Figure 3.

Characterization of the physical properties of fibers formed in vitro.

(a) A representative experiment monitoring the relative growth rates of WT and PNM2 SupNM. Polymerization of SupNM was performed with 5% (w/w) WT seed of the specified conformation, and the rate of addition of SupNM monomers was monitored by Thioflavin T fluorescence. Data were normalized to initial and final intensities. (b) The growth rates of Sc4 and Sc37 PNM2 SupNM normalized to those of WT SupNM polymerized on the relevant seed. Initial time points were fitted to a line and the slope (initial growth rate) was calculated. Values represent the mean ± s.d. for three experiments. (c,d) Thermal stability of WT and PNM2 fibers in Sc4 and Sc37 conformations. WT and PNM2 SupNM fibers in the Sc4 or Sc37 conformations were incubated at increasing temperatures, and samples were subjected to SDS-PAGE. Band intensities (susceptibility of aggregates to thermal solubilization) were plotted against temperature and fitted to a sigmoidal function. (e) Relative seeding efficacy of WT or PNM2 fibers in the Sc4 or Sc37 conformation before and after stirring for 30 or 60 min. Seeding efficacy was determined by monitoring the initial fiber growth rates of polymerization reactions using stirred samples as seeds. Values represent mean ± s.e.m. for three experiments.