Extended Data Fig. 5: Analysis of allosteric effects in the formation of the Rb-E1A complex.
From: Conformational buffering underlies functional selection in intrinsically disordered protein regions
Measurements were performed by loading the cell with Rb or with a pre-assembled complex of Rb with peptide/proteins containing one of the interacting motifs and titrating with peptide/proteins containing the complementary motif loaded into the syringe. Panels show heat exchanged as a function of time, (upper panel) and the enthalpy per mole of injectant plotted as a function of [peptide or protein]/[Rb] molar ratio (Lower panel, black circles) along with the corresponding fit using a single site binding model (Lower panel, black lines). Binding traces correspond to: a, Rb (30 μM, cell) titrated with E1AE2F (300 μM, syringe) at 10 °C; b, [E1ALxCxE:Rb] (30 μM, cell) titrated with E1AE2F (300 μM, syringe) at 10 °C; c, Rb (30 μM, cell) titrated with E1AΔL(300 μM, syringe) at 10 °C; d, [E1ALxCxE:Rb] (30 μM, cell) titrated with E1AΔL (300 μM, syringe) at 10 °C; e, Rb (15 μM, cell) titrated with E1ALxCxE (150 μM, syringe) at 20 °C; f, [E1AE2F:Rb] (15 μM, cell) titrated with E1ALxCxE (150 μM, syringe) at 20 °C; g, [E1AΔL:Rb] (15 μM, cell) titrated with E1ALxCxE (150 μM, syringe) at 20 °C. Thermodynamic parameters derived from the fitting are shown in Supplementary Table 1. A schematic representation of each titration design is shown above the ITC traces: Rb: grey double circle, E2F motif: blue oval, LxCxE motif: red oval. The E1A linker is depicted as a black line. h, ITC measurements of E1AE2F and E1AΔL at different temperatures. The heat capacity change (ΔCp) was calculated from the slope of the plot of ΔH vs temperature. E1AE2F: filled blue circles; E1AΔL: open blue circles. Thermodynamic parameters are reported in Supplementary Data Table 5.
