FIGURE 5.
Microscale thermophoresis and surface plasmon resonance confirm a direct interaction between PREP and aSyn. The interactions between aSyn and PREP (A) and between aSyn and PREP(S554A) (B) were studied by MST. Equal amounts of fluorescently labeled (NT-647) recombinant PREP or PREP(S554A) were titrated by recombinant unlabeled aSyn at various concentrations. Similar to PCA results, aSyn binds to PREP and PREP(S554A) with a Kd of ∼2.96 and ∼1.41 μm, respectively. Fluorescence time traces taken from single experiment suggest low or no aggregation had occurred in both sets of experiments (insets in both graphs). The curve and Kd values were calculated by averaging Kd curves assimilated using NTanalysis software from three independent experiments (mean ± S.E.). C, a time versus signal plot of SPR measurement is shown, normalized by Bmax of the first order analysis. Three doses of aSyn were used in the injections (0.5, 5, and 50 μg/ml). Kd determined by SPR was 3.61 μm, and it was the mean of three independent experiments.