Specificity and Kinetics of Α-synuclein Binding to Model Membranes Determined with Fluorescent Esipt Probe*

Alpha-Synuclein was expressed in Escherichia coli using plasmid pT7-7 encoding for the protein (courtesy of the Lansbury laboratory, Harvard Medical School, Cambridge, MA). Following transformation, BL21competent cells were grown in LB in the presence of ampicillin (100 mg/ml). Cells were induced with IPTG, cultured at 37 °C for 4 hours and harvested by centrifugation in an Avanti J25 centrifuge with a JA-20 rotor at 5000 rpm (Beckman Coulter). The cell pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM PMSF, and lysed by multiple freeze–thaw cycles and sonication. The cell suspension was boiled for 20 min and centrifuged at 13500 rpm with a Beckman JA-20 rotor. Streptomycin sulfate was added to the supernatant to a final concentration of 10 mg/ml and the mixture was stirred for 15 min at 4 °C. After centrifugation at 13500 rpm, the supernatant was collected and ammonium sulfate was added (to 0.36 g/ml). The solution was stirred for 30 minutes at 4 °C and centrifuged again at 13500 rpm. The pellet was resuspended in 25 mM Tris-HCl, pH 7.7, and loaded onto an HQ/M-column on a BIOCAD (Applied Biosystems) workstation. AS eluted at 300 mM NaCl using a salt gradient from 0-600 mM NaCl and was further purified and desalted with a Superdex 200 column (GE Healthcare) equilibrated with 25 mM Na-PO4, pH 6.2. The protein was concentrated with an Amicon Ultracel filter (10 kDa), and its purity was assessed by PAGE and electrospray ionization mass spectrometry (ESI-MS). The protein concentration was estimated from the absorbance at 275 nm using a molar extinction coefficient of 5600 M cm. The cysteine variants of AS A18C, A90C and A140C were constructed using the Quick-Change sitedirected mutagenesis kit (Stratagene), and the introduced modification was verified by DNA sequencing. Cysteine mutants were prepared similarly to WT AS but with addition of 1 mM TCEP as a reducing agent to the buffer during purification and in the final buffer in order to prevent oxidation.