Extended Data Fig. 2: Unfolding of DHFR fusions and effect of GroEL trapping mutant (related to Fig. 3).
From: Targeted substrate loop insertion by VCP/p97 during PP1 complex disassembly
(a) Unfolding reactions with Eos–DHFRI3 with or without methotrexate (Mtx). Reactions without the p37 adapter served as control as indicated. SDS22–PP1–Eos-DHFRI3 (35 nM) or variants thereof were mixed with p97 (175 nM hexamer) and p37 (500 nM), and the reaction started by addition of ATP (2 mM). Red Eos fluorescence was recorded. Mean ± SD, n=3 independent experiments. (b) Inhibition of DHFR activity in SDS22–PP1–Eos-DHFRI3 by Mtx. Conversion of dihydrofolate to tetrahydrofolate was followed spectrometrically by measuring the absorbance of NADPH at 340 nm, at indicated Mtx concentrations. Mean ± range, n=2 independent experiments. (c) Unfolding reactions of circular SPEosI3 with indicated concentrations of GroELD87K mutant that prevents refolding of green Eos. Green Eos fluorescence was recorded. Mean ± SD, n=3 independent experiments.
