Table 1 Amino acid ROCK inhibitor sequence of C-terminal region of syn-wt and truncated or mutated mutants All mutant proteins were expressed in E. coli BLR(DE3)
and purified according to the method for Syn-wt (Yagi et al. 2005), with the exception of Syn118, Syn103, Syn119-140CF, and the Syn119-140CF/Y136A mutant. Syn118, Syn119-140CF, and Syn119-140CF/Y136A were purified utilizing Q-Sepharose anion-exchange chromatography at a pH different from the wild type (8.5 instead of 7.5). Regarding Syn103, due to the replacement Inhibitors,research,lifescience,medical of acidic amino acids in the C-terminal region, this mutant was unable to bind to Q-Sepharose. Syn103 was therefore purified as follows: After ultrasonic disruption, removal of nucleic acids by addition of streptomycin, heat-treatments, and fractionation
by ammonium sulfate, protein solution desalting with a Cellulofine CH-25m Inhibitors,research,lifescience,medical (Seikagaku Kogyo, Tokyo, Japan) gel-filtration column, which had been equilibrated with purification buffer (50 mmol/L Tris–HCl, pH 7.5, containing 1 mmol/L EDTA, 0.1 mmol/L dithiothreitol, and 0.1 mmol/L phenylmethylsulfonyl fluoride). Protein fractions were pooled and loaded onto an SP-Sepharose cation-exchange column (GE Healthcare Life Sciences, Chalfront St. Giles, U.K.), which had been equilibrated with the purification buffer and eluted with a linear salt gradient (0–1 mol/L NaCl) at a flow rate of 1 mL/min. Inhibitors,research,lifescience,medical The purified Syn103 protein was desalted using a gel-filtration column equilibrated with 2.5 mmol/L ammonium bicarbonate. The three C-terminal truncation mutants, Syn130-140CF, and Syn119-140CF were lyophilized and stocked at 4°C until use. Protein concentrations of Syn-wt was Inhibitors,research,lifescience,medical determined by using a molar absorption coefficient of ε 0.1%280 nm = 0.354 (Narhi Inhibitors,research,lifescience,medical et al. 1999), and protein concentrations of the other mutant proteins were determined by using individual calculated absorption coefficients (see Table 1) estimated from amino acid content (Pace et al. 1995). Amyloid fibril formation and ThioT binding assay α-syn proteins (1 mg/mL) were induced L-NAME HCl to form fibrils at 37°C in fibrillation
buffer (25 mmol/L Tris–HCl buffer, pH 7.5), containing either 0, 0.15, or 1 mol/L NaCl by linear (back and forth) shaking at a rate of 170 repetitions/min. Fibril formation was monitored by ThioT (Wako, Osaka, Japan) binding assay, using a HITACHI F-4500 (Hitachi Hightechnologies, Tokyo, Japan) or JASCO FP-6300 (JASCO, Tokyo, Japan) fluorescence spectrophotometer. Fibril samples were mixed with 25 μmol/L ThioT in phosphate-buffered saline, and fluorescence intensity was monitored at 480 nm upon excitation at 440 nm. In certain experiments, to boost the sensitivity as well as to facilitate fibrillation, a multiwell plate reader, ARVO X4 (Perkin Elmer, Waltham, Massachusetts), was used in measurements.