Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

Binding behavior of Snu114-EGFP fusion protein to the spliceosome analyzed by dcFCCS. (A) Affinity-purified B^actΔPrp2 complexes assembled on Atto647N-M3Act, carrying Snu114-EGFP (column 1), were complemented with Prp2, Spp2, and ATP (column 2) and Cwc25 (column 3). After incubation (45 min at 23°C; see Materials and Methods), dcFCCS measurements were then performed at complex concentrations of 1.0 nM. Cross-correlation amplitudes derived from two independent preparations are shown for each complex. Error bars indicate the standard deviation from two independent measurements. (B) Salt-resistance of Snu114-EGFP binding to the spliceosome during catalytic activation. Affinity-purified B^actΔPrp2 complexes assembled on Atto647N-M3Act, carrying Snu114-EGFP (columns 1–3), were complemented with Prp2, Spp2, and ATP (columns 4–6). After the standard incubation, increasing concentrations of KCl were added to the samples, which were then subjected to dcFCCS measurements.