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BIBTEX RIS

Biophysical Properties of the Synucleins and Their Propensities to Fibrillate

2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês

10.1074/jbc.m109541200

ISSN

1083-351X

Autores

Vladimir N. Uversky, Jie Li, Pierre O. Souillac, Ian S. Millett, Sebastian Doniach, Ross Jakes, Michel Goedert, Anthony L. Fink,

Tópico(s)

Ginkgo biloba and Cashew Applications

Resumo

The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous α-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, β- and γ-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. β-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas α- and γ-synucleins were slightly more compact and structured. γ-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to α-synuclein, β- And γ-synucleins inhibit α-synuclein fibril formation. Complete inhibition of α-synuclein fibrillation was observed at 4:1 molar excess of β- and γ-synucleins. No significant incorporation of β-synuclein into the fibrils was detected. The lack of fibrils formed by β-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed. The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous α-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, β- and γ-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. β-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas α- and γ-synucleins were slightly more compact and structured. γ-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to α-synuclein, β- And γ-synucleins inhibit α-synuclein fibril formation. Complete inhibition of α-synuclein fibrillation was observed at 4:1 molar excess of β- and γ-synucleins. No significant incorporation of β-synuclein into the fibrils was detected. The lack of fibrils formed by β-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed. Synucleins belong to a family of closely related presynaptic proteins that are encoded by three distinct genes, described only in vertebrates (reviewed in Ref. 1.Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1112) Google Scholar). They are soluble, relatively small, intracellular and especially abundant in neural tissues. Synucleins are characterized by the presence of acidic stretches within the COOH-terminal region and a repetitive, degenerative amino acid motif KTKEGV throughout the first 87 residues, causing a variation in hydrophobicity with a strictly conserved periodicity of 11 amino acids (2.George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (732) Google Scholar). Such a periodicity is characteristic of the amphipathic helices of apolipoproteins (2.George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (732) Google Scholar). The name "synuclein" was first given to a protein that was isolated from the electric organ of Torpedo californica, where it was found in both nerve terminals and the nuclear envelope (3.Maroteaux L. Campanelli J.T. Scheller R.H. J. Neurosci. 1988; 8: 2804-2815Crossref PubMed Google Scholar). The family of synuclein proteins includes: α-synuclein, which was also called non-amyloid component precursor protein, or synelfin (2.George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (732) Google Scholar, 3.Maroteaux L. Campanelli J.T. Scheller R.H. J. Neurosci. 1988; 8: 2804-2815Crossref PubMed Google Scholar, 4.Ueda K. Fukushima H. Masliah E. Xia Y. Iwai A. Yoshimoto M. Otero D.A. Kondo J. Ihara Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11282-11286Crossref PubMed Scopus (1239) Google Scholar, 5.Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (908) Google Scholar, 6.Tobe T. Nakajo S. 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It has been estimated to account for as much as 1% of the total protein in soluble cytosolic brain fractions (11.Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. de Silva H.A. Kittel A. Saitoh T. Neuron. 1995; 14: 467-475Abstract Full Text PDF PubMed Scopus (1134) Google Scholar). α-Synuclein is located in close vicinity to, and is loosely associated with, synaptic vesicles and thus may play a role in the release of neurotransmitters (5.Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (908) Google Scholar, 11.Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. de Silva H.A. Kittel A. Saitoh T. Neuron. 1995; 14: 467-475Abstract Full Text PDF PubMed Scopus (1134) Google Scholar). It belongs to the rapidly growing family of natively unfolded proteins, which have little or no ordered structure under physiological conditions (12.Weinreb P.H. Zhen W. Poon A.W. Conway K.A. Lansbury Jr., P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1326) Google Scholar, 13.Uversky V.N. Li J. Fink A.L. J. Biol. Chem. 2001; 276: 10737-10744Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Natively unfolded proteins are characterized by a unique combination of low overall hydrophobicity and large net charge (14.Uversky V.N. Gillespie J.R. Fink A.L. Proteins. 2000; 41: 415-427Crossref PubMed Scopus (1783) Google Scholar). Human β-synuclein is a 134-amino acid neuronal protein showing 78% identity to α-synuclein. α- And β-synucleins share a conserved COOH terminus, with three identically placed tyrosine residues. However, β-synuclein lacks 11 residues within the middle region (residues 73–83) (1.Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1112) Google Scholar). Like α-synuclein, it is expressed predominantly in the brain (5.Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (908) Google Scholar, 15.Shibayama-Imazu T. Okahashi I. Omata K. Nakajo S. Ochiai H. Nakai Y. Hama T. Nakamura Y. Nakaya K. Brain Res. 1993; 622: 17-25Crossref PubMed Scopus (109) Google Scholar). The third member of the human synuclein family is the 127-amino acid γ-synuclein, which shares 60% similarity with α-synuclein at the amino acid sequence level (1.Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1112) Google Scholar). It lacks the tyrosine-rich COOH-terminal signature of α- and β-synucleins. γ-Synuclein is abundant in spinal cord and sensory ganglia (10.Buchman V.L. Hunter H.J. Pinon L.G. Thompson J. Privalova E.M. Ninkina N.N. Davies A.M. J. Neurosci. 1998; 18: 9335-9341Crossref PubMed Google Scholar). Interestingly, it is more widely distributed within the neuronal cytoplasm than α-and β-synucleins, being present throughout cell bodies and axons (10.Buchman V.L. Hunter H.J. Pinon L.G. Thompson J. Privalova E.M. Ninkina N.N. Davies A.M. J. Neurosci. 1998; 18: 9335-9341Crossref PubMed Google Scholar). The presence of synucleins has also been established in other cell types: thus, α-synuclein was found in platelets (16.Hashimoto M. Yoshimoto M. Sisk A. Hsu L.J. Sundsmo M. Kittel A. Saitoh T. Miller A. Masliah E. Biochem. Biophys. Res. Commun. 1997; 237: 611-616Crossref PubMed Scopus (107) Google Scholar), β-synuclein in Sertoli cells of the testis (17.Nakajo S. Tsukada K. Kameyama H. Furuyama Y. Nakaya K. Brain Res. 1996; 741: 180-184Crossref PubMed Scopus (25) Google Scholar), and γ-synuclein in the epidermis (18.Ninkina N.N. Privalova E.M. Pinon L.G. Davies A.M. Buchman V.L. Exp. Cell Res. 1999; 246: 308-311Crossref PubMed Scopus (26) Google Scholar) and in metastatic breast cancer tissue (8.Ji H. Liu Y.E. Jia T. Wang M. Liu J. Xiao G. Joseph B.K. Rosen C. Shi Y.E. Cancer Res. 1997; 57: 759-764PubMed Google Scholar). Several observations have implicated α-synuclein in the pathogenesis of Parkinson's disease (PD) 1The abbreviations used are: PDParkinson's diseaseLBLewy bodiesLNLewy neuritesThTthioflavin TFTIRFourier transform infraredSAXSsmall angle x-ray scatteringRSStokes radius and several other neurodegenerative disorders. Clinically, PD is a movement disorder characterized by tremor, rigidity, and bradykinesia. These symptoms are attributed to the progressive loss of dopaminergic neurons from the substantia nigra. Surviving neurons contain cytosolic filamentous inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs) (19.Lewy F.H. Lewandowski M. Handbuch der Neurologie. Springer, Berlin1912: 920-933Google Scholar, 20.Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1256) Google Scholar). In addition to the substantia nigra, LBs and LNs are also found in other brain regions, such as the dorsal motor nucleus of the vagus, the nucleus basalis of Meynert, and the locus coeruleus (20.Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1256) Google Scholar). α-Synuclein has been shown to be a major fibrillar component of LBs and LNs (21.Spillantini M.G. Schmidt M.L. Lee V.M.Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6267) Google Scholar, 22.Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar). Moreover, two different missense mutations in the α-synuclein gene, resulting in the A53T and A30P substitutions, have been identified in a small number of kindreds with autosomal dominantly inherited, early onset PD (23.Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar, 24.Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3344) Google Scholar). In addition, the expression of wild type (WT) α-synuclein in transgenic mice (25.Masliah E. Rockenstein E. Veinbergs I. Mallory M. Hashimoto M. Takeda A. Sagara Y. Sisk A. Mucke L. Science. 2000; 287: 1265-1269Crossref PubMed Scopus (1572) Google Scholar) or of WT, A30P, and A53T α-synuclein in transgenic flies (26.Feany M.B. Bender W.W. Nature. 2000; 404: 394-398Crossref PubMed Scopus (1725) Google Scholar) leads to motor deficits and neuronal inclusions reminiscent of PD. Abundant α-synuclein-positive LBs and LNs in the cerebral cortex are also neurophathological hallmarks of dementia with Lewy bodies, a common late-life dementia that is clinically similar to Alzheimer's disease (21.Spillantini M.G. Schmidt M.L. Lee V.M.Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6267) Google Scholar, 22.Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar, 27.Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M.Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathology. 1998; 152: 879-884PubMed Google Scholar). Furthermore, LBs and aggregated α-synuclein have been detected in diffuse Lewy body disease (28.Iseki E. Marui W. Kosaka K. Akiyama H. Ueda K. Iwatsubo T. Neurosci. Lett. 1998; 258: 81-84Crossref PubMed Scopus (65) Google Scholar, 29.Takeda A. Mallory M. Sundsmo M. Honer W. Hansen L. Masliah E. Am. J. Pathol. 1998; 152: 367-372PubMed Google Scholar), some cases of of Alzheimer's disease (30.Trojanowski J.Q. Goedert M. Iwatsubo T. Lee V.M. Cell Death. Differ. 1998; 5: 832-837Crossref PubMed Scopus (248) Google Scholar) and Down's syndrome (31.Lippa C.F. Schmidt M.L. Lee V.M. Trojanowski J.Q. Ann. Neurol. 1999; 45: 353-357Crossref PubMed Scopus (275) Google Scholar), as well as some cases of Hallervorden-Spatz disease (32.Arawaka S. Saito Y. Murayama S. Mori H. Neurology. 1998; 51: 887-889Crossref PubMed Scopus (159) Google Scholar). Finally, α-synuclein is the major component of the filamentous neuronal and glial inclusions of multiple system atrophy (33.Spillantini M.G. Crowther R.A. Jakes R. Cairns N.J. Lantos P.L. Goedert M. Neurosci. Lett. 1998; 251: 205-208Crossref PubMed Scopus (835) Google Scholar). Parkinson's disease Lewy bodies Lewy neurites thioflavin T Fourier transform infrared small angle x-ray scattering Stokes radius It has recently been reported that in addition to α-synuclein-containing LBs and LNs, the development of PD and dementia with Lewy bodies is accompanied by the appearance of non-filamentous α-, β-, and γ-synuclein-positive accumulations in axon terminals of the hippocampus (34.Galvin J.E. Uryu K. Lee V.M. Trojanowski J.Q. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13450-13455Crossref PubMed Scopus (373) Google Scholar). This implicates β- and γ-synucleins, in addition to α-synuclein, in the progression of PD and dementia with Lewy bodies. The aggregation of recombinant α-synuclein and its A30P and A53T mutants has been studied in vitro. It has been established that all three proteins, as well as carboxyl-terminal truncated forms thereof, readily assemble into filaments, with morphologies and staining characteristics similar to those of filaments extracted from disease-affected brain (13.Uversky V.N. Li J. Fink A.L. J. Biol. Chem. 2001; 276: 10737-10744Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 35.Crowther R.A. Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1998; 436: 309-312Crossref PubMed Scopus (354) Google Scholar, 36.Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 37.El-Agnaf O.M.A. Jakes R. Curran M.D. Wallace A. FEBS Lett. 1998; 440: 67-70Crossref PubMed Scopus (241) Google Scholar, 38.Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 39.Narhi L. Wood S.J. Steavenson S. Jiang Y. Wu G.M. Anafi D. Kaufman S.A. Martin F. 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Sci. 2000; 920: 42-45Crossref PubMed Scopus (179) Google Scholar, 45.Giasson B.I. Murray I.V. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2001; 276: 2380-2386Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). Fibrillation is believed to occur via a nucleation-dependent mechanism (40.Wood S.J. Wypych J. Steavenson S. Louis J.C. Citron M. Biere A.L. J. Biol. Chem. 1999; 274: 19509-19512Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 46.Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1347) Google Scholar) with the critical primary stage being the structural transformation of the protein from the unfolded conformation to a partially folded intermediate (13.Uversky V.N. Li J. Fink A.L. J. Biol. Chem. 2001; 276: 10737-10744Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Under these experimental conditions, β- and γ-synucleins failed to assemble into filaments (41.Serpell L.C. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Crossref PubMed Scopus (661) Google Scholar, 43.Biere A.L. Wood S.J. Wypych J. Steavenson S. Jiang Y. Anafi D. Jacobsen F.W. Jarosinski M.A. Wu G.M. Louis J.C. Martin F. Narhi L.O. Citron M. J. Biol. Chem. 2000; 275: 34574-34579Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 45.Giasson B.I. Murray I.V. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2001; 276: 2380-2386Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). Comparatively little is known about the structural properties of β- and γ-synucleins. The question of reciprocal effects of individual family members on the aggregation of other synucleins has not been considered as yet. Here, we compare the structural properties of α-synuclein and its propensity to form fibrils with those of β- and γ-synucleins. Data on the in vitro fibrillation of α-synuclein in the presence of β- or γ-synuclein are also presented. Thioflavin T (ThT) and porcine intestinal heparin (Grade I-A, molecular weight 18,000) were obtained from Sigma. All other chemicals were of analytical grade from Fisher Chemicals. Human recombinant α-, β-, and γ-synucleins were expressed and purified as described (5.Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (908) Google Scholar, 22.Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar, 47.Jakes R. Crowther R.A. Lee V.M. Trojanowski J.Q. Iwatsubo T. Goedert M. Neurosci. Lett. 1999; 269: 13-16Crossref PubMed Scopus (90) Google Scholar). Assay solutions contained proteins at a concentration of 70–280 μm (1–4.0 mg/ml) in 20 mm sodium phosphate, 0.1 m NaCl, pH 7.5, buffer, containing 10 μm ThT. A volume of 100 μl of the mixture was pipetted into a well of a 96-well plate (white plastic, clear bottom) and a 1/8 th-inch diameter Teflon sphere (McMaster-Carr, Los Angeles, CA) was added. Each sample was run in triplicate or quadruplicate. The plate was loaded into a fluorescence plate reader (Fluoroskan Ascent) and incubated at 37 °C with shaking at 600–900 rpm with a shaking diameter of 1 mm. The fluorescence was measured at 30-min intervals with excitation at 444 nm and emission at 485 nm, with a sampling time of 40 ms. Data from replicate wells were averaged before plotting fluorescence versus time. The kinetics of α-synuclein fibrillation are sigmoidal, defined by an initial lag phase, a subsequent growth phase in which ThT fluorescence increased, and a final equilibrium phase, where ThT fluorescence reached a plateau indicating the end of fibril formation. ThT fluorescence measurements were plotted as a function of time and fitted to a sigmoidal curve using the empirical approach described in Ref. 48.Nielsen L. Khurana R. Coats A. Frokjaer S. Brange J. Vyas S. Uversky V.N. Fink A.L. Biochemistry. 2001; 40: 6036-6046Crossref PubMed Scopus (990) Google Scholar. CD spectra were obtained with an AVIV 60DS spectrophotometer (Lakewood, NJ) using protein concentrations of ∼35 μm. Spectra were recorded in a 0.01-cm path length cell from 250 to 190 nm with a step size of 0.5 nm, a bandwidth of 1.5 nm, and an averaging time of 10 s. For all spectra, an average of 5 scans was obtained. CD spectra of the appropriate buffers were recorded and subtracted from the protein spectra. Attenuated total reflectance data were collected on a Nicolet 800SX FTIR spectrometer equipped with an MCT (mercury-cadmium-telluride) detector. The IRE (72 × 10 × 6 mm, 45° germanium trapezoid) was held in a modified SPECAC out-of-compartment attenuated total reflectance apparatus. The hydrated thin films were prepared as described previously (49.Oberg K. Chrunyk B.A. Wetzel R. Fink A.L. Biochemistry. 1994; 33: 2628-2634Crossref PubMed Scopus (197) Google Scholar, 50.Oberg K.A. Fink A.L. Anal. Biochem. 1998; 256: 92-106Crossref PubMed Scopus (136) Google Scholar). Previous investigations have shown that comparable secondary structure analyses are obtained for native proteins from thin film attenuated total reflectance-FTIR as from transmission mode FTIR and x-ray crystallography (50.Oberg K.A. Fink A.L. Anal. Biochem. 1998; 256: 92-106Crossref PubMed Scopus (136) Google Scholar, 51.Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (517) Google Scholar). Typically 1024 interferograms were co-added at 4 cm−1 resolution. Data analysis was performed with GRAMS32 (Galactic Industries). Secondary structure content was determined from curve fitting to spectra deconvoluted using second derivatives and Fourier self-deconvolution to identify component band positions. Hydrated thin film samples were prepared by drying 50 μl of 70 μm α-synuclein solution on a ZnSe crystal with dry N2. The IR spectra were collected, followed by Fourier transformation using the spectrum of the clean crystal as a background. Water (liquid and vapor) components were subtracted from the protein spectrum. Fluorescence measurements were performed in semimicro quartz cuvettes (Hellma) with a 1-cm excitation light path using a FluoroMax-2 spectrofluorometer (Instruments S.A., Inc., Jobin Yvon-Spex). The light source was a 150-W xenon lamp. Transmission electron micrographs were collected using a JEOL JEM-100B microscope operating with an accelerating voltage of 80 kV. Typical nominal magnifications ranged from ×20,000 to 50,000. Samples were deposited on Formvar-coated 300-mesh copper grids and negatively stained with 1% aqueous uranyl acetate. Small angle x-ray scattering (SAXS) measurements were made using Beam Line 4-2 at Stanford Synchrotron Radiation Laboratory (52.Wakatsuki S. Hodgson K.O. Eliezer D. Rice M. Hubbard S. Gillis N. Doniach S. Rev. Sci. Instrum. 1992; 63: 1736-1740Crossref Scopus (35) Google Scholar). X-ray energy was selected at 8980 eV (Cu edge) by a pair of Mo/B4C multilayer monochromator crystals (53.Tsuruta H. Brennan S. Rek Z.U. Irving T.C. Tompkins W.H. Hodgson K.O. J. Appl. Cryst. 1998; 31: 672-682Crossref Scopus (54) Google Scholar). Scattering patterns were recorded by a linear position-sensitive proportional counter, which was filled with an 80% Xe, 20% CO2 gas mixture. Scattering patterns were normalized by incident x-ray fluctuations, which were measured with a short length ion chamber before the sample. The sample-to-detector distance was calibrated to be 230 cm, using a cholesterol myristate sample. To avoid radiation damage of the sample in SAXS measurements, the protein solution was continuously passed through a 1.3-mm path length observation flow cell with 25-μm mica windows. Background measurements were performed before and after each protein measurement and then averaged before being used for background subtraction. All SAXS measurements were performed at 23 ± 1 °C. The radius of gyration (Rg) was calculated according to the Guinier approximation (54.Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, London1982Google Scholar), lnI(Q)=lnI(0)−Rg2Q23Equation 1 where I(0) is the forward scattering amplitude, Q is the scattering vector given by Q = (4π sin θ)/λ, where 2θ is the scattering angle, and λ is the wavelength of x-ray. The hydrodynamic dimensions (Stokes radii, RS) of synucleins were measured by size-exclusion chromatography. Size-exclusion measurements were performed on a Superose-12 column using a Amersham Bioscience FPLC chromatographic system. A set of globular proteins (Gel Filtration Chromatography Standards from Bio-Rad Laboratories) with known RS values was used to create a calibration curve, 1000/Vel versus RS(55.Ackers G.K. Adv. Protein Chem. 1970; 24: 343-446Crossref PubMed Scopus (342) Google Scholar, 56.Corbett R.J. Roche R.S. Biochemistry. 1984; 23: 1888-1894Crossref PubMed Scopus (183) Google Scholar, 57.Uversky V.N. Biochemistry. 1993; 32: 13288-13298Crossref PubMed Scopus (451) Google Scholar). Hydrodynamic dimensions of native and completely unfolded globular protein with known molecular mass, M, were calculated from empirical equations (57.Uversky V.N. Biochemistry. 1993; 32: 13288-13298Crossref PubMed Scopus (451) Google Scholar), log(RSN)=0.369·log(M)−0.254Equation 2 log(RSU)=0.533·log(M)−0.687Equation 3 based on the intrinsic viscosity data (58.Tanford C. Adv. Protein Chem. 1968; 23: 121-282Crossref PubMed Scopus (2437) Google Scholar). Here RSN and RSU are the Stokes radii of native (N) and unfolded (U) protein. α-Synuclein fibrils were grown for 1 week in a 2-fold molar excess of β-synuclein at pH 7.5, 37 °C. Fibrillar material was spun down using a Beckman Microfuge 18. The pellet was washed twice with distilled water then dissolved in alkaline solution, and the resulting solution was analyzed by MS using a MicroMass Quattro-2 instrument. Fig. 1A represents the far-UV CD spectra of human recombinant α-, β-, and γ-synucleins measured at pH 7.5 and 3.0 at 20 °C. At neutral pH, all three proteins show far-UV CD spectra typical of an essentially unfolded polypeptide chain. This includes the characteristic minimum in the vicinity of 196 nm and the absence of bands in the 210–230-nm region. Interestingly, α- and γ-synucleins possess almost indistinguishable spectra, whereas the far UV-CD spectrum of β-synuclein shows a slightly increased degree of disorder, manifested by a small increase in negative ellipticity in the vicinity of 196 nm and somewhat lower intensity in the vicinity of 222 nm (see Table I). This suggestion was further confirmed by hydrodynamic studies (see below). As the pH is decreased changes were observed in the spectral shape for all three proteins. Fig. 1 shows that the decrease in the minimum at 196 nm is accompanied by an increase in negative intensity around 222 nm, reflecting pH-induced formation of secondary structure. All three proteins possess almost identical far-UV CD spectra at acidic pH (see also Table I). Importantly, the pH-induced changes in the far-UV CD spectra of the synucleins were completely reversible. Previously we have shown that the pH-induced increase in the structure of WT α-synuclein represents an intramolecular process involving the formation of a partially folded intermediate, and not self-association (13.Uversky V.N. Li J. Fink A.L. J. Biol. Chem. 2001; 276: 10737-10744Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Thus, this also appears to be the situation for the β- and γ-synucleins.Table IMajor structural characteristics of the human recombinant α-, β-, and γ-synucleinsProtein, conditionsMRNS (E)aRS values were calculated for a native globular protein with a given molecular mass, M, from the equation (57): log (RS)N = 0.369 log M − 0.254.RUS (E)bRS values were calculated for an unfolded protein with a given molecular mass, M, from the equation (57): log (RS)U = 0.533 log M − 0.682.RS(E)cMeasured by calibrated SEC column. Protein concentration was 0.02 mg/ml.Rg(E)dEstimated from the Guinier region of SAXS data. These measurements have been performed in solution containing 4.0 mg/ml of a given protein.I(0)dEstimated from the Guinier region of SAXS data. These measurements have been performed in solution containing 4.0 mg/ml of a given protein.M/MαdEstimated from the Guinier region of SAXS data. These measurements have been performed in solution containing 4.0 mg/ml of a given protein.M/MαeCalculated from theoretical molecular masses (see second column of this table).[θ]222, deg cm2 dmol−1Daα-Synuclein pH 7.5, 100 mmNaCl14,460.119.134.331.8 ± 0.441 ± 10.03152 ± 0.000081.0001.000−2220 ± 50 pH 3.0, 100 mm NaCl27.9 ± 0.430 ± 1−2860 ± 60 8m urea34.5 ± 0.4−1500 ± 90β-Synuclein pH 7.5, 100 mm NaCl14,276.919.034.133.9 ± 0.449 ± 10.03072 ± 0.000070.9750.987−1890 ± 50 pH 3.0, 100 mm NaCl27.5 ± 0.4−2890 ± 60 8 murea−1500 ± 90γ-Synuclein pH 7.5, 100 mmNaCl13,300.818.532.830.4 ± 0.461 ± 10.07874 ± 0.000152.4980.920−2180 ± 50 pH 3.0, 100 mm NaCl26.5 ± 0.4−2880 ± 60 8 murea−1500 ± 90a RS values were calculated for a native globular protein with

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