Figure 3.

Kinetics of spontaneous and seeded PHF assembly. (A) Dimeric tau protein (construct K19) was incubated in assembly buffer at a concentration of 10 μM in the presence of poly-Glu (MW 600, 80 μM) and varying concentrations of seeds obtained by sonication of preassembled PHFs. Assembly was followed by ThS fluorescence. Note that in the presence of added seeds the assembly curves can be fitted by a simple exponential approach to equilibrium, while in the absence of seeds (bottom curve) there is a lag phase because of the rate-limiting spontaneous nucleation. (B) The maximum assembly rates obtained from A increase linearly as a function of seed concentration. (C) Concentration dependence of maximum velocities for dimeric tau protein (K19) with either heparin at various concentration ratios [K19/heparin, 1:1, (○), 1:0.5 (▿), 1:0.25 (□)], or polyglutamate [MW 1,000; 1:4 (•)] without added seeds (spontaneous nucleation). The size of the nucleus (n = 2x) can be estimated from the slope of the curve (x) (24). The slope varies between 2 and 3.5, depending on the polyanion and the stoichiometry, so that the nucleus contains about 4–7 subunits (= tau dimers) or 8–14 tau monomers.