Fig. 7
Human/mouse mismatches at positions 121 and 122 affect membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. a Solutions of recombinant aSyn variants were incubated with increasing concentrations of SUVs composed of egg PG:PC (1:1 mol:mol) and analyzed by far-UV CD to determine [θ]MR,222. The data were fit to eq. 3 (see ‘Experimental Procedures’), and values for Kd, minimum [θ]MR,222, and maximum α-helical content were determined from the values of the fit parameters (Table S1). b Monomeric aSyn variants were incubated with PG:PC SUVs (protein-to-lipid ratio, 1:20 mol/mol) at 37 °C for 72 h. Left: Representative Western blot image (the vertical line to the right of the image shows the region of the blot with bands corresponding to oligomeric forms of aSyn). Right: Bar graph showing the ratio of total oligomer band intensity to monomer band intensity determined for each sample. c Calcein-loaded SUVs were incubated in the absence (‘ctrl’) or presence of aSyn variants. Calcein release was monitored via fluorescence measurements using excitation and emission wavelengths of 485 nm and 515 nm, respectively. Left: graph showing % dye release versus time, with 100% release determined as the signal obtained from vesicles treated with Triton X-100. Right: table listing P values determined for vesicle permeabilization data obtained at the 120 h time point (a more complete listing of P values at different time points is presented in Table S2). The data in (b) and (c) are presented as the mean ± SEM, n = 4 (B) or n = 3 (C). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA (b) or two-way ANOVA (c) followed by Tukey’s multiple comparisons post hoc test (a square root transformation was carried out on the data in panel c)