Cup horn sonicator

A. spirocauda genomic DNA was pulse-sonicated using a cup-horn sonicator (QSonica, Newton, CT) for 2.5 min to generate fragmented DNA. Fragmentation was confirmed using 1% agarose gel electrophoresis. The resulting fragment sizes ranged between 200 and 1500 base pairs. Fragmented total genomic DNA was then prepared for sequencing using the NEBNext Ultra^® II kit (New England BioLabs, Ipswich, MA) following the manufacturer's instructions. Total input DNA for library construction was 28 ng. Library quality was validated using the Agilent Bioanlyzer High Sensitivity DNA kit (#5076-4626, Agilent, Santa Clara, CA) and was sequenced using MiSeq v2 500 cycle reagents (MS-102-2003, Illumina, San Diego, CA) and a 1% PhiX control.

We developed a sensitive assay that uses the fluorescein arsenical dye FlAsH-EDT₂ (Invitrogen TC-FlAsH^™ in-cell tetracysteine tag detection kit, catalog no. T34561) to measure fibril growth rates in the presence of C2-WT and mutant C2-Asyn monomer. The SPARK assay was similar to FlAsH seed growth experiments demonstrated in our previous publication^{39 (link)}. Amplified fibril samples at 1.5 μM concentration in fibril buffer plus 0.1% Triton X-100 were sonicated for 5 min at amplitude 50 in the cup horn sonicator (Qsonica) and then mixed with 0.5 mg/ml of C2-Asyn monomer (C2-WT-Asyn and mutant C2-Asyn) in a total volume of 25 μl. The seeds plus monomer mixture were quiescently incubated for 3 h at 37 °C in Corning Black 96-well plates (Fisher, catalog no. 07-200-762). After 3 h, FlAsH assay mixture consisting of 3.5 mm tris(2-carboxyethyl)phosphine, 1 mm EDT, 1 mm EDTA, 25 nm FlAsH-EDT₂, and 200 mM Tris-HCl, pH 8.0 was added and the mixture incubated for additional 1 h at room temperature. FlAsH fluorescence was detected in a BioTek plate reader using a 485/20-nm excitation filter, a 528/20-nm emission filter, top 510-nm optical setting, and gain setting 100. Data was normalized to fibril growth rates with WT-C2-Asyn monomer. We selected 6^th cycle LBD and MSA amplified fibrils (case LBD1, LBD2, LBD3, LBD4, MSA1, MSA2, MSA3 and MSA4) for SPARK assays.

Reconstitution of Fluc was carried out as described previously (14 (link)). Briefly, 25 mg/mL chloroform stocks of EPLs (Avanti Polar Lipids Inc. #100600C) were dried by evaporation under a stream of nitrogen gas until a thin dried film of lipids appeared. The film was washed with pentane (Sigma-Aldrich #236705-1L). Then, lipids were resolubilized in the required dialysis buffer with 35 mM CHAPS (Anatrace #C316S) for a final concentration of 20 mg/mL lipids. The lipid-detergent mixture was solubilized using a cup-horn sonicator (Qsonica, Newtown, CT) until the sample was transparent. Protein was added to the solubilized lipid-detergent mixture and placed into 3,500 MWCO dialysis cassettes (Thermo Scientific #66330), and then the samples were then dialyzed in the dark at room temperature in 1,000× sample volume with 4 buffer changes every 4 to 12 h. At the end of dialysis, the samples were freeze-thawed to form large paucilamellar vesicles. This involved 3 repetitions of freezing the samples at –80 °C and thawed in a room-temperature water bath. Samples were stored at room temperature, in the dark for the desired amount of time or frozen at –80 °C until used.