Materials and Methods s20

Supporting Information

Instantaneous Amyloid Fibril Formation of α-Synuclein from the Oligomeric Granular Structures in the Presence of Hexane

Jung-Ho Lee, Ghibom Bhak, Sang-Gil Lee, and Seung R. Paik *

School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Gwanak-Ku, Seoul 151-744, Korea

E-mail: [email protected]

General consideration

Materials. Recombinant α-synucleins cloned in pRK172 were overexpressed in E.coli

BL21 (DL3) and purified according to the procedures described previously 1,2. Briefly, the heat-treated cell lysate containing -synuclein was loaded onto a DEAE-Sephacel anion-exchange column which had been equilibrated with an equilibrium buffer containing 0.1 M NaCl in 20 mM Tris-Cl, pH 7.5, and eluted with 0.4 M NaCl in the

Tris buffer under a linear gradient. Subsequently, Sephacryl S-200 size-exclusion chromatography was performed with 20 mM Mes, pH 6.5, containing 0.1 % NaN3. The elutent was applied onto S-Sepharose cation-exchange chromatography equilibrated with 20 mM Mes, pH 6.5. The protein was obtained with a linear gradient between 0 M and 0.5 M NaCl. The purified α-synuclein was dialyzed against 20 mM Mes, pH 6.5, and stored at -80ºC in aliquots. Materials and reagents including n-hexane, thioflavin-T,

S1 and 8-Anilino-1-naphthalenesulfonic acid (ANS) were provided from Sigma in the highest purity unless otherwise mentioned.

Amyloid fibrillation kinetics and granule preparation. For the amyloid fibrillation kinetics of α-synuclein, the protein was aged at a fixed concentration of 1 mg/ml in 20 mM Mes, pH 6.5, at 37ºC under continuous shaking at 200 rpm with an orbit shaker

(Vision Scientific Co, Korea). Aliquots (20 μl) collected during the aggregation were combined with 2.5 μM thioflavin-T in 50 mM glycine, pH 8.5, to a final volume of 200

μl, and thioflavin-T binding fluorescence of the amyloid fibrils was monitored at 482 nm with an excitation at 450 nm. The kinetics consisted of a lag phase for approximately 25 hours and an elongation phase for 35 hour before it reached the stationary phase, and the granules were collected at 12 hr-point during the lag phase of aggregation kinetics.

Atomic Force Microscope (AFM). To obtain morphological information of the granular or fibrous protein aggregates, aliquots (5 µl) of incubation mixture were transferred to freshly cleaved mica. Samples adsorbed on the mica were washed with distillated water to remove unbound protein aggregates and dried for 10 min. AFM images were obtained with Dimension 3100 in tapping mode. Measurements were carried out using silicon cantilevers with a spring constant of 21-78 N/m (Nanoscience) and a resonance frequency of 260-410 kHz. Operating conditions for AFM were scan rate = 0.5 Hz.

Transmission Electron Microscope (TEM). Aliquot (10 l) containing the protein aggregates of -synuclein was adsorbed onto a carbon-coated copper grid (300-mesh) and air-dried for 1 min. For clear negative staining, we used the supernatant of 2%

S2 uranyl acetate (Electron Microscopy Sciences) following centrifugation at 13200 rpm for 2 min. Then, the aggregates were examined with TEM.

Characterization of the oligomeric protein aggregate, granule

AFM analysis. By employing AFM, the granular structure of -synuclein was characterized. The granules were homogeneous in terms of their spherical morphology and molecular dimension. Analysis of the granules in the AFM image gave rise to mean- width, Wm, and mean-height as 51.65 nm and 4.08 nm, respectively (Figure S1).

Considering molecular dimensions of the hemispheres, actual size of the round-shape granules was estimated as 16 nm in diameter, which matches to an average thickness of normal amyloid fibrils.3

Dynamic Light Scattering (DLS). The hydrodynamic diameter, DH was determined by dynamic light scattering (DLS) at 25°C with an Otsuka Electronics DLS-7000 photometer. The granules of α-synuclein were filtered through 0.22 μm membrane filter

(Millipore) before measuring with DLS. The operating conditions were k = 488 nm, angle = 90° with a protein concentration at 1.23 mg/ml. Then the hydrodynamic diameter was calculated by cumulative analysis from DLS data (Figure S2).

CD analysis. In order to evaluate secondary structure content of the protein aggregates,

CD analyses were conducted for monomers, oligomeric granules, and amyloid fibrils of

α-synuclein. The spectra were obtained between 195 and 250 nm with a step resolution of 0.1 nm and scan speed of 10 nm/min by using a 1 mm pathlength quartz cell. All of the spectra were obtained from an average of five scans.

S3 Investigation of n-hexane effect on the granules of α-synuclein

8-Anilino-1-naphthalenesulfonic acid (ANS) binding fluorescence. Fluorescence of

ANS was measured with Perkin Elmer US/LS-55 luminescence spectrometer. Samples contained 25 μM ANS and 43.4 μM α-synuclein in 20 mM Mes buffer, pH 6.5. An excitation wavelength of 375 nm was used, and the excitation and emission slits were set at 5 nm. Structural transition of the protein upon n-hexane treatment was evaluated with the ANS binding fluorescence spectra in the presence and absence of 5% n-hexane.

Fibrillation of -synuclein through the granular assembly. α-Synuclein was incubated in a state of either granule-only (1 mg) or an 1:1 mixture between granule (0.5 mg) and monomer (0.5 mg) in the presence and absence of 5% hexane within 1 ml of 20 mM

Mes, pH 6.5, under a continuous shaking condition at room temperature. The aggregations were assessed with the thioflavin-T binding fluorescence. For separation of the soluble and the particulate fractions, the 1:1 (granule:monomer) mixture was centrifuged at 13,000 rpm for 1 hour following the hexane treatment at 5% for 5 min.

Protein content in the supernatants was estimated with BCA assay kit (Pierce). Fibrillar content in the precipitates was monitored with the thioflavin-T binding fluorescence

(see above).

S4 Figure S1. Size-characterization of the granule obtained at 12 hr-point by AFM analysis.

S5 Figure S2. The hydrodynamic diameter, DH, of the granule showing a homogeneous size-distribution.

Reference

(1) Lee, D.; Lee, S.-Y.; Lee, E.-N.; Chang, C.-S.; Paik, S. R. J. Neurochem. 2002, 82, 1007. (2) Paik, S. R.; Lee, J. H.; Kim, D. H.; Chang, C. S.; Kim, J. Arch. Biochem. Biophys. 1997, 344, 325. (3) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333.