Figure 3. Binding of Nb_Roco1 induces (nucleotide-dependent) monomerization of CtRoco.

(A) Influence of Nb_Roco1 on the stopped-flow FRET traces of labeled CtRocCOR upon mixing with different nucleotides to follow monomerization over time. The FRET signal of Cy3/Cy5-labeled CtRocCOR (S928C) over time is shown after rapid mixing with buffer (left panel), or 100 μM GDP (middle panel) or GppNHp (right panel), either in the absence or presence of an excess of Nb_Roco1. Monomerization is observed through a decrease in FRET signal. (B) Single-molecule FRET histograms for A555/A647-labeled CtRocCOR in the absence of nucleotide, and in the presence of GDP or GppNHp, and in the absence (left panels) and presence of Nb_Roco1 (center panels). The right panels show the superposition of the two individual histograms. The population of dimeric CtRocCOR (90% FRET efficiency) decreases in the presence of Nb_Roco1, indicating monomerization. The GppNHp-bound state of CtRocCOR (lower panel) is predominantly monomeric in the absence and presence of Nb_Roco1. Signals below 20% FRET efficiency belong to molecules carrying only a donor dye whose signals are not completely suppressed by PIE.