Catalytic activation by Prp2 creates a high-affinity binding site for Cwc25 in the spliceosome. (A) BactΔPrp2 complexes assembled on Atto647N-M3Act (column 1) were complemented on the amylose matrix with C-terminally labeled Cwc25-Alexa488 (column 2) or Cwc25-Alexa488 together with Prp2, Spp2 plus ATP (column 3). After incubation, the complexes were washed and eluted with maltose, and dcFCCS measurements were performed. (B) BactΔPrp2 complexes assembled on Atto647N-M3Act were directly eluted from the amylose matrix or activated on the column by Prp2 and Spp2 in the presence of ATP to generate B* before the elution. Increasing amounts of complexes were then supplemented with 0.3 nM Cwc25-Alexa488, and the resulting cross-correlation amplitude was measured. Cross-correlation amplitudes from two independent measurements were plotted against the respective complex concentration (Hill plot, semilogarithmic scale). Curve-fitting revealed a dissociation constant of Cwc25-Alexa488 to complex B* of Kd = 0.03 nM. The lower curve was obtained under similar conditions but by adding complex BactΔPrp2 instead of B*.
