Fig. 2. Interaction of PSMα3 with different αS species by dcFCCS.

a–d Representative auto-correlation curves for α-synuclein (αS) (blue line) and PSMα3 (red line) and cross-correlation curves for interacting molecules (purple line). The amplitude (G) error is shown as faint colored area for the corresponding correlation curves. Samples contained (a) ~15 nM αS monomer and ~15 nM PSMα3, (b) 1 nM type A* and ~5 nM PSMα3, (c) 1 nM type B* oligomers and ~5 nM PSMα3 or (d) ~5 nM sonicated fibrils and ~5 nM PSMα3. e Titration binding curves for the interaction of PSMα3 with type A* oligomers (red circles), type B* oligomers (blue circles) or sonicated fibrils (gray circles) obtained by dcFCCS, showing their corresponding analysis assuming a model of n identical and independent binding sites (referred in equation 7 as N_max) per αS aggregated species (solid lines). N_P represents the number of bound peptides per aggregate. f Auto-correlation curves (αS in blue, PSMα3 peptide in red) and cross-correlation curve for the interacting molecules (in purple) obtained in samples containing ~1 nM αS type B* oligomers and ~2 nM PSMα3 in the absence (solid lines) or presence (dashed lines) of a 500-molar excess of unlabeled monomer with respect to the particle concentration of oligomers. The inset shows the number of bound peptides (N_P) per aggregate in both conditions. For αS aggregated species, each consisting of several tens of monomers, the species concentrations are in the picomolar range and, as further explained above, single-particle conditions are ensured throughout the experiments.