Fig. 3. Metastability and RF seeding incompetence of gO/CFs. (A) HewL RF kinetics below the COC (38 μM hewL, 400 mM NaCl) without seeding (black), or after seeding with isolated gO/CFs (blue) or RFs (orange) at either 3.5 or 7 μM each, and incubated at pH 2, T = 52 °C. (B) ThT-detected dissociation of pre-assembled dimAβ upon addition of the binding protein ZAβ3 (grey), which sequesters monomeric dimAβ in a β-hairpin conformation (red, see ref. 65 and 67). The complex is shown in ribbon representation, hydrophobic side chains of ZAβ3 in direct contact with the Aβ40 β-hairpin are shown as spheres (Protein Data Bank entry ; 2OTK). The assembly of 6 μM dimAβ was monitored by ThT fluorescence, with addition of 14 μM ZAβ3 at different time points, indicated by red lines. Addition of ZAβ3 during the gO/CF formation-dominated time regime results in nearly complete loss of ThT fluorescence, while ZAβ3 addition during the RF-dominated time regime leads to a progressive decrease in the amplitude of the fluorescence drop. (C) Energy diagram illustrating the order of thermodynamic stability of different states of Aβ, above the COC and under the present experimental conditions.