A low-affinity binding site is created for Prp16-EGFP during catalytic activation of the spliceosome by Prp2 as analyzed by dcFCCS. Affinity-purified Bact complexes assembled on Atto-Actwt were complemented on the matrix with recombinant Prp16-EGFP plus ATP (column 1, Bact) and additionally with the proteins indicated on the left. After incubation, complexes were washed with GK75 buffer (A) or GK150 buffer (B) and then eluted from the matrix with maltose. dcFCCS measurements were then performed at complex concentrations of 1.0 nM. C*/PC indicates that upon Slu7/Prp18 binding to the C* complex, the latter is transformed into the post-catalytic complex (PC). (C) The B* complex was complemented on the matrix with Prp16-EGFP and incubated at 23°C, the matrix was washed with GK75 buffer, and supplemented with increasing amount of untagged Prp16 (columns 2–4) or Slu7/Prp18 (columns 5–7). The matrix was washed, complexes were eluted with maltose, and cross-correlation amplitudes were measured by dcFCCS. (D) The B* complex was complemented on the matrix with Slu7-EGFP and Prp18 and incubated at 23°C. The matrix was then washed with GK75 buffer, and the complexes were supplemented with an increasing amount of untagged Slu7 and Prp18 (columns 2–4) or Prp16 (columns 5–7). After washing, complexes were eluted with maltose and cross-correlation amplitudes were measured by dcFCCS. Cross-correlation amplitudes derived from two independent experiments are shown for each complex. Error bars indicate the standard deviation from two independent measurements.
