Figure 8.

SOS autoactivation-mediated SOS cooperativity. SOS^cat titration with the lipid vesicle-tethered wt Ras•GDP* and reverse titration were performed in the presence of excess GTP. When necessary, we used 5% activated SOS^cat instead of 0% activated SOS^cat. Unlike with 0% activated SOS, the partly activated SOS (e.g. 5% activated SOS, see the Materials and Methods section for its preparation) initiates SOS autoactivation without wt Ras•GTP, the reaction initiator. (a). 0% or 5% activated SOS^cat (1 μM) was titrated with various densities of the lipid vesicle-tethered Ras•GDP* (0–8.0 × 10³ Ras•GDP molecules/μm²) in the presence of excess free GTP (1 mM). The resultant SOS-mediated Ras nucleotide exchange of each titration was monitored as indicated in Figure 5. GraphPad Prism software was used to calculate and plot the apparent k_cat values of each titration that reflected Ras•GTP production (s⁻¹) against Ras•GDP (molecules/μm²) and fit them to a hyperbolic saturation or a Hill plot. A hyperbolic saturation curve was used to determine the K_m of SOS^cat for the lipid vesicle-tethered as 2.4 × 10³ molecules/μm.² The Hill coefficient and half-maximal effective substrate concentration (K_0.5) values of the hysteretic saturation curvature titration were, respectively, 2.1 and 0.9 × 10³ molecules/μm.² (b). The lipid vesicle-tethered Ras•GDP* (1.0 × 10³ Ras•GDP molecules/μm²) was titrated with various amounts of 0% or 5% activated SOS^cat (0–10 μM) in the presence of excess free GTP (1 mM); the corresponding apparent k_cat (s⁻¹) values were calculated as indicated in A and plotted against SOS^cat (μM). GraphPad Prism software was also used to fit the titration plots to a hyperbolic saturation or a Hill plot. The SOS concentration needed to yield one half of the maximal velocity for the catalysis of the lipid vesicle-tethered Ras•GDP* (1.0 × 10³ Ras•GDP molecules/μm²) was determined to be 1.9 μM. The hyperbolic saturation curvature was used for this determination. Intriguingly, the estimated Hill coefficient of the hysteretic saturation curvature titration of B also converges to 2.1. Insets: Expansions of the hysteretic saturation curvature in both A and B are shown