α syn

Bovine insulin was purchased from Sigma-Aldrich (CAS 11070–73-8, St. Louis, MO, USA), α-Syn was purchased from AnaSpec (Cat. N. AS-55555–1000, Fremont, CA, USA), (5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenoic acid (ARA) from Spectrum chemical MFG Corp. (CAS 506–30-9), and (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid (DHA) from Acros organics (CAS 6217–54-5).

α-Syn is purchased from AnaSpec, CA, USA. The preparation of α-Synuclein follows a group protocol by Zhou and Kurouski.^{32 (link)} Briefly, α-Syn was dissolved to final concentration of 150 μM in 1X PBS buffer, pH at 7.5 stock. Next the stock was mixed with DMPC or DMPS lipid unilamellar vesicles (LUVs) and reach final protein concentration at 45 μM. For early stage oligomers study, the solution was kept under room temperature without any agitation.
The preparation of lipid unilamellar vesicles (LUVs) was repeated by using previous protocol by Dou and Kurouski.^{33 (link)} After LUVs were collected through the extruder, the sizes of lipid LUVs were checked by using dynamic light scattering (DLS). In this experiment, we investigate the lipid to protein ratio 1:2. Samples for AFM-IR, CD, and FT-IR measurements were collected on day 2, day 8, and day 15, denoted as D2, D8, and D15.