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Molecular Brain Research

Research report

Effects of nitration on the structure and aggregation of a-synuclein

Vladimir N. Uverskya,b,*, Ghiam Yamina, Larissa A. Munishkinaa, Mikhail A. Karymovc,

Ian S. Millettd, Sebastian Doniachd, Yuri L. Lyubchenkoc, Anthony L. Finka,*

aDepartment of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USAbInstitute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia

cSchool of Life Sciences, Arizona State University, Tempe, AZ 85287, USAdDepartments of Physics and Chemistry, Stanford University, Stanford, CA 94305, USA

Accepted 17 November 2004

Available online 3 February 2005

Abstract

Substantial evidence suggests that the aggregation of the presynaptic protein a-synuclein is a key step in the etiology of Parkinson’s

disease (PD). Although the molecular mechanisms underlying a-synuclein aggregation remain unknown, oxidative stress has been implicated

in the pathogenesis of PD. Here, we report the effects of tyrosine nitration on the propensity of human recombinant a-synuclein to fibrillate in

vitro. The properties of nitrated a-synuclein were investigated using a variety of biophysical and biochemical techniques, which revealed that

nitration led to formation of a partially folded conformation with increased secondary structure relative to the intrinsically disordered

structure of the monomer, and to oligomerization at neutral pH. The degree of self-association was concentration-dependent, but at 1 mg/mL,

nitrated a-synuclein was predominantly an octamer. At low pH, small-angle X-ray scattering data indicated that the nitrated protein was

monomeric. a-Synuclein fibrillation at neutral pH was completely inhibited by nitrotyrosination and is attributed to the formation of stable

soluble oligomers. The presence of heparin or metals did not overcome the inhibition; however, the inhibitory effect was eliminated at low

pH. The addition of nitrated a-synuclein inhibited fibrillation of non-modified a-synuclein at neutral pH. Potential implications of these

findings to the etiology of Parkinson’s disease are discussed.

D 2004 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Degenerative disease: Parkinson’s

Keywords: a-synuclein;Oxidative stress; Nitration;Aggregation; Amyloid fibrils; Inhibition of fibrillation; Partially folded intermediate; Natively unfolded protein

1. Introduction

Proteins are targets for numerous oxidative modifica-

tions induced by their interaction with reactive oxygen

Abbreviations: ROS, reactive oxygen species; RNS, reactive nitrogen

species; PD, Parkinson’s disease; AD, Alzheimer’s disease; LB, Lewy

bodies; LN, Lewy neuritis; TNM, tetranitromethane; CD, circular dichro-

ism; SAXS, small angle X-ray scattering; FTIR, Fourier transform infrared;

TFT, thioflavin T; DLS, dynamic light scattering; SEC, size-exclusion

chromatography; EM, electron microscopy; AFM, atomic force micro-

scopy; GAG, glycosoaminoglycan

* Corresponding authors. Department of Chemistry and Biochemistry,

University of California, Santa Cruz, CA 95064, USA. Fax: +1 831 459

2935.

E-mail address: enzyme@ucsc.edu (A.L. Fink).

0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.molbrainres.2004.11.014

species (ROS) or reactive nitrogen species (RNS), produced

as a result of different physiological and non-physiological

processes. The spectrum of oxidative modifications in

proteins is very wide. For example, interaction of a protein

with nitric oxide can lead to the S-nitrosylation of reduced

cysteine residues, the nitration of tyrosine and tryptophan

residues and the formation of nitric oxide-iron heme

adducts [28]. Here we focus on tyrosine nitration, which

is a covalent protein modification resulting from the

addition of a nitro (�NO2) group onto one of the two

equivalent ortho carbons of the aromatic ring of tyrosine

residues [26].

It is known that several nitrating species, including

nitrogen dioxide, peroxynitrite, and nitrous acid, can be

134 (2005) 84–102

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 85

formed in vivo either concurrently or at different times

depending on the cell type and the stimulus; consequently,

there are several potential pathways leading to the nitration of

tyrosine residues in proteins [26]. For example, the generation

of nitric oxide from arginine is catalyzed by nitric oxide

synthase; under pathological circumstances, the formation of

peroxynitrite by the reaction of nitric oxide and superoxide

takes place and the subsequent reaction of peroxynitrite with

CO2 leads to the formation of an intermediate capable of

nitrating tyrosine residues in proteins [31]. Interestingly, not

all proteins are uniformly susceptible to oxidative damage

and only a few proteins undergo nitration. Recently, it has

been shown that there were only about 40 nitrated proteins out

of 1000 during an inflammatory challenge. These included a

large number of mitochondrial proteins, which regulate

cellular energy metabolism [2]. In many cases, nitration has

been shown to affect protein function, for example, man-

ganese superoxide dismutase has been shown to be inacti-

vated by selective nitration [38], and peroxynitrite-mediated

nitration of lymphocyte-specific tyrosine kinase inhibits the

ability of this protein to phosphorylate tyrosine residues [32].

Creatine kinase is another key intracellular enzyme regulating

energy metabolism that is nitrated and inactivated by

peroxynitrite [65].

Oxidatively modified proteins have been implicated in the

pathogenesis of both normal aging and neurodegenerative

diseases, including Alzheimer’s and Parkinson’s diseases

[3,27,28,30]. PD is the second most common age-related

neurodegenerative disorder, and is diagnosed in more than

50,000 Americans each year. It is manifested as a movement

disorder associated with the progressive loss of dopaminer-

gic neurons in the substantia nigra. The histopathologic

hallmark of PD is eosinophilic cytoplasmic inclusions in the

surviving nigral dopaminergic neurons known as Lewy

bodies (LBs) and Lewy neurites (LNs) [18,35]. The cause of

PD is unknown, but considerable evidence suggests a

multifactorial etiology involving genetic and environmental

factors [76]. Several lines of evidence implicate the

presynaptic protein a-synuclein in PD pathogenesis. Fibrillar

a-synuclein is a part of Lewy bodies [64]; familial early-

onset PD has been associated with the missense mutations

A53T, A30P, and E46K in a-synuclein [33,49,81]; tripli-

cation of the a-synuclein gene locus also causes familial

early-onset PD [60]; the production of a-synuclein in

transgenic mice [40] or flies [14] leads to the motor deficits

and neuronal inclusions reminiscent of PD.

a-Synuclein is a small (14 kDa), highly conserved

protein that is abundant in various regions of the brain

[39]. This protein has been estimated to account for as much

as 1% of the total protein in soluble cytosolic brain

fractions. a-Synuclein has four tyrosines, Tyr39, Tyr125,

Tyr132, and Tyr135 and is a natively unfolded protein

[13,74,78], which has little or no ordered structure under

physiological conditions, due to a unique combination of

low overall hydrophobicity and large net charge [72]. In

vitro, a-synuclein forms fibrils with morphologies and

staining characteristics similar to those extracted from

disease-affected brain [6,7,9,19,36,42].

Oxidative injury has been implicated in the pathogenesis

of PD [12,20,29,67]. The existence of extensive and wide-

spread accumulation of nitrated a-synuclein (i.e., protein

containing the product of the tyrosine oxidation, 3-nitro-

tyrosine) in Lewy bodies has been demonstrated, using

antibodies to specific nitrated tyrosine residues ina-synuclein

[12,20]. It was proposed that the selective and specific

nitration of a-synuclein in these disorders may directly link

oxidative and nitrative damage to the onset and progression of

neurodegenerative synucleinopathies [20]. We have previ-

ously reported that a-synuclein fibril formation at neutral pH

in vitro was completely inhibited by tyrosine nitration [80],

and that the addition of sub-stoichiometric concentrations of

nitrated a-synuclein led to a substantial inhibition of

fibrillation of non-modified a-synuclein. The fact that

fibrillation is relatively unaffected by nitrative oxidation of

a Tyr-free mutant of a-synuclein demonstrates that Tyr and

Tyr nitration are not required for fibrillation [45]. Here, we

present the results of a detailed structural and conformational

analysis of nitrated a-synuclein, as well as some new details

of its aggregation, in order to shed light on the underlying

molecular mechanisms of this inhibitory effect.

2. Materials and methods

2.1. Expression and purification of human a-synuclein

Human wild type a-synuclein was expressed using

Escherichia coli BL21 (DE3) cell line transfected with

pRK172/a-synuclein WT plasmid (generously donated by

M. Goedert, MRC Cambridge). Expression and purification

of human recombinant a-synuclein and its mutants from E.

coli were performed as previously described [77].

2.2. Supplies and chemicals

Thioflavin T (ThT), 1-anilinonaphthalene-8-sulfonic acid

(ANS), and uranyl acetate dihydrate were obtained from

Sigma (St. Louis, MO). Titanium (III) sulfate and tetranitro-

methane (TNM) were obtained from Aldrich Chemical Co.

Aluminum (III) chloride and heparin were obtained from

Mallinckrodt Chemical Works and GibcoBRL, respectively.

Zinc (II) sulfate, lead (II) nitrate, calcium (II) chloride, and

all other chemicals were of analytic grade from Fisher. All

buffers and solutions were prepared with nanopure water

and stored in plastic vials.

2.3. Nitration of a-synuclein by tetranitromethane

Nitration of a-synuclein was induced by adding a 50 ALaliquot of 1% tetranitromethane in ethanol to 500 AL of 4–5

mg/mL protein solution. The reaction mixture was stirred

vigorously at room temperature for 10 min. The procedure

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–10286

was repeated with addition of another 50 AL aliquot of 1%

TNM solution under the same conditions [54]. After 10 min,

urea was added to a final concentration of 2M and this

protein mixture was dialyzed with four changes of

appropriate buffer at pH 7.8 to completely remove unreacted

TNM. Success of oxidation was confirmed by MS analysis

(MicroMass Quattro II). Samples for MS analysis were

prepared by diluting 2 AL of protein solution in 200 AL of

50% acetonitrile/50% pH 2.0 HCl mixture.

2.4. UV absorbance spectroscopy

UV absorbance spectra were determined with semimicro

quartz cuvettes (Hellma) with a 5-mm light path using a UV-

2401 spectrophotometer (Shimadzu, Japan). Protein quanti-

fication was determined using absorption maximum of 275

for control and 428 nm for oxidized a-synuclein. Molar

absorptivity of oxidized protein was calculated according to

Sokolovsky et al. [62].

2.5. Circular dichroism (CD) measurements

CD spectra were obtained on an AVIV 60DS spectropho-

tometer (Lakewood, N.J.) using a-synuclein concentrations

of 1.0 mg/mL in 40 mM phosphate/100 mM NaCl or Tris/

NaCl buffer. Spectra were recorded in a 0.1-mm pathlength

cylindrical quartz cell (Starna, Atascadero, CA) from 250 to

190 nm with a step size of 1.0 nm, band width of 1.5 nm, and

an averaging time of 2 s. For all spectra, an average of 5 scans

was obtained. CD spectra of the appropriate buffers were

recorded and subtracted from the protein spectra.

2.6. FTIR spectra

Attenuated total reflectance (ATR) Fourier Transform

Infrared Spectroscopy data was collected on a Nexus 670

FTIR instrument (ThermoNicolet, Madison, WI). Protein

samples at 0.5 mg/mL, 50 AL volume were measured using

the hydrated thin film technique, as described previously

[46,47].

2.7. Dynamic light scattering

Stokes radii were determined using a DynaPro Molecular

Sizing Instrument (Protein Solutions, Lakewood, NJ) at a-

synuclein concentration of 0.1 mg/mL using a 1.5 mm

pathlength 12 AL quartz cuvette. Prior to measurement,

solutions were filtered with a 0.1-Am Whatman Anodisc-13

filter.

2.8. Fluorescence measurements

Fluorescence measurements were determined using a

FluoroMax-3 spectrofluorometer (Instruments S.A., Jobin

Yvon Horiba, France) with a 1-mL semimicro quartz

cuvette (Hellma) having 1-cm excitation light pathlength.

Tyrosine fluorescence was monitored in the 290 to 350 nm

range after excitation at 275 nm. Protein concentration was

kept at 0.1 mg/mL. ANS binding measurements were

performed using the spectrofluorometer and 1-mL semi-

micro cuvette previously described. Emission spectra were

recorded from 400 to 600 nm with an excitation at 350 nm

in increments of 1 nm, an integration time of 1 s, and a slit

width of 1 nm. Exact concentration of ANS was

determined by its absorption at 350 nm. The molar ratio

of ANS : a-synuclein was kept equal to 5.

2.9. Small-angle X-ray scattering experiments

SAXS measurements were made using Beam Line 4-2

at Stanford Synchrotron Radiation Laboratory. X-ray

energy was selected at 8980 eV (Cu edge) by a pair of

Mo/B4C multilayer monochromator crystals [68]. Scatter-

ing patterns were recorded by a linear position-sensitive

proportional counter, which was filled with an 80% Xe/

20% CO2 gas mixture. Scattering patterns were normal-

ized by incident X-ray fluctuations, which were measured

with a short length ion chamber before the sample. The

sample-to-detector distance was calibrated to be 230 cm,

using a cholesterol myristate sample. The measurements

were performed in a 1.3-mm path length observation

static cell with 25 Am mica windows. To avoid radiation

damage of the sample in SAXS measurements, the

protein solution was continuously passed through a 1.3-

mm path length observation flow cell with 25 Am mica

windows. Background measurements were performed

before and after each protein measurement and then

averaged before being used for background subtraction.

All SAXS measurements were performed at 23 F 1 8C. Theradius of gyration (Rg) was calculated according to the

Guinier approximation [22]:

ln I Qð Þ ¼ ln I 0ð Þ � R2g Q

2=3

where Q is the scattering vector given by Q = (4k sinu)/k,where 2u is the scattering angle, and k is the wavelength of

X-ray. I(0), the forward scattering amplitude, is proportional

to molecular mass of the scattering profile.

2.10. Size exclusion chromatography (SEC)

Hydrodynamic dimensions (Stokes radius, RS) of non-

modified and nitrated a-synucleins at different fibrillation

stages were measured by gel filtration. SEC data were

collected on a Waters 2695 Separation Module (Milfold,

MA) with a Waters 996 Photodiode Array Detector and

Millennium 32 software. Protein (~0.05 mg/mL) was

loaded onto a Tosoh Bioscience G2000SWXL column.

The elution was carried out isocratically at a flow rate of

0.4 mL/min and monitored by the absorbance at 280 nm.

Prior to measurement, solutions were filtered with a 0.1 AmWhatman Anodisc-13 filter. All measurements were made

at 25 8C. A set of globular proteins (Gel Filtration

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 87

Chromatography Standards from Bio-Rad Laboratories)

with known RS values was used in order to create a

calibration curve, 1000/Vel versus RS [8,69,70]. The

accuracy of determination of RS by this equation is about

5%. Relative areas of chromatographic peaks were esti-

mated from the elution profiles by their deconvolution

using LabCalc. The accuracy of such deconvolution was

~10%.

Hydrodynamic dimensions of natively unfolded protein

and the pre-molten globule-like partially folded protein were

calculated from empirical equations [75]:

log RNUS

� �¼ � 0:551F 0:032ð Þ þ 0:493F 0:008ð Þ:log Mð Þ

log RPMGS

� �¼� 0:239F 0:055ð Þ þ 0:403F 0:012ð Þ:log Mð Þ

where M is the molecular mass, whereas RSNU and RS

PMG are

the Stokes radii of natively unfolded (NU) and pre-molten

globule-like (PMG) protein, respectively.

2.11. Fibril formation assay

Fibril formation of oxidized and non-oxidized a-

synuclein in presence of various metals and heparin was

monitored in a fluorescence plate reader (Fluoroskan

Ascent). Protein solutions contained 20 AM ThT and

protein concentration of 1.0 mg/mL (70 AM) at pH 7.5.

Buffers used to assay oxidized a-synuclein in presence of

control or heparin and metals were 40 mM phosphate,

100 mM NaCl buffer and 25 mM Tris, 50 mM NaCl,

respectively. Final heparin concentration was 75 AM while

those of various metals tested were 1 or 2 mM. Samples

were run in quadruplicate or quintuplicate with 150 ALsample volumes along with a 1/8th in. diameter Teflon

sphere (McMaster-Carr, Los Angeles) in wells of a 96-well

plate (white plastic, clear bottom). The plate was loaded

into a fluorescence plate reader and incubated at 37 8Cwith shaking at 120 rpm with a shaking diameter of 20 mm.

Fibril formation was detected by measurement of ThT

fluorescence at 30-min intervals with excitation at 444 nm

and emission at 485 nm; curve fitting was implemented as

described previously [44].

2.12. Lowry assay

Lowry assay was performed on well samples obtained

from the fibril formation assay: 50 AL samples were

centrifuged at 13,000 rpm for 20 min, the supernatant was

removed and mixed with 50 AL of buffer while the pellet

was re-dissolved in 100 AL of buffer for each sample tested.

Supernatant and re-dissolved pellet were added separately to

a mixture of 2 mL reagent A and 0.2 mL 50% Folin reagent

[37]. After vigorous mixing, samples were incubated for 2 h

to complete the reaction.

2.13. Electron microscopy

Negative staining electron microscope (EM) images

were taken using Formvar/carbon grids (Ted Pella, Redd-

ing, CA). Grids were prepared by incubating 2-fold diluted

protein samples for 10 min, washing three-times with

water and then negatively staining with 1% uranyl acetate

for 5 min.

2.14. Atomic force microscopy imaging

1-(3-Aminopropyl)silatrane (APS)-modified mica was

used as an AFM substrate [58,59]. 5 AL of the sample were

placed on APS-mica for 2 min, rinsed with deionized

water, and dried with argon. Images were acquired in air

using MAC mode AFM (Molecular Imaging, Phoenix, AZ)

and MultiMode SPM NanoScope IIIa system (Veeco/

Digital Instruments, Santa Barbara, CA) operating in

Tapping Mode. Type II MAC levers (Molecular Imaging,

Phoenix, AZ) with spring constant ~2.8 N/m were used

with MAC mode AFM. Silicone cantilevers from Olympus

(Asylum Research, Santa Barbara, CA) with spring

constant ~42 N/m were used with the MultiMode system.

The typical resonant frequency was 60–70 kHz for Type II

Mac tips and 360–400 kHz for the Olympus tips. The

width and height measurements were determined from the

AFM images using Femtoscan software (Advanced Tech-

nologies Center, Moscow, Russia). The diameters of

spherical-shaped oligomers were estimated from the height

measurements.

2.15. Limited proteolysis

Non-modified and nitrated a-synucleins (1 mg/mL) were

digested with trypsin. Trypsin (5 pmol) digestion was

carried out in a total volume of 20 AL with 20 mM Tris–

HCl (pH 7.5) at 37 8C for various time intervals. The

reactions were stopped by 1:1 dilution with a sample buffer

containing 8% SDS, 24% glycerol, 0.015% Coomassie blue

G, and 0.005% Phenol red in 0.9 M Tris–HCl (pH 8.45).

The cleaved products were analyzed with the precast High

Density acrylamide gels from PhastSystem (Amersham

Biosciences, Piscataway, NJ). The gel was run according

to the manufacturer’s procedure and visualized with

Coomassie brilliant blue staining. Experiments were run at

least in duplicate, and the data averaged. The rate of

digestion was proportional to the trypsin concentration.

3. Results

3.1. Nitration of a-synuclein in vitro

The conversion of Tyr to Tyr(NO2) using TNM is well

known [61,62]. The most probable scheme for the reaction

of TNM with Tyr is indicated in the following scheme [62]:

Scheme 1.

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–10288

The modified protein was subjected to MS and spectro-

scopic analysis to determine the extent of nitration. ESI-MS

analysis of a-synuclein prior to the oxidative modification

revealed one major peak, with the expected molecular mass

of 14460 F 3 kDa. The conditions used for the in vitro

modification of a-synuclein resulted in nitration of all four

tyrosines, as evidenced by the major MS peak positioned at

14642F 3 kDa, which corresponds to the mass of human a-

synuclein with four tyrosines nitrated (14460 + 4� 46� 4�1 = 14640 kDa, see Scheme 1 above).

The degree of nitration can be further measured by specific

changes in the spectral properties of the protein solution. At

pH 8.0, the Tyr absorption maximum at 275 nm has a molar

absorptivity, e, of 1360. Nitration increases e to 4000 and, inaddition, generates a new absorption band with maximal

absorption at 428 nm and e = 4100 [62]. Fig. 1 compares the

absorption spectra of non-modified and nitrated a-synucleins

and illustrates the increase in the absorption in the vicinity of

275 nm and the appearance of a new band at 428 nm

corresponding to the absorption of Tyr(NO2). Another

important feature of the Tyr(NO2) is the lack of the character-

istic tyrosine fluorescence [54,55]. In agreement with this

observation, the inset to Fig. 1 shows the fluorescence spectra

of the nitrated and non-modified a-synucleins. As expected,

the nitration quenches the protein’s intrinsic fluorescence.

Fig. 1. Comparison of the absorbance spectra of non-modified (solid line) an

fluorescent spectra at excitation at 275 nm. Measurements were performed at pH 8

and fluorescence measurements, respectively.

Both MS and absorbance data indicate that the nitrated

a-synuclein is, within experimental error, fully nitrated.

3.2. Effect of nitration on structural properties of

a-synuclein

3.2.1. Secondary structure from FTIR

Fig. 2A shows the FTIR (amide I region) spectra measured

for non-oxidized (solid line) and nitrated forms (dashed line)

of human recombinant a-synuclein at pH 7.5. The FTIR

spectrum of non-modified a-synuclein at pH 7.5 is typical of

a substantially unfolded polypeptide chain, whereas nitration

leads to significant spectral changes, indicative of increased

ordered structure. The most evident change is the decrease of

the band in the vicinity of 1655 cm�1, which corresponds to

the disordered conformation, accompanied by the appearance

of a new band in the vicinity of 1625 cm�1, which

corresponds to h-sheet. These observations are further

illustrated by Fig. 2B, which represents the difference FTIR

spectrum and clearly shows that the depletion in signal in the

vicinity of 1655 cm�1 occurs concomitantly with the rise of

the signal around 1625 cm�1. This means that the nitrated a-

synuclein contained increased amounts of h-structure in

comparison with the non-modified protein. We have pre-

viously observed increased h-sheet content in soluble

d nitrated a-synucleins (dashed line). Inset represents the corresponding

.0 (23 8C). Protein concentration was 0.35 and 0.05 mg/mL for absorbance

Fig. 2. Secondary structure analysis of the non-modified (solid line) and nitrated a-synucleins (dashed line) by ATR-FTIR spectroscopy. (A) FTIR spectra in

the amide I region measured at pH 7.5. Measurements were carried out at 23 8C. Protein concentration was 0.5 mg/mL. (B) Nitration-induced changes in a-

synuclein secondary structure as detected by difference FTIR spectra. The FTIR spectrum of non-modified a-synuclein has been subtracted from the spectrum

of the nitrated protein. Thus, the difference spectrum reports the excess (positive values) or deficit of particular structure (negative values) in the nitrated protein

as compared to the non-modified protein.

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 89

oligomeric forms of human a-synuclein analyzed by FTIR

[36,41,74,75,77,80], which were attributed to the formation

of the oligomers (see below).

3.2.2. Secondary structure from far-UV CD

In agreement with FTIR data, the far UV-CD spectrum of

nitrated a-synuclein measured at pH 7.5 shows a slightly

increased degree of order in comparison with the non-

oxidized protein (Fig. 3). This is manifested by a small

decrease in negative ellipticity in the vicinity of 196 nm and

somewhat larger intensity in the vicinity of 222 nm. Thus,

nitration leads to some increase in the amount of secondary

structure compared to natively unfolded monomeric a-

synuclein. Lowering the pH of a solution of a-synuclein

results in the transformation of natively unfolded a-

synuclein into a partially folded conformation [74]. Interest-

ingly, the low pH spectrum of unmodified a-synuclein were

comparable with those induced by nitration (Fig. 3).

Another important observation is that the shape of far-UV

CD spectrum measured for the nitrated protein is not

affected by decrease in pH.

3.2.3. ANS fluorescence

Non-native partially folded conformations of proteins

are characterized by the presence of solvent-exposed

hydrophobic clusters to which ANS binds. This interaction

Fig. 3. Far-UV CD spectra of non-modified and nitrated a-synuclein. Control, pH 7.5 (solid line), nitrated, pH 7.5 (dotted), control, pH 3.0 (short dashes),

nitrated, pH 3.0 (dash two dots), fibrils (long dashes), nitrated oligomers (dash single dot). Measurements were performed at 23 8C using a cell with 0.1 mm

pathlength. Protein concentration was 0.5 mg/mL.

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–10290

results in a considerable increase in the ANS fluorescence

intensity and in a pronounced blue shift of the fluorescence

emission maximum [15,57]. Therefore, we analyzed the

changes in ANS fluorescence to monitor the gain of

partially folded structure in nitrated a-synuclein. Fig. 4

shows that nitration led to a large enhancement of the dye

fluorescence intensity, which paralleled the gain of

secondary structure (see Figs. 2 and 3) and the oligome-

rization (see below). The large blue shift in the ANS

fluorescence maximum from ~515 nm to ~475 nm clearly

reflects a nitration-induced transformation of a-synuclein

from the natively unfolded state to a partially folded

conformation.

Fig. 4. ANS fluorescence spectra measured for the non-modified (2) and nitrated

Measurements were carried out at 23 8C. Protein concentration was kept at 0.05

3.2.4. SAXS studies on non-modified and nitrated

a-synucleins at neutral and acidic pH

Analysis of SAXS scattering curves using the Guinier

approximation gives information about the radius of

gyration, Rg. Presentation of the scattering data in the form

of Kratky plots provides information about the globularity

(packing density) and conformation of a protein [11,22]. For

a native globular protein, this plot has a characteristic

maximum, whereas unfolded and partially folded polypep-

tides have significantly different-shaped Kratky plots.

Guinier analysis confirmed that non-modified a-synuclein

at neutral pH has Rg of 41 F 1 2 (Fig. 5A). This parameter

decreases to 30 F 1 2 at pH 3.0, reflecting considerable

a-synuclein (3). The spectrum of free ANS is shown for comparison (1).

mg/mL.

Fig. 5. Guinier (A) and Kratky plot (B) representation of the results of small angle X-ray-scattering analysis of a-synuclein: 1—non-modified protein at pH 7.5;

2—nitrated protein at pH 7.5; 3—non-modified protein at pH 3.0; 4—nitrated protein at pH 3.0. Measurements were carried out at 23 8C. Protein concentrationwas 1.0 mg/mL.

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 91

compaction of the protein. These Rg values are in a good

agreement with those reported earlier [36,74]. Note that the

observed Rg for a-synuclein at neutral pH is smaller than

that estimated for a random coil (52 2), indicating that the

molecule is more compact. On the other hand, the Rg for the

partially folded conformation stabilized by low pH is much

larger than that of a folded globular protein of the size of

a-synuclein (15 2) [74].Nitrated a-synuclein possessed an Rg of 69 F 1 2 at pH

7.5, whereas this value dropped to 33 F 1 2 when pH was

decreased to pH 3.0 (Fig. 5A). Thus, the hydrodynamic

dimensions of the nitrated and non-modified proteins were

rather comparable at acidic pH, whereas at neutral pH, there

was a considerable increase in Rg associated with the nitrated

protein. Aswill be shown later, this increase inRg is due to the

specific association of the modified protein to form

oligomers. The Guinier plot for a homogeneous system is

known to be linear at small angles [11,22]. Thus, the linearity

of Guinier plots indicates that both forms of a-synuclein,

non-modified and nitrated, were homogeneous under the

conditions used for both neutral and acidic solutions.

Analysis of the X-ray scattering in the form of a

Kratky plot shows that non-modified a-synuclein lacks a

well-developed globular structure at both pH 7.5 and pH

3.0 (Fig. 5B). The profile of the Kratky plot at neutral

pH is typical for a random coil conformation, whereas

that at pH 3 shows changes consistent with the develop-

ment of the beginnings of a tightly packed core.

Interestingly, nitration does not significantly affect the

shape of Kratky plot at pH 3.0 but results in a dramatic

change of the corresponding profile measured at pH 7.5

(Fig. 5B). The changes observed indicate the presence of

a relatively tightly packed core in the nitrated a-synuclein

molecules in the oligomers (see below).

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–10292

3.2.5. Limited proteolysis

Limited proteolysis is frequently used to characterize the

structure and dynamics of native proteins and partially

folded folding intermediates [17,43,50]. Generally, this

approach is based on the observation that proteolysis of a

protein substrate can occur only if the polypeptide chain

can bind and adapt to the specific stereochemistry of the

protease active site [16,24]. Obviously, this is difficult to

achieve with native tightly packed protein structures,

whereas unfolded or partially folded proteins are degraded

much faster.

We have compared the accessibility of non-modified and

nitrated a-synuclein to proteolysis by trypsin at neutral pH.

Results of this analysis revealed that the non-modified a-

synuclein degraded relatively fast under the conditions

studied (10 min), whereas nitration increased the resistance

of this protein to trypsinolysis and nitrated a-synuclein was

digested significantly slower under the same conditions

(Fig. 6). Thus, oxidative modification increases conforma-

tional stability and decreases structural flexibility of a-

synuclein, most likely due to the formation of stable soluble

oligomers (see below).

3.3. Effect of nitration on the oligomerization state of

human a-synuclein

Specific self-association has been shown to occur during

early stages of a-synuclein fibrillation both in vitro and in

vivo [10,56,73]. We have shown that a partially folded

conformation is stabilized at neutral pH as a result of specific

oligomerization of non-modified a-synuclein [73]. In fact,

the formation of oligomers by this protein coincides with a

small, but reproducible, change in the circular dichroism

spectrum and an increase in the 1-anilinonaphthalene-8-

Fig. 6. Proteolytic susceptibilities of non-modified (black circles) and nitrated a-

Materials and methods.

sulfonic acid (ANS) binding (cf. Figs. 2–4). In order to

ascertain the oligomerization status of nitrated a-synuclein,

the modified and non-modified forms of protein were further

compared using SAXS, size-exclusion chromatography

(SEC) and DLS.

3.3.1. SAXS analysis

The SAXS forward-scattering intensity values give

information on the degree of protein association. In fact,

I(0), the forward scattering amplitude, is proportional to

n � qc2 � V2, where n is the number of scatters (protein

molecules) in solution; qc is the electron density differ-

ence between the scatterer and the solvent; and V is the

volume of the scattering particle. This means that the

value of forward-scattered intensity, I(0), is proportional

to the square of the molecular weight of the molecule

[22]. Thus, I(0) for a pure N-mer sample will therefore be

N-fold that for a sample with the same number of

monomers since each N-mer will scatter N2 times as

strongly as monomer, but in this case the number of

scattered particles (N-mers) will be N times less than that

in the pure monomer sample. The results of analysis of

I(0) values for non-modified a-synuclein indicate that a

decrease in pH is not accompanied by noticeable changes

of this parameter, confirming that the pH-induced partial

folding of non-modified a-synuclein is an intramolecular

process (cf. [74]). I(0) values determined for nitrated a-

synuclein at neutral and acidic pH were correspondingly

about 8- and 1.3- to 1.5-fold greater than I(0) of non-

modified protein, directly indicating the presence of

oligomers at neutral pH. This means that at 70 AMconcentration nitrated a-synuclein predominantly forms

octamers at neutral pH, and a mixture of monomers and

dimers at acidic pH.

synuclein (open circles). Experimental procedure employed is described in

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 93

3.3.2. Hydrodynamic properties of non-modified and

nitrated a-synucleins at neutral pH from SEC

The unfolding of a protein is associated with a dramatic

increase in its hydrodynamic volume. Thus, hydrodynamic

parameters are very useful in estimation of the degree of

folding. The hydrodynamic radius of globular proteins

increases by ~15–20% as they transform into the molten

globule state [52,69]. The hydrodynamic volume of the pre-

molten globule is even larger [53,75]. Furthermore, native,

molten globule, pre-molten globule, and unfolded confor-

Fig. 7. SEC analysis of non-modified (A) and nitrated a-synuclein (B). Experime

lines, whereas component peaks are shown by dashed lines. Profile of the non

correspond to a species with RS of 31.3 F 0.5 2. The elution profile of the nit

corresponding to species with RS of 31.8 F 0.5 2 (I), 36.0 F 0.5 2 (II), 45.3 F

mations of globular proteins possess very different molec-

ular mass dependencies of their hydrodynamic radii, RS

[66,71]. Thus, equilibrium conformations of a globular

protein (native, molten globule, pre-molten globule, and

unfolded states) can easily be discriminated by the degree of

compactness of the polypeptide chain.

To obtain information about the hydrodynamic dimen-

sions of non-modified and nitrated a-synucleins and their

aggregation intermediates, the size exclusion chromato-

graphy behavior of the proteins under different experimental

ntal profiles are shown by solid lines. Curve fit data are present by dotted

-modified protein is satisfactorily described by a single peak (A), which

rated protein can be deconvoluted using a set of 5 log-normal peaks (B),

0.9 2 (III), and 60.2 F 1.5 2 (IV).

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–10294

conditions was studied. It is known that SEC separates

proteins by differences in their hydrodynamic dimensions

rather than by their molecular masses [1]. This approach has

been successfully applied to determine the Stokes radius

(RS) values for proteins in different conformational states

[69,70]. Fig. 7 compares the elution profiles corresponding

to the non-modified and nitrated forms of a-synuclein. Non-

modified a-synuclein elutes as a single peak (Fig. 7A),

whose elution volume corresponds to a protein with a RS of

31.3 F 0.5 2, in good agreement with the SAXS data. In

contrast, Fig. 7B shows that the nitrated a-synuclein elution

profile represents a mixture of at least four different species

with RS of 31.8 F 0.5, 36.0 F 0.5, 45.3 F 0.9 and 60.2 F

Fig. 8. Kinetics of fibrillation of non-modified (A) and nitrated a-synuclein (B

Fibrillation was studied at pH 7.5 (filled circles), pH 3.0 (open circles), pH 7.5 in t

ZnSO4 (open triangles), in 20 mM Na-phosphate buffer containing 100 mM NaC

450 nm, and the emission wavelength was 482 nm, using the plate reader. The am

order to facilitate comparison of the kinetics.

1.5 2. These coincide with the dimensions expected for a

polypeptide chain of 14640 kDa, if it would be a natively

unfolded monomer (RS = 31.8 2), pre-molten globule-like

dimer (RS = 36.4 2), pre-molten globule-like tetramer (RS =

47.9 2) and pre-molten globule-like octamer (RS = 63.6 2),respectively. As discussed subsequently, the concentrations

of the protein in the SEC experiments are much lower than

those in the SAXS experiments.

3.3.3. DLS analysis of non-modified and nitrated

a-synucleins at neutral pHThe hydrodynamic radii, RS, of the non-modified and

nitrated a-synucleins were further analyzed using DLS.

) monitored by the enhancement of Thioflavin T fluorescence intensity.

he presence of 100 AM heparin (filled triangles) or in the presence of 2 mM

l. Measurements were performed at 37 8C. ThT fluorescence was excited at

plitude of the ThT signals has been normalized to a common final value in

Fig. 9. Soluble and insoluble a-synuclein, measured by Lowry analysis of

the products of non-modified and nitrated a-synuclein fibrillation. pH 7.5

(A) and pH 3.0 (B). Plot (B) also represents the results of inhibition studies

on non-modified protein fibrillation in the presence of different concen-

trations of nitrated a-synuclein at pH 7.5. Numbers in (B) show the molar

ratios of the nitrated over non-modified protein. Black and open bars

represent the relative protein contents in the supernatant and pellet,

respectively. Protein was incubated at 37 8C for 300 h.

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 95

The majority of the non-modified protein (~95%) was

characterized by an RS of (~32 F 3 2), whereas the

majority of the nitrated a-synuclein (96%) formed small

oligomers with RS of 62 F 3 2. Thus, there is excellent

agreement between DLS and SEC data for both non-

oxidized and modified a-synucleins. The fact that the

nitrated protein existed predominantly as a single species

(octamer) in DLS (and SAXS) and as a mixture of

monomers, dimers, tetramers, and octamers in SEC

experiments can be explained by the difference in protein

concentration (0.1 and 0.05 mg/mL for DLS and SEC,

respectively). Furthermore, the protein undergoes a sub-

sequent ~10- to 20-fold dilution in SEC experiments

during its migration through the column, leading to the

dissociation of the octamers.

The combined analysis of spectroscopic and hydro-

dynamic data, as well as the results of the limited

proteolysis, revealed that nitrated a-synuclein is partially

folded, and does not have a well-packed compact structure.

The data indicate that the partially folded conformation of

nitrated a-synuclein is stabilized at neutral pH as a result of

a selective self-assembly process, giving rise to the

appearance of relatively stable octamers. On the other hand,

non-modified and nitrated a-synuclein behave similarly at

acidic pH, where both forms adopt predominantly mono-

meric partially folded conformations.

3.4. The effect of nitration on the fibrillation of a-synuclein

3.4.1. Fibrillation at neutral pH

Thioflavin T (ThT) is a fluorescent dye that interacts with

amyloid fibrils leading to an increase in the fluorescence

intensity in the vicinity of 480 nm. Fig. 8 compares

fibrillation patterns of non-modified (panel A) and nitrated

a-synuclein (panel B) monitored by ThT fluorescence.

Fibril formation for the non-oxidized a-synuclein at neutral

pH is characterized by a typical sigmoidal curve. In contrast,

there was no evidence of fibril formation by nitrated a-

synuclein at neutral pH even after incubation for 300 h.

After incubation at 37 8C for 300 h with stirring, the

solutions of non-modified and nitrated a-synuclein were

subjected to centrifugation and subsequent analysis of

supernatants and pellets by the Lowry assay. Results of this

analysis are shown in Fig. 9A as relative contents of soluble

and insoluble materials in each sample. Fig. 9A shows that

the largest portion of the non-modified protein was

insoluble, whereas the vast majority of the nitrated a-

synuclein was observed in supernatant; that is, was soluble.

This observation excludes the formation of amorphous

aggregates by the nitrated a-synuclein upon incubation for

prolonged times at elevated temperatures.

Further evidence that the nitration inhibits a-synuclein

aggregation is obtained from AFM imaging. After incuba-

tion at 37 8C for 10 days with agitation, AFM images of

non-modified a-synuclein indicate the presence of fibrils

having diameters of 7.3 F 0.7 nm (Fig. 10A), while the

sample of nitrated protein incubated under the same

conditions for 10 days did not show any traces of fibrillar

material. In contrast, AFM images of nitrated a-synuclein

reveal the presence of small spheroid-like aggregates (Fig.

10B) with an average diameter of 8.9 + 3.5 nm, but

consisting of a mixed population with diameters of ~4, 8,

12, and 16 nm (see Fig. 10C).

3.4.2. Fibrillation at acidic pH or at neutral pH in the

presence of heparin or metal ions

The acceleration of a-synuclein fibrillation at low pH is

attributed to the decrease in net charge of the molecule,

leading to the stabilization of an amyloidogenic partially

folded intermediate [74]. Similarly, the addition of heparin

and certain metal cations accelerate the rate of fibrillation of

non-modified protein [5,76].

Fig. 8 shows that the non-modified (panel A) and nitrated

forms of a-synuclein (panel B) have similar kinetics of

Fig. 10. AFM images of fibrils and aggregates formed by non-modified (A) and nitrated a-synuclein (B). The scale bars represent 100 nm and 1000 nm

respectively; the height scales are for (A) 20 nm and (B) 100 nm. Panel (C) shows the height distribution of spherical oligomers.

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–10296

fibrillation at pH 3. This means that the inhibitory effect of

nitration is eliminated under these conditions. However, the

addition of heparin or metals did not eliminate the inhibitory

effect of nitration at pH 7.5. The results of Lowry assays

(Fig. 9B) show that after incubation of non-modified a-

synuclein the protein is mostly insoluble at acidic pH, or at

neutral pH in the presence of heparin or metals, whereas the

nitrated protein is insoluble at acidic pH, but is mostly in the

supernatant at neutral pH even in the presence of heparin or

metals. This provides additional support for the comparable

aggregation propensities of both forms of a-synuclein under

conditions of low pH, and demonstrates the strong anti-

fibrillation effects of nitration at neutral pH.

3.4.3. Nitrated a-synuclein inhibits fibrillation of the

non-modified protein

Fig. 11A represents data on the effect of nitrated a-

synuclein on the fibrillation kinetics of the non-modified

protein. As previously reported [80], the addition of nitrated

protein to a solution of non-modified a-synuclein leads to

inhibition of the non-modified protein’s fibrillation, and a

notable decrease in the ThT fluorescence was observed

when the ratio of modified protein over the non-modified a-

synuclein was as low as 1:10. Fig. 11B illustrates that both

lag-time and elongation rate of non-modified a-synuclein

fibrillation are considerably affected by the addition of

nitrated protein. The extent of these effects depends on the

relative content of the modified protein, with larger

concentrations showing stronger inhibition effects. In fact,

we did not observe fibrillation (at least within the time scale

studied) when the ratio of modified over the non-modified

a-synuclein was 2:1 and higher. The data in Fig. 9B

confirms that the amount of insoluble material decreases as

the relative concentration of the nitrated protein is increased.

Fig. 12 shows EM images of fibrils and aggregates

induced in non-modified and nitrated a-synuclein after

incubation at 37 8C for 10 days with agitation. These data

give further support to the effective inhibition of the non-

modified a-synuclein fibrillation by the addition of different

molar equivalents of the nitrated protein. Fig. 12A shows

that the non-modified protein formed typical amyloid fibrils,

whereas nitrated a-synuclein stayed predominantly in the

Fig. 11. Inhibition of fibrillation of non-modified a-synuclein in the presence of nitrated protein. (A) The fibrillation kinetics were studied for 70 AM non-

modified a-synuclein solution in 20 mM Na-phosphate buffer, 100 mM NaCl, pH 7.5 in the absence (filled circles) or presence of 2.0 (open triangles), 1.0

(open squares), 0.50 (open diamonds), 0.25 (open triangles), and 0.10 molar equivalents of the oxidized protein (open hexagons). Fibrillation kinetics of the

oxidized proteins is shown for comparison (open circles). (B) Dependence of the nucleation (filled circles) and elongation times (open circles) of the non-

modified protein fibrillation on the relative concentration of nitrated a-synuclein.

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 97

form of small oligomers (Fig. 12B). Similarly, when the

nitrated protein was added to the non-modified a-synuclein,

the predominant species were small oligomers (Figs. 12C

and D). Interestingly, no insoluble amorphous aggregates

were detected in the solutions containing nitrated protein.

3.4.4. Analysis of non-modified and nitrated a-synucleinfibrillation by DLS and SEC

In order to illuminate the changes in oligomerization

state during the fibrillation of the non-modified and nitrated

a-synucleins, the hydrodynamic radii of the both protein

forms have been estimated by DLS. These DLS experiments

were performed in parallel with the analysis of the reaction

mixtures by SEC. As already noted, RS for the non-modified

protein is 32 F 2 2 prior to the initiation of fibrillation: at

early incubation times (i.e., well before the beginning of

fibril formation as detected by the increase in the ThT

fluorescence intensity), the majority of the non-modified

protein (~95%) was characterized by a slightly increased RS

value (~35–37 2). This increase was due to the appearance

of the dimeric form, which according to the SEC analysis,

accounts for ~3% of total protein (see Fig. 13A). Further-

more, at these pre-fibrillation time points, another small

fraction of non-modified protein (~2%) formed large

oligomers, the size of which increased monotonically from

~200 to ~2000 2, confirming a previous report [34]. Finally,

almost no monomeric and small oligomeric species have

been detected when fibrils start to form, and according to

DSL, the majority of protein in the soluble fraction of the

reaction mixture possessed RS of N400 2.Nitrated a-synuclein has an increased propensity to

oligomerize (see above). In fact, even at time zero (i.e.,

before the beginning of agitation) the majority of this

protein (96%) formed small oligomers with the RS of 62 F

Fig. 12. Negatively stained transmission electron micrographs of different a-synuclein aggregates and fibrils induced in non-modified (A) and nitrated proteins

alone (B) as well as in non-modified a-synuclein in the presence of 2.0 (C) and 0.5 molar equivalents of the nitrated protein (D).

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–10298

3 2, whereas the remainder of the protein was assembled

into larger oligomers (RS of 160 F 10 2). Parallel SECanalysis revealed that octamers, tetramers, dimers, and

monomers are present in the reaction mixture (see above).

Fig. 13B and DLS data show that the size and relative

population of small oligomers was almost constant during

the incubation. On the other hand, DLS revealed that large

oligomers showed a time-dependent increase in size from

160 to 350 2.

Fig. 13. SEC analysis of the non-modified (A) and nitrated a-synuclein (B)

during incubation. Protein samples were collected after the indicated time

of incubation at 37 8C at pH 7.5 with stirring, filtered with a 0.1-AmWhatman Anodisc-13 filter and loaded onto a Tosoh Bioscience

G2000SWXL column.

4. Discussion

Oxidative stress is believed to be a contributing factor to

the etiology of Parkinson’s disease [12,20,29,48,51,63,67].

It is also accepted that aggregation and/or fibrillation of a-

synuclein is involved in the etiology of this disorder and

number of other neurodegenerative disorders. We have

previously shown that methionine-oxidized a-synuclein,

which is expected to represent one of the most common

products of oxidative damage to a-synuclein, rather

surprisingly, fails to form fibrils and inhibits fibrillation of

non-oxidized a-synuclein [25,77]. Similarly, in a prelimi-

nary report, we showed that another product of oxidative

Fig. 14. Models of non-modified and nitrated a-synuclein aggregation

under the different experimental conditions. (See the text for explanations).

(A) Aggregation of non-modified protein at neutral pH. (B) Aggregation of

the nitrated protein at neutral pH. (C) Aggregation of non-modified and

nitrated proteins at acidic pH. UN = monomeric a-synuclein, I = partially-

folded intermediate, O = soluble oligomer, A = amorphous precipitate, F =

fibrils, * indicates nitrated.

Fig. 15. Model of the inhibition of non-modified a-synuclein fibrillation in

the presence of nitrated protein. (See Fig. 14 and text for explanations.)

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102 99

modification, nitrated a-synuclein, also does not fibrillate,

and inhibits fibril formation of non-modified protein [80].

In previous studies, we have shown that the formation

of a partially folded intermediate is a critical initial step

of the a-synuclein fibrillogenesis [74], and that a-

synuclein fibrillation is accelerated under conditions that

populate such an intermediate state. These conditions

include acidic pH [74], the presence of metal cations

[76], or heparin and other glycosaminoglycans [23]. The

significant stabilization of the natively unfolded confor-

mation has been shown to represent a contributing factor

to the inhibition of methionine-oxidized a-synuclein

fibrillation [77]. Furthermore, in the mixtures of non-

oxidized and oxidized proteins, methionine-oxidized a-

synuclein affected primarily the duration of the lag-time,

and did not effect the rate of elongation. Thus, interaction

of methionine-oxidized a-synuclein with the non-oxidized

protein inhibited the nucleation process, but not the

elongation of fibrils [77].

Data presented in this paper show that nitration of a-

synuclein has significant effects on its structural properties

and propensity for aggregation. Nitration leads to formation

of a partially folded conformation, which is stabilized by

formation of specific oligomers, most likely octamers.

Importantly, these octamers are stable and possess

decreased conformational flexibility, as seen in the increase

in stability toward limited proteolysis. Since this nitration-

induced oligomerization inhibits fibrillation of human

recombinant a-synuclein in vitro, the stable oligomers

must be located off the fibrillation pathway. The fact that

the nitrated protein inhibits fibrillation of non-modified a-

synuclein at sub-stoichiometric concentrations suggests that

interaction between nitrated and non-modified forms of

human a-synuclein leads to formation of stable off-pathway

hetero-oligomers.

Models for the aggregation of non-modified (A) and

nitrated (B) a-synuclein at neutral pH are shown in Fig. 14.

The model for aggregation of both forms of the protein at

acidic pH is also shown (Fig. 14C). In the model O, F and A

represent soluble oligomers, fibrils, and amorphous aggre-

gates, respectively; UN and UN* are the natively unfolded

states of non-modified and nitrated a-synuclein, respectively,

whereas I and I* represent the productive (leading to fibrils)

and non-productive partially folded conformations. The

Roman numerals indicate the major stages of the aggregation

process. Taking into account the observations presented in

the current study, we propose that nitration of tyrosines leads

to partial folding of a-synuclein; that is, leads to a shift in the

equilibrium in stage I for the nitrated protein, UN* X I*, in

favor of the partially folded conformation I*. This facilitates

formation of soluble oligomers, stage IV, whereas stages II,

III, and Vare arrested (or at least strongly inhibited) at neutral

pH (Fig. 14B). We assume that the partially folded

conformation I* is different from that for the non-modified

protein, I, as the formation of I is known to accelerate rather

than inhibit the fibrillation of a-synuclein [74].

Both non-modified and nitrated forms of a-synuclein

show similar aggregation behavior at acidic pH (Fig. 14C).

Here, the non-modified and nitrated a-synucleins are more

prone to form the productive partially folded conformation I

(thicker arrow at stage I) and, as a consequence, show a

faster rate of fibrillation (stages II–V). We assume that the

pathways of nitrated a-synuclein to I* and to stable

V.N. Uversky et al. / Molecular Brain Research 134 (2005) 84–102100

oligomers are considerably diminished at low pH due to the

pH-induced destabilization of these complexes and stabili-

zation of productive partially folded conformation I (Fig.

14C). A model for the inhibitory effect of nitrated a-

synuclein on the non-oxidized protein fibrillation is shown

in Fig. 15, in which the conformational equilibrium is

shifted toward the formation of soluble hetero-oligomers.

Importantly, neither the presence of metals nor the

addition of heparin was able to overcome the nitration-

induced inhibition of the a-synuclein fibrillation at neutral

pH. This is contrary to results for the methionine-oxidized

protein, which was shown to fibrillate effectively in the

presence of certain metals even at neutral pH [79].

Interaction of non-modified a-synuclein with metals

induces the formation of the amyloidogenic partially

folded species, I. On the other hand, nitration induces

partial folding of the protein to the non-amyloidogenic

species I*, which accelerates formation of the non-

fibrillating oligomers. Most likely, these oligomers are

further stabilized by the interaction with metals and thus

do not fibrillate. It has been shown that different GAGs

(heparin, heparan sulfate) and other highly sulfated

polymers (e.g., dextran sulfate) are able bind to a-

synuclein and stimulate its fibrillation in vitro [5]. Heparin

binding sites on a variety of proteins are invariably shown

to contain clusters of basic amino acid residues capable of

binding to the negatively charged heparin polymer [4]. The

N-terminal region of a-synuclein contains multiple repeats

of the consensus sequence KTKEGV, and pairs of lysine

residues spaced two residues apart occur at positions 10/

12, 21/23, 33/35, 42/44, and 58/60. It is likely that this

region of a-synuclein constitutes the GAG binding motif

[5]. Our data suggest that this GAG-binding region is

buried in the oligomeric form of nitrated a-synuclein or

that the affinity of the nitrated protein for heparin is lower

than that for self-association.

Finally, we would like to make a few comments about

the implication of our findings to the etiology of PD. The

extensive and widespread presence of nitrated a-synuclein

has been shown in the signature inclusions of such

synucleinopathies as Parkinson’s disease, dementia with

Lewy bodies, the Lewy body variant of Alzheimer’s

disease, and multiple system atrophy. Interestingly, nitrated

a-synuclein was detected in the major filamentous building

blocks of these inclusions, as well as in the insoluble

fractions of the affected brain regions of the different

synucleinopathies [20]. This led to the conclusion that the

nitration of a-synuclein reflects a direct link between

oxidative and nitrative damage and the onset and progres-

sion of these neurodegenerative disorders [21]. However,

we have shown that nitration effectively inhibits fibrillation

of a-synuclein. Furthermore, the ability of the non-modified

a-synuclein to form fibrils is effectively inhibited by the

addition of the sub-stoichiometric concentrations of nitrated

a-synuclein. These findings suggest that the nitration

detected in the proteinaceous deposits accumulated in the

synucleinopathy-affected brain most likely occurred after,

rather than before, fibril formation.

Acknowledgment

This research was supported by Grant NS39985 (to ALF)

from the National Institutes of Health.

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