Figure 2.

Real-time detection of PhAs^III binding by Hh using an optical reporter. A) Effect of PhAs^III on the fluorescence of FRET-active Hh precursor protein. Spectra of precursor, C-H-Y, in aqueous buffered solution (pH 7.1) with and without 1 equiv of added PhAs^III (λ_ex=400 nm, λ_em=420–600 nm). Inset: Schematic of FRET-active construct with cyan fluorescent protein fused to the N terminus of the HhC domain and yellow fluorescent protein fused to the C terminus. B) Kinetics of PhAs^III binding by C-H-Y measured by FRET change. Time traces showing loss of FRET signal from C-H-Y after addition of the indicated concentration of PhAs^III. Solid lines show first-order exponential decay (k_obs), calculated by using best-fit values. C) Bimolecular kinetics of PhAs^III binding by C-H-Y is saturable. Rate constants (k_obs [s⁻¹]) for FRET loss plotted as a function of increasing PhAs^III concentration. Solid line shows the expected behavior, assuming reversible formation of a noncovalent encounter complex, followed by irreversible formation of the PhAs^III/C-H-Y complex. D) Fluorometric titration of binding of PhAs^III to C-H-Y. Plot shows the overall FRET change (FRET_○–FRET_f) as a function of PhAs^III concentration. Solid line shows the expected behavior for an inhibitor with a EC₅₀ value of 0.4 μM. Error bars indicate data range (n=3)