Fig 1.
a) Overall reaction pathways and intermediates of α-synuclein aggregation shown schematically. Blue spheres: monomers within oligomers (shapes of oligomers are arbitrary). Red stars: R1 nitroxide spin label (see b). b) Molecular structure of the R1 spin label attached to the protein. No specific kinetic pathways are shown.
Fig 2.
Room temperature EPR spectra of α-synuclein (R1-αS) at different time points of aggregation.
Full spectra: Inset, the box shows the region zoomed into. Zoomed-in spectra: Amplitude expanded four-fold with respect to inset. a) Start of aggregation (t = 0). b) 9 hours of aggregation. c) 24 hours of aggregation. d) 42 hours of aggregation. Black: Experimental spectra. Red: Simulated spectra. Arrow: feature of broad spectral component (see text).
Fig 3.
Aggregation of α-synuclein as a function of time derived from EPR.
Amount of fast fraction (green dots) caused by monomers. Amount of medium fraction (blue dots) assigned to oligomers. Amount of slow fraction (red dots) assigned to fibrils. The solid lines are the fractions of monomer, oligomer and fibrils predicted from the best fit of the model, with a reaction order of n = 7.
Table 1.
Rotation correlation time (τr) of R1-αS in the three fractions observed by EPR.
Fig 4.
Schematic of the kinetic model used to fit the experimental data, showing species, rate constants and processes considered in the kinetic model.
Because monomers and oligomers reach pre-equilibrium on a timescale significantly faster than that of the measurement intervals, altering this model to make oligomers off-pathway does not affect the fit quality. As such, the oligomers cannot be resolved as on- or off-pathway and must instead be considered part of the reactant ensemble at this experimental time resolution [42]. Left: Oligomer formation (ko) and dissociation (kd) interconvert monomers (m) and oligomers (O). Conversion (kc) or nucleation (kn), green arrow, leads to fibrils (right). Elongation, (k+) grows existing fibrils, M denotes the monomer equivalent fibril concentration and P the number concentration of fibrils.