, the protein expressed

in mammalian cell lines undergoes N-terminal acetylation, which may affect the

conformation of the protein [13].

In this work, we explored the oligomeric state of recombinant α-syn using different

strategies to lyse cells (osmotic shock versus ultrasound sonication) or to purify the

protein (heating versus acidification). Independent of the method used for protein

purification, α-syn was obtained predominantly as a disordered monomer in equilibrium

with a minor population of fibrillogenic, non-covalent dimers, which display a mixture of

random-coil and β-sheet secondary structures. These data were corroborated by the

analysis of the structure of α-syn using 1H-15N-heteronuclear single quantum coherence

(HSQC) experiments that revealed that neither the method used for cell lysis nor the

exposure to high temperature or low pH during purification changed the conformation of

the protein. Collectively, these data reinforce the idea that α-syn is expressed in E. coli as

an unfolded monomer that likely exists in equilibrium with variable amounts of

oligomeric species, notably a dynamic dimer prone to fibrillization.

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Results and Discussion

Purification of recombinant α-syn. To verify the effect of the purification protocol on

the conformation of α-syn, we produced recombinant α-syn using different strategies:

osmotic shock versus ultrasound sonication to lyse cells and heating versus acidification

for protein purification. Table 1 compares the α-syn yield obtained using either

ultrasound sonication or osmotic shock to lyse cells. For both methods, the protein

purification was based on two steps: acidification with HCl (pH 3.5) of the cell lysate to

precipitate non-α-syn proteins, followed by precipitation of α-syn with (NH4)2SO4 (50%

saturation). After precipitation of non-α-syn proteins, the supernatant contained 70-74%

α-syn when either ultrasound sonication or osmotic shock was used. Afterwards,

precipitation with 50% saturated (NH4)2SO4 and dialysis resulted in approximately 25-31

mg per L culture of highly pure α-syn, which represented a yield of 58-65% with a purity

of approximately 95-98% as estimated by SDS-PAGE. In this protocol, no

chromatographic steps were used and all purification steps can be easily performed in a

single day. Collectively, our data did not indicate significant differences between osmotic

shock and sonication with respect to the yield or purity of α-syn, though osmotic shock

was previously suggested to be the best choice for release of α-syn-containing periplasm

fraction because the protein is located inside the periplasm instead of the cytoplasm

[14,15]. In addition, the use of MgCl2 in the osmotic shock protocol should be carefully

evaluated because Mg2+ ions were described to interact with α-syn and induce

conformational changes that might inhibit protein aggregation [16].

Most of the current protocols for α-syn purification are based on the boiling of the

cell lysate fraction to remove non-α-syn proteins by precipitation [17,18]. Considering

that α-syn is an unfolded protein, sample heating is not expected to have any effect on the

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structure of α-syn that remains in solution, whereas natively folded cytoplasmic proteins

thermally denature and precipitate. Thus, heating of the cell lysate has been extensively

used as a simple and high-yield protocol for the purification of α-syn, though the

formation of degradation products after α-syn solution is exposed to 90-100 °C has been

reported [19]. To avoid α-syn degradation by heating, the use of acidification with HCl as

an alternative method to purify α-syn was suggested [20]. Figure 1A shows the SDS-

PAGE analysis of samples from each purification step in which either the acidification or

heating of cell lysate was used to remove the majority of non-α-syn proteins. For both

methods, ultrasound sonication rather than osmotic shock was used. The purified α-syn

[Figure 1A, lanes 4 and 4´] displayed a predominant band with an apparent molecular

mass of ~17 kDa corresponding to α-syn monomers. Although α-syn has a molecular

mass of 14 kDa, it migrates on SDS-PAGE with an apparent mass of ~17 kDa, which is

likely due to the poor binding of the N-terminal acidic tail of α-syn to SDS molecules

[14,19]. Curiously, in our experiments, degradation bands were not observed after heat

treatment.

Table 1 shows that a single step based on acid precipitation resulted in α-syn with

a purity of 70%, whereas heating at 90 °C for 15 min resulted in α-syn with only 39%

purity, as estimated by SDS-PAGE. However, the yield from acid precipitation was

significantly lower than obtained by heating (71% compared with 94% yield,

respectively). Though the protein yield was 20% lower with acid precipitation than with

the heating process, both methods provided highly purified protein samples. After the

ammonium sulfate/dialysis steps, the yields were 58% and 78% for heating and acid

precipitation, respectively. Together, these data indicate that precipitation with acid is

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more efficient at removing non-α-syn proteins from the cell lysate, although acidification

resulted in a lower yield than the boiling procedure.

Both heat- and acid-purified α-syn displayed a CD spectrum with a negative band

at 200 nm that is typical of disordered structures, although a slight band at approximately

220 nm was verified for the α-syn obtained using the heating protocol (Figure 1B).

Additionally, the α-syn purified by both the acidification and heating methods displayed

similar fibrillogenic properties; typical sigmoidal curves for the kinetics of fibrillization

were observed, with an initial lag phase, associated with the nucleation process, followed

by an exponential growth phase (Figure 1C).

Oligomeric state of α-syn. To determine the oligomeric state of the recombinant α-syn

purified with either the heating or the acidification protocol, the protein was cross-linked

with GHD and then analyzed by SDS-PAGE (Figure 2). After cross-linking, a small

population of α-syn was found as dimers (3-5% of total α-syn) with an apparent

molecular mass of ~34 kDa, as determined using the Totallab software (Figures 2A and

2B). These species were also revealed by western blot using the monoclonal antibody

anti-human α-syn 14H2L1. We also observed a very small population of α-syn running at

~60 kDa. Because α-syn monomers and dimers exhibit respective molecular masses of 18

and 34 kDa according to SDS-PAGE, this 60 kDa protein may be either a tetrameric or

trimeric form of α-syn. Mass spectrometry (MALDI-TOF/TOF) was used to confirm the

oligomeric state of cross-linked α-syn. A molecular mass of 14,458 Da was obtained

from the MALDI-TOF/TOF spectrum of non-cross-linked α-syn purified using the

heating protocol (Figure 2C), and no differences were observed in comparison with α-

syn purified using the acidification protocol (data not shown). After cross-linking, both

heat- and acid-purified α-syn exhibited a minor population of dimers and trimers with the

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predominant monomer. For the α-syn purified using the heating protocol, a very small

population of tetramers (64,269 Da) was also observed. Together, the analysis of cross-

linked α-syn by both SDS-PAGE and mass spectroscopy indicated that the protein is

mostly monomeric with a small population of oligomers, notably dimers.

Uversky and colleagues demonstrated that a reversible folding of α-syn, with a

gain in β-sheet structure, occurs under high temperature or at low pH, resulting in the

formation of partially folded intermediates with similar conformations [21]. However, the

formation of heat- or acid-induced stable α-syn oligomers has not been reported. Considering that α-syn has a pI=4.7 and. hence, an excess of negative charge at neutral

pH, acidification decreases protein-protein repulsion, thus favoring protein aggregation.

Therefore, one could expect that a small fraction of dimers could be produced by the

acidification of cell lysate during α-syn purification. However, SAXS experiments

indicated that α-syn is monomeric at pH 3.0; hence, the gain in β-sheet structure was not

associated with α-syn oligomerization/fibrillization. More importantly, at pH 3.0,

essentially all of the molecules of the protein rather than just a small fraction behave as

partially folded intermediates. Therefore, the exposure of α-syn to low pH induces a

reversible folding of α-syn mediated by an intramolecular process and not by self-

association/oligomerization of the protein. Collectively, these data indicate that the α-syn

dimers observed here did not result from either HCl or heat treatment. Indeed, dimers

were found at similar concentrations in recombinant α-syn produced by both protocols.

The existence of α-syn dimers in equilibrium with monomers has also been observed

using NMR paramagnetic relaxation enhancement experiments [22] and ESI-MS-ESI

[23,24].

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Using an in vitro cross-linking protocol, α-syn dimers were detected in HEK293T

cells expressing high levels of the protein [12]. However, α-syn from diverse cell lines

migrates as a single band in native electrophoresis gels. In this context, Lashuel and

colleagues suggested that the α-syn dimers detected in intact cells using a cross-linking

protocol may be the result of a diffusion-controlled process rather than actual oligomers

[12]. Although a random association among α-syn monomers that specifically produces

dimers but not other oligomeric forms appears improbable, a series of experiments were

performed to rule out the diffusion-controlled hypothesis. Although only the α-syn

purified by the acidification protocol was used for these experiments, the presence of

dimers was also found in the protein purified using the heating protocol. First, we

evaluated the effect of the concentration of GHD on the pattern of α-syn oligomers.

Notably, the formation of non-specific oligomers induced by cross-linking was expected

to be highly dependent on the concentration of cross-linker. Figure 3A shows an increase

in the band running at the expected molecular weight of dimers when the GHD

concentration was increased from 0.1 to 5 mM (i.e., a protein:GHD ratio of 1:2 to 1:100).

Tetramers were visualized only at high GHD concentrations (25 mM). By fixing the

GHD concentration (5 mM) and varying the protein concentration from 10-100 µM, α-

syn dimers appeared with an intensity proportional to the protein concentration.

Importantly, a ladder-like pattern of α-syn oligomers such as what would be produced by

non-specific cross-linking were not observed at high concentrations of either GHD or α-

syn. A ladder-like pattern of α-syn oligomers, including dimers, was reported for

recombinant α-syn subjected to in vitro cross-linking with discuccinimidyl glutarate

(DSG) [11]. In this case, the cross-linking of α-syn in the presence of lysozyme resulted

in the formation of hetero-oligomers, suggesting that the dimers were products of

diffusion-controlled cross-linking. However, a direct interaction between lysozyme and

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α-syn that may perturb the equilibrium among the diverse oligomeric states of α-syn in

solution cannot be discounted. It should be kept in mind that lysozyme is capable of

forming fibrils in solution similarly to α-syn [25]. In our experiments, we cross-linked α-

syn with GHD in the presence of bovine serum albumin (BSA). Figure 3C shows that

BSA did not interfere with the oligomeric pattern of α-syn. Cross-linking the protein in

the presence of BSA trapped monomers and dimers similarly to α-syn cross-linked alone.

The additional bands correspond to BSA (66 kDa) and its higher-order oligomers.

Therefore, α-syn dimers trapped by GHD are likely actual oligomers rather than an

artifact associated with diffusion-controlled cross-linking.

To verify whether the cross-linking of α-syn produced higher-order molecular

species not detected by SDS-PAGE, the protein was cross-linked with different

concentrations of GHD and then analyzed by SEC in a Superdex 200 column. Due to its

disordered structure, the α-syn monomer has a larger Stokes radius than a folded globular

protein with the same molecular weight, and it thus elutes in SEC as a 60-70 kDa protein

[26]. SEC analysis of non-crosslinked α-syn revealed two distinct populations: a major

population corresponding to the unfolded monomer (elution volume 14.3 mL) along with

the presence of a small population (12.3 mL) that eluted at close proximity to the

unfolded monomer (Figure 3D). The A140C α-syn dimeric mutant exhibits two distinct

peaks in SEC that correspond to the monomeric and the dimeric forms with elution

volumes nearly identical to those observed here [12]. Higher-order oligomers (elution

volume ~7.5 mL) are eluted in or just after the void volume, and they were only

visualized at high GHD concentrations. The low proportion of dimers in the non-cross-

linked sample may be attributed in part to the dilution-induced dissociation of the dimers.

Thus, an increase in GHD concentration resulted in an increase in peak area

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corresponding to the dimer (Figure 3D, inset). Interestingly, cross-linking increased the

elution volume of both the monomeric and the dimeric forms (Figure 3E), which might

be associated with a compaction of the structure of these conformers. It has been

described that monomers and dimers exhibit dynamic conformations and that they may

exist in both extended and compact conformations [24]. For the monomer, a slight

compaction occurs due the contact between the C-terminal region and both the NAC and

N-terminal portions of the protein [27]. In this context, cross-linking with GHD appeared

either to trap compact forms of the monomer and dimer or, more likely, to compact their

structures by linkage-induced approximating amine groups that are distant of each other.

To verify whether α-syn dimers exist in equilibrium with monomers, we isolated

the putative dimer by SEC (elution at 12.3 mL) and then cross-linked with GHD (Figure

3F). Whereas the sample immediately cross-linked appeared as a single ~34 kDa species

by western blot using the monoclonal antibody anti human-α-syn (14H2L1), the sample

that was not cross-linked exhibited a molecular mass corresponding to α-syn monomers

(~17 kDa). Importantly, the presence of the monomer (elution volume of 14.3 mL) with

the dimer was verified when the fraction collected at 12.3 mL was re-chromatographed in

SEC, indicating that the dimers readily dissociated to monomers upon dilution.

Collectively, these data provide convincing evidence that α-syn dimers are highly

dynamic and exist in equilibrium with the monomeric species.

To determine whether α-syn dimers are irreversibly dissociated by heating as

found for the helicoidal tetramer [9], α-syn samples were boiled for 5 min, cooled in an

ice bath and then cross-linked immediately (labeled as 1 min) or 10 or 30 min after the

boiling procedure (Figure 3G). The cross-linking was performed at 25 °C. Whereas α-

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syn boiled but not cross-linked appeared as a single band corresponding to the monomer,

α-syn dimers were visualized in all cross-linked samples, and their intensity was

enhanced with increased recovery time. These data support the idea that the dimers are

not products of non-specific cross-linking and, more importantly, that they are reversibly

dissociated by heat treatment. This effect contrasts with that of the α-syn helicoidal

tetramer, which irreversibly dissociates into unfolded monomers after heating [9].

Structural and fibrillogenic properties of α-syn dimers. To verify whether α-syn

dimers exist on a pathway to fibril formation, aliquots were withdrawn at specific times

from an α-syn solution under aggregation conditions, cross-linked with GHD and then

analyzed by SDS-PAGE (Figure 4A). Only the α-syn purified using the acidification

protocol was used for these experiments. We verified that α-syn dimers were readily

consumed during fibrillization. In fact, the initial population of dimers completely

disappeared after 3 days of aggregation, suggesting that they are an intermediate of the

fibrillization pathway. Unlike the putative helicoidal tetramer that resists fibril formation,

the population of tetramers/trimers found at the initial time appeared to be substrates for

fibrillization because they were consumed during aggregation. In addition, these data

suggest that these species were not produced by diffusion-controlled cross-linking. It is

worth mentioning that a GST-tagged construct of α-syn expressed in E. coli under non-

denaturing conditions fails to form fibrils [10]. However, this construct retained an extra

10 residues at the N-terminus after the removal of the GST tag, and the impact of this

additional sequence on fibrillization behavior was not determined. Only the similarity

between the 1H-15N-HSQC fingerprint of this construct and in vivo α-syn [28] was used to

confirm that this extra sequence did not influence the structural properties of α-syn.

Although our findings agree, in part, with previous works that indicated that existence of

an in vitro and in vivo equilibrium between the disordered monomer and different

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oligomeric forms of α-syn, the inability of these oligomers to evolve to fibrils defended

by some studies remains obscure.

A conversion of α-syn monomer from random-coil to β-rich conformation is a

critical step during protein fibrillization. To verify the secondary structure of α-syn

dimers, dimers were isolated by SEC and analyzed using CD spectroscopy. Figure

4B shows that α-syn dimers displayed a mix of random-coil and β-sheet structure.

The negative band at 200 nm associated with disordered structure was reduced for

the dimer compared with the monomer and accompanied by a rising signal at 218

nm, which corresponds to β-sheet conformation. Based on the CD data, the

hypothesis that α-syn dimers are intermediates in the formation of α-helix-rich

tetramers appears to be improbable, even though our data also suggested the presence

of a minor population of tetramers/trimers in equilibrium with the unfolded

monomers and dimers. Unfortunately, we were not able to isolate and characterize

the secondary structure and fibrillogenic properties of these higher-order oligomers

because of their very low abundance in our experiments.

An important rate-limiting step in fibrillization is the formation of stable covalent

dimers of α-syn, which is favored by oxidative modifications of α-syn such as tyrosine

nitration and the formation of di-tyrosines [29]. In our studies, we observed the presence

of a population of non-covalent dimers of α-syn that was consumed prior to the monomer

during the formation of fibrils. Regarding these data, we speculate that the reversible

dimerization of α-syn may represent an important step in the formation of fibrils, similar

to that reported for the formation of stable covalent dimers under oxidative conditions.

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Conformation of α-syn expressed into E. coli. To determine whether different protocols

for α-syn purification produced conformational changes in the protein, we compared the

1H-15N-HSQC spectra of the protein under different conditions. A similar strategy was

used by Lashuel and colleagues to confirm that α-syn purified using either non-

denaturing or denaturing conditions present nearly identical conformations [12].

However, for these studies, 15N-α-syn (denaturing protocol) was prepared using reverse-

phase HPLC but not the boiling protocol. In our work, cell lysate obtained from either

sonication or osmotic shock was subjected to two sequential precipitations with

ammonium sulfate, at 30% and 50% saturation, to provide an α-syn-rich fraction in

crowded conditions. Figure 5A shows that the 1H-15N-HSQC spectrum of α-syn-

containing cell lysate from osmotic shock (in grey) superimposed completely with the

spectrum of highly pure α-syn (95-98% purity) (in black), which was obtained using the

sonication/acidification protocol. Although α-syn in crowded conditions has 45-50%

purity, the contaminants did not appear to perturb the conformation of the protein. For the

cell lysate obtained by ultrasound sonication, a number of deviations were observed when

its spectrum was compared with that of the pure protein (Figure 5B). These deviations

may be due to either the lower proportion of α-syn in the cell lysate from sonication

compared with osmotic shock (28% vs. 45%) or minor conformational changes of the

protein in the crowded conditions. Nevertheless, we observed that the NMR spectra of α-

syn from the cell lysates and pure protein are similar, and both exhibit a dense cluster of

cross-peaks over a narrow range that indicates a highly disordered conformation. The

Selkoe group demonstrated that the α-syn folded tetramer may undergo destabilization

and the formation of unfolded monomers after cell lysis, which was attributed to the heat

and/or shearing induced by sonication [11]. However, no significant differences in α-syn

structure were observed when the α-syn-containing cell lysates obtained by osmotic

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shock and sonication were compared. Osmotic shock does not produce heat and is hence

a less destabilizing method to for cell lysis than sonication.

Figures 5C and 5D show the effect of boiling on the conformation of highly pure

α-syn and α-syn-containing cell lysate, respectively. Therefore, unlike the previous

experiments described by Lashuel and colleagues [12], the effect of heating on protein

conformation was evaluated on both α-syn alone or in crowded conditions. We observed

that boiling the sample did not produce any effect on the structure of either α-syn sample

as verified by the superposition of the 1H-15N-HSQC spectra before (black) and after

(grey) the treatment. Studies on the effect of macromolecular crowding on α-syn structure

by NMR experiments in vitro and in living E. coli revealed that the crowded environment

in the bacterial periplasm stabilizes the disordered monomer, preventing structural

changes of the protein induced by temperature [28]. Because conformational differences

were not observed between α-syn-containing cell lysate and purified α-syn after heating

to 100 °C and then cooling, we suggest that the conformational changes produced by

heating are most likely completely reversible when the protein returns to 15 °C.

Supporters of the idea that α-syn exists as a helically folded tetramer under native

conditions have noted that the unfolded monomer may be a product of either problems in

generating a correctly folded protein in E. coli or the use of denaturing conditions such as

heating or other chemical denaturants in the purification protocol. However, an

alternative non-denaturing protocol for the production of α-syn generated the helically

folded α-syn tetramer in E. coli [10], suggesting that the reasons for the structural

differences were associated with the protocol used for protein purification rather than its

expression in bacteria. We have shown that the purified α-syn obtained using either

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heating or acidic conditions has essentially the same conformation as the α-syn found in

cell lysate, independent of the use of ultrasound sonication or osmotic shock to lyse

bacteria.

Divergences in the α-syn native structure. Since the helical tetramer was first proposed

to be the predominant physiological species of α-syn in neuronal and non-neuronal cells

[9], a number of studies corroborating this idea have been published in parallel with other

studies contradicting it [10-13]. Curiously, all of these studies are based on well-

established protocols and present compelling evidence to sustain their conclusions. Table

2 summarizes some of the data about the oligomeric state of native α-syn. In light of

these findings, we attempted to examine the source of these contradictions. Upon

purification under non-denaturing conditions and using an in vitro cross-linking protocol,

native α-syn was found to be predominantly a helically folded tetramer in neuronal

(M17D) and non-neuronal cells (HEK293, HeLa, COS-7), frontal cortex mouse tissue

and human red blood cells [9]. An in vivo cross-linking protocol was used to demonstrate

that endogenous α-syn forms an ~60 kDa species in intact neuronal and non-neuronal

cells (HEL cells, M17D, rat primary neurons) [11]. In this same study, it was suggested

that cell lysis (ultrasound sonication) results in a destabilization of the putative 60 kDa

tetramer to form monomers. Therefore, according to this hypothesis, the production of

unfolded monomers may be associated with a lysis sensitivity of α-syn folded tetramers.

Conversely, several lines of evidence support the idea of α-syn as a disordered

monomer. First, the native state of α-syn, alone or tagged with GST, in E. coli was found

to be a disordered monomer even with the use of non-denaturing conditions to purify the

protein. The same results were obtained for α-syn from diverse mammalian cell lines and

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homogenates of mouse, rat and human brains [12]. Second, in-cell NMR studies indicated

that α-syn behaves as a monomer in intact E. coli cells [30], reinforcing the idea that the

disordered monomer is not an artifact associated with either the use of denaturing

methods or cell lysis. It is worth noting that Selkoe and colleagues also identified varying

amounts of monomers, dimers and trimers in certain types of cells [9]. These apparent

conflicting findings may be reconciled by the idea of α-syn monomers existing in

equilibrium with variable amounts of oligomeric species, including the helical tetramer.

In this context, the differences observed in the native structure of α-syn in different cell

lines might arise from the relative abundance of the helical fraction at the time of analysis

as well as the influence of the purification protocols on this subtle equilibrium (see reply

of Bartels and Selkoe in [31]).

Another aspect that may be relevant for understanding the divergence of findings

about the native structure of α-syn is the use of bacterially expressed α-syn instead of α-

syn isolated from higher organisms. Post-translational modifications of α-syn may occur

in mammalian cells that are not present when the protein is bacterially expressed that may

interfere with the native structure of the protein [32]. For example, α-syn purified from

human erythrocytes is N-α-acetylated, a modification commonly found on human

proteins. The acetylation increases the hydrophobicity of the N-terminal portion of the

protein, facilitating protein-protein interactions, and it is also considered a stabilizing

factor for the α-helical structure of proteins [33,34]. However, the impact of N-

acetylation of α-syn (NAc-α-syn) on its physical and functional properties, including

fibrillization kinetics, interaction with lipid membranes and secondary structure, remains

unclear.

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It was reported that the binding properties of α-syn to small unilamellar vesicles are

strongly impacted by its acetylation; NAc-α-syn has an approximately 2-fold higher

affinity for the negatively charged lipid vesicles than the non-acetylated protein does

[35]. However, the N-terminal acetylation appears to not have any effect on the ability of

α-syn to bind to synaptosomal membranes in vitro and in HeLa cells [36]. Differences

were also observed for the role of N-acetylation on the kinetics of fibrillization; some

studies have indicated no effect [35,36], whereas others pointed to a slower growth rate

for the formation of fibrils with NAc-α-syn [37]. Additionally, some studies suggest that

NAc-α-syn exhibits an increased transient helical propensity in comparison with the

intrinsically disordered monomer [35, 37], whereas other studies indicate that N-terminal

acetylation does not significantly affect the protein structure in vivo and in intact cells

[36]. Moreover, the influence of the current protocols for α-syn purification on the

properties of NAc-α-syn is still under investigation. In this context, NAc-α-syn, but not

unmodified α-syn, purified under mild conditions in the presence of the non-ionic

detergent n-octyl-β-glucopyranoside (BOG), forms α-helical tetramers along with

monomers [13]. However, NAc-α-syn behaves as an unfolded monomer when it is

purified in the absence of BOG, even if the detergent is subsequently added to pure α-

syn. These data suggest that the N-acetylation of α-syn alone is not sufficient to promote

the stabilization of helically folded oligomers.

In conclusion, our findings strongly indicate that the disordered monomer is the

major α-syn form expressed in E. coli, although the existence of less-stable oligomeric

forms cannot be ruled out. Importantly, the α-syn disordered monomer is not an artifact

of the production of the protein in E. coli at least in relation to either the method of lysis

or the use of heating/acidification to purify the protein. Additionally, we demonstrated

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that the disordered monomer exists in equilibrium with a dynamic dimer, which is not a

product of diffusion-controlled cross-linking as previously suggested. Moreover, the α-

syn dimer is prone to fibrillate, and thus it might be considered an interesting target to

anti-fibrillogenic molecules.

Materials and Methods

Expression and purification of α-syn. The pT7-7-wt plasmid containing wildtype

human α-syn (kindly provided by D. Foguel) was transformed into BL21(DE3)pLysS E.

coli, and when the absorbance at 600 nm reached 0.5, its expression was induced with the

addition of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 15-18 h at 20 °C.

Cells were harvested by centrifugation (8,000 g) for 30 min, resuspended in 20 mM Tris-

HCl, 5 mM EDTA and 1 mM PMSF pH 8.0 and then lysed by ultrasonication for 1 min

intervals from 1 min to 30 min at 300 Watts. Alternatively, α-syn from the periplasm was

released by osmotic shock as previously described [14]. The non-α-syn proteins in the

crude extract were removed using two well-established protocols: acidic or heat

treatment. In the former, the pH of the crude extract was adjusted to 3.5 with 9% HCl and

then centrifuged to 16,000 g for 20 min. The pH of the supernatant was then raised to 7.5.

In the heating protocol, crude extract was heated at 90 °C for 15 min and then centrifuged

at 16,000 g for 20 min. Then, α-syn was precipitated by the addition of ammonium

sulfate to 50% saturation, followed by centrifugation (16,000 g, 20 min). The pellet was

resuspended in 20 mM Tris buffer pH 8.0 with 1 mM EDTA and dialyzed overnight

against 2 L of the same buffer. After a dialysis against MilliQ water (2 L, 2 times), α-syn

was lyophilized and characterized by circular dichroism, SDS-PAGE and size-exclusion

chromatography (SEC) in a Superdex 200 10/300 GL column (GE Healthcare, Uppsala,

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Sweden). The percentage of α-syn in each fraction of the purification was estimated by

the relative intensity of its band after SDS-PAGE using the Totallab program.

Cross-linking. To determine the α-syn oligomeric state, 50 µM of protein in 20 mM

HEPES pH 7.0 was cross-linked by incubation with varying concentrations of

glutaraldehyde (GHD) for 10 min (25 °C), and the reaction was stopped by the addition

of 1 M Tris buffer.

Immunoblotting. A total of 5-10 μg per lane of protein, cross-linked or untreated, was

loaded onto a 12% SDS-PAGE gel, blotted onto a nitrocellulose membrane and incubated

with rabbit monoclonal anti-human α-syn (14H2L1) (1:1,000) (Life Technologies, USA)

followed by incubation with the secondary antibody anti-rabbit IgG tagged with alkaline

phosphatase; the bands were visualized using Sigma-Fast™ BCIP/NBT (Sigma-Aldrich,

USA).

α-Syn fibrillization. α-Syn fibrils were formed by the incubation of 50 μM α-syn in 10

mM sodium phosphate buffer (NaPB) with 100 mM NaCl pH 7.5 in a 96-well plate

(Corning NBS 96-well white plate) at 37 °C under stirring (350 rpm) in a Thermomixer

Comfort apparatus (Eppendorf, Germany) in the presence of one 3-mm glass bead per

well. The fibrillization was monitored in situ by performing the assay in the presence of 5

µM Thioflavin-T (Thio-T). Thio-T binding was evaluated by fluorescence in a Cary

Eclypse Fluorimeter (Varian Inc.) by exciting at 446 nm and collecting the emission at

485 nm (slits: 5 nm).

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Circular dichroism (CD) spectroscopy. CD spectra were recorded in a Jasco-715

spectropolarimeter (Jasco, Tokyo). A buffer containing 10 mM NaPB and 100 mM NaCl

pH 7.5 was used as baseline and subtracted from the protein spectra. The spectra were

recorded from 195 to 260 nm.

Mass spectrometry analysis of GHD-cross-linked α-syn. Mass spectra were obtained

using a Bruker Daltonics Ultraflex Speed MALDI-TOF/TOF. Samples were diluted to

approximately 10 mg/mL in 0.1% TFA and mixed 1:1 with sinapinic acid at 10 mg/mL.

Samples from cross-linked α-syn (from the heating or the acidification protocol) were

centrifuged at 13,400 rpm for 5 min to pellet insoluble material. Non-cross-linked α-syn

was diluted with 3 M guanidine hydrochloride with 0.1% TFA and cleaned using a

ZipTip (Millipore, USA) prior to mixing with sinapinic acid. Appropriate calibrants

were measured every four unknown samples.

Two-dimensional NMR. NMR measurements were performed at 15 °C on a Bruker

Avance III (Bruker Biospin GmbH, Reinstetten, Germany) operating at a 1H frequency of

800 MHz. The sample contained 200 µM of purified 15N-α-syn in 10 mM NaPB pH 7.5

and 100 mM NaCl [10% D2O (v/v)]. For the analysis of cell lysate containing 15N-α-syn,

the fraction from the sonication step was subjected to two sequential precipitations with

ammonium sulfate: the first at 30% saturation to precipitate non-α-syn proteins, followed

by a second precipitation at 50% saturation in which α-syn was recovered in the pellet.

This procedure provided α-syn with approximately 60-70% purity. The 1H-15N-HSQC

experiments were acquired with echo-antiecho using 1,024 points in the direct dimension

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and 320 points in the indirect dimension. Data were processed with TOPSPIN 3.1

(Bruker Biospin Corporation, USA) and analyzed using the CARA software package

(http://www.nmr.ch). The 1H-15N-HSQC spectra of α-syn were assigned according to Rao

et al. [38].

Acknowledgements:

This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de

Janeiro (FAPERJ) and the International Foundation for Science (IFS).

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Table 1. Purification of α-syn from E. coli.

Osmotic shock/acidification Sonication/ acidification Sonication/BoilingPurification steps

Total protein, mg

α-syn purity, %

α-syn, mg

Yield, %

Total protein

α-syn purity, %

α-syn, mg

Yield, %

Total protein

α-syn purity, %

α-syn, mg

Yield, %

Crude extract 85 45 38 100 191 28 53 100 195 26 51 100

Acid/ boiling 45 74 33 86 55 70 38 71 123 39 48 94

(NH4)2SO4 31 90 28 74 38 91 35 66 56 80 45 88

Dialysis 27 95 25 65 32 98 31 58 42 95 40 78

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Table 2. Summary of some of the main findings about the native oligomeric state of α-syn.

Source Experimental approach α-Syn oligomeric state Reference

Neuronal (M17D) and non-neuronal cells (HEK293, HeLa, COS-7); frontal cortex mouse tissue; human red blood cells

Native gel electrophoresis; analytical ultracentrifugation; in vitro cell cross-linking

Mostly helical folded tetramer 50-60 kDa; variable amounts of monomer

[9]

Bacterially expressed recombinant α-syn (E. coli; GST-tag)

Mild-purification conditions (use of BOG); NMR; in vitro cross-linking

Stable helical tetramers [10]

Intact neuronal and non-neuronal cells (HEL cells, M17D, rat primary neurons)

In vivo cross-linking Mostly oligomers of ~ 60 kDa; free monomers produced by cell lysis

[11]

Bacterially expressed recombinant α-syn (E. coli; with or without GST-tag); mammalian cell lines; homogenates of mouse, rat and human brains

Native and denaturing electrophoresis; oligomer-specific ELISA

Mostly disordered monomers [12]

Bacterially expressed recombinant α-syn (intact E. coli cells)

In-cell NMR Mostly disordered monomers [30]

Bacterially expressed recombinant NAc-α-syn Mild-purification conditions (use of BOG); analytical ultracentrifugation

α-helical tetramers and monomers [13]

Semisynthetic and bacterially expressed recombinant NAc-α-syn

Native and denaturing electrophoresis; in-cell NMR

Disordered monomers [36]

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Figure 1. Recombinant α-syn produced using either a heating or acidification

protocol. (A) The fractions from each step of purification of α-syn were analyzed by

denaturing electrophoresis (12% SDS-PAGE): 1: crude cell extract; 2 and 2´:

supernatants after boiling (10 min) or acidification (pH 3.5), respectively; 3 and 3’:

pellets from 50% (NH4)2SO4 saturation (precipitation of α-syn) of fractions from boiling

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and acidification procedures, respectively; 4 and 4´ correspond to the fractions 3 and 3´,

respectively, after the dialysis step. (B) and (C) display the CD spectra and the kinetics of

fibrillization, respectively, of α-syn purified using the heating or acidification protocol.

The kinetic assays were performed in sextuplicates and the results are represented as

mean ± standard deviations.

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Figure 2. Oligomeric states of α-syn purified using either heating (A) or acidification

(B). α-Syn at a concentration of 50 µM, boiled for 5 min or untreated with heat, was in

vitro cross-linked with 5 mM GHD, run on a 12% SDS-PAGE gel and stained by silver

staining (on the left). The corresponding immunoblots (on the right) were performed

using a monoclonal human α-syn antibody (14H2L1) (Life Technologies, USA). The

MALDI-TOF/TOF analysis of α-syn before and after cross-linking with GHD is shown in

(C). Observed molecular weights are depicted above their respective peaks.

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Figure 3. The α-syn dimer is not produced by diffusion-controlled cross-linking. (A)

Pure recombinant α-syn (50 µM) was cross-linked with GHD concentrations of 0, 0.1,

0.5, 1 and 5 mM and run on a 12% SDS-PAGE gel (revealed by silver staining). (B) In

vitro cross-linking was performed by fixing the GHD concentration at 5 mM and using α-

syn concentrations of 10, 20, 50, 70 and 100 µM. (C) α-Syn at a concentration of 50 µM

was cross-linked with 5 mM GHD in the absence or presence of an equimolar

concentration of BSA; on the right, the cross-linking of 50 µM α-syn was performed in

the presence of varying concentrations of BSA (25, 50 and 100 µM). (D) 50 µM α-syn

was in vitro cross-linked with 0, 1, 5 or 25 mM GHD and then run in a Superdex 200

10/300 GL column. The elution volumes of the unfolded monomer and dimer, cross-

linked or left untreated, were plotted in (E); a standard curve was generated using the

proteins lysozyme (14.4 kDa), carbonic anhydrase (30 kDa), ovalbumin (45 kDa), BSA

(66 kDa) and phosphorylase B (97 kDa). (F) The fraction corresponding to the α-syn

dimer (elution at 12.3 mL) was separated from the monomer (elution at 14.3 mL) using

SEC (Superdex 200 10/300 GL column), cross-linked with 5 mM GDH or left untreated

and then analyzed by western blotting using monoclonal human α-syn (14H2L1). In

addition, the non-cross-linked dimer fraction was re-chromatographed in SEC (showed in

red, on the right). (G) To verify whether α-syn dimers were irreversibly dissociated by

heating, 50 µM of protein was boiled for 5 min, cooled in an ice bath and then cross-

linked immediately (labeled as 1 min), 10 or 30 min after the boiling procedure. The non-

cross-linked protein is shown on the left.

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Figure 4. The α-syn dimer displays a mix of random coil and β-sheet secondary

structure and is a precursor of fibril formation. (A) To verify whether α-syn dimers

exist on the fibril formation pathway, aliquots were withdrawn at 0, 1 and 3 days from 50

µM α-syn solution under aggregation conditions (37 °C, 350 rpm), cross-linked with

GHD and then analyzed on a 12% SDS-PAGE gel. (B) α-Syn dimers were isolated by

SEC and analyzed by CD spectroscopy.

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Figure 5. NMR 1H-15N-HSQC spectra of α-syn under different conditions.

Comparison between the 1H-15N-HSQC spectra of α-syn-containing cell lysate from

osmotic shock (~45% α-syn) (A) or ultrasound sonication (28% α-syn) (B), both shown

in grey, and the highly pure α-syn (95-98% purity) (in black) obtained using the

sonication/acidification protocol. (C) and (D) show the effect of boiling on the

conformation of pure α-syn and α-syn-containing cell lysate (sonication), respectively.

All experiments were performed at 15 °C and using 200 µM 15N-α-syn in PBS buffer.

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