Fig. 5: ATP-driven HT-aggr disaggregation by BiP in vitro.

a Protein absorbance traces (280 nm) of BiP WT (50 µM) alone (grey), with native HT-aggr (dark blue) or native HT-aggr, fused to J-domain (HT-aggr-JD, 10 µM) (light blue), resolved by gel filtration chromatography (10–600 kDa fractionation range). b Traces as in (a) of native HT-aggr (green), native HT-aggr-JD (light blue) and heat-aggregated HT-aggr-JD (53 °C, 20 min, 10 µM) (magenta). c, Traces as in (a, b) of aggregated as in (b) HT-aggr-JD, incubated with BiP WT/ATP (magenta) or BiP V461F/ATP (orange), a substrate-binding deficient BiP mutant. Note the increase in the main peak of the aggregated HT-aggr-JD (pale red) upon the addition of BiP WT/ATP only, indicative of its recruitment to the aggregates. d Traces as in (a, b) of aggregated as in (b) HT-aggr-JD, incubated with BiP WT/ATP for the indicated periods (4 h, magenta; 24 h, orange; 48 h, light blue). e Fluorescence traces of chromatograms as in (d) this time exclusively detecting HT-aggr-JD (pre-aggregated) through the fluorescence of its P2-halo ligand label (HT-aggr-JD, pale red; 4 h, magenta; 24 h, orange; 48 h, light blue). f Fluorescence traces of chromatograms for the P2-labelled HT-aggr-JD (pre-aggregated) in the absence of BiP for the same time course and trace’s colours as in (e). Fluorescence was measured for discrete data points, 0.3 mL each. Dynamic light scattering measurements showing the hydrodynamic diameter (Dh) of P2-labelled HT-aggr-JD (pre-aggregated) for the same time course were plotted in the inset to unveil the growth of the aggregates in the absence of BiP and explain why aggregates are retained in the pre-filter of the column leading to a decreased amount eluted over time. The measurements in (a–f) were performed in the presence of ATP (3–9 mM), which elutes at 20.2 mL, fractions omitted from the chromatogram.