Figure 2.

Mutant hS2 proteins display increased affinity by decreasing K_dto phosphorylated hC0-C2 protein by SPBA. The binding curve between hS2^Wt and hC0-C2 (solid green), hS2^Wt and hC0-C2p (dashed green), (A) hS2^R870H and hC0-C2 (solid blue), hS2^R870H and hC0-C2p (dashed blue), (B) hS2^E924K and hC0-C2 (solid orange), hS2^E924K and hC0-C2p (dashed orange), (C) hS2^E930Δand hC0-C2 (solid red), and hS2^E930Δand hC0-C2p (dashed red). D, relative maximal binding capacity (B_max) was determined for each listed combination and compared against hS2^Wt and C0-C2 proteins. E, binding affinity or dissociation constant, K_d, compared against hS2^Wt and hC0-C2 proteins. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparison test with single pooled variance. n = 3 with triplicates for each concentration. Data are expressed as the mean ± SEM. See Table 1 for analysis of main factors and interactions. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus hS2^Wt/hC0-C2 controls. hS2^E924K, human recombinant proximal S2 protein with E924K mutation; hS2^E930Δ, human recombinant proximal S2 protein with E930Δ mutation; hS2^R870H, human recombinant proximal S2 protein with R870H mutation; hS2^Wt, human recombinant proximal S2 WT protein; S2, subfragment 2 region; SPBA, solid-phase binding assay.