Quantamaster 400

Protein melting temperatures (T_m) were measured using thermal shift analysis. 0.1 μM NS1 was mixed with Sypro Orange (S5692, Sigma-Aldrich) in 20 mM sodium phosphate (pH 7), 100 mM NaCl, and 2 mM TCEP. Data were recorded from 20 °C to 80 °C using a Quantamaster 400 fluorometer (Photon Technology International). T_m was calculated by fitting data to Boltzman sigmoid equation (GraphPad Prism).

The progression of PrP^c conversion to fibrils during shaking was monitored by Thioflavin T (ThT) fluorescence enhancement. MoPrP ^90–231 and MoPrP ^23–231 were converted by shaking at 250 rpm and 37°C as described above. PrP samples were then removed at different time points and mixed with 20 mM sodium acetate pH 5.5 and 100 µM ThT (Sigma) to give 10 µM PrP (monomeric concentration). Emission spectra were acquired on a QuantaMaster 400 spectrofluorimeter (Photon Technology International Inc., London, ON, Canada) with an excitation wavelength of 440 nm. The fluorescence intensity at the emission maximum of 480 nm was then plotted over the entire sampling period. The ThT fluorescence time course was fit to a sigmoidal function as described above for the RENAGE fibril peak area.

An appropriate volume from a stock solution of CCVJ (in DMSO) was added to LS x solutions to yield 5–10 wt% LS x solutions containing 0.07 wt% CCVJ. n-SiO₂ wafers were washed following the protocol described in the “Thin-Film Preparation” section. The dye-ionomer solutions were then spin-coated on these prewashed n-SiO₂ wafers at 3,000 rpm to prepare ~100 and ~250 nm-thick films. The films were annealed following the same procedure mentioned earlier in the “Thin-Film Preparation” section. The films were then placed inside a quartz humidity chamber (2.25″ × 2.25″ × 2.875″) and exposed to air with varied %RH to measure fluorescence spectra using steady-state fluorescence spectroscopy (PTI Quantamaster 400, Horiba, NJ). The excitation wavelength (λ_exc) of CCVJ was 440 nm, while the emission wavelength (λ_em) ranged between 470 and 560 nm. Same spectroscopic parameters (excitation/emission slit width = 1 mm; step size = 10 under excitation correction and zero bias) were used to measure fluorescence of all samples.

Fluorescently labeled SOPG/C12-NBD-PG LUVs in PBS were incubated at room temperature with individual α-synuclein constructs for 1 hr. Permeabilization of the LUV membranes was monitored by adding reductant to the buffer to bleach the fluorescence of solvent-exposed C12-NBD-PG. C12-NBD-PG fluorescence was measured using a Photon Technology International QuantaMaster400 spectrofluorometer (HORIBA Scientific (Canada), London, ON) at an excitation wavelength of 466 nm, quantifying the fluorescence emission at 535 nm (2-nm bandwidths). Bleaching was initiated by adding 1 M sodium dithionite to the buffer, and the fluorescence was measured over the next 2 min. Measurements were repeated with different protein construct concentrations (0.3–7 µM for each construct) to achieve protein:lipid molar ratios ranging from 1:300 to 1:10, and compared to a positive control in which LUVs were lysed by 0.1% (v/v) Triton X-100 (SigmaAldrich, Oakville, ON, Canada).
To determine the total extent of membrane leakage, the fluorescence intensity was measured 2 min after dithionite addition. The fractional amount of permeabilization was calculated as $\left|I_{\alpha S}-I_{PBS}\right|/I_{PBS}$ , where I_αS is the fluorescence intensity 2 min after adding dithionite to vesicles incubating with α-synuclein and I_PBS is the fluorescence intensity 2 min after adding dithionite to vesicles in PBS without any α-synuclein present.