Figure 5.

Estimating binding constants from the titration curves of matched pairs. (A) Plot showing normalized fluorescence curves obtained from titrating an increasing amount of Venus+^CDD to its cognate ^NDD+mTurquoise2. The trace obtained from ^NDD+mTurquoise2 is shown as the thick blue line; traces obtained with increasing Venus+^CDD concentration are shown as thinner purple to red lines. The turquoise box outlines the peak ^NDD+mTurquoise2 (donor) fluorescence at 475–480 nm. (B) Plots of the FRET efficiency E by Venus+^CDD concentration for all matched pairs. Each color corresponds to a different matched pair: Venus+^CDD_BaeM/^NDD_BaeN+mTurquoise2, red; Venus+^CDD_DifF/^NDD_DifG+mTurquoise2, orange; Venus+^CDD_MlnB/^NDD_MlnC+mTurquoise2, green; Venus+^CDD_MlnC/^NDD_MlnD+mTurquoise2, light blue; Venus+^CDD_MlnD/^NDD_MlnE+mTurquoise2, blue; Venus+^CDD_MlnF/^NDD_MlnG+mTurquoise2, purple. The data were fit well by a single-site binding curve function, as shown by the solid lines. Each point is the average of three replicates. Error bars correspond to standard deviations. (C) Plots of estimated fraction of ^NDD+mTurquoise2 bound at each Venus+^CDD concentration using the data shown in panel B. (D) Estimated dissociation constant (K_d) values of matched pairs and R² of fit to eq 3. Standard deviations are indicated.