TR-F IAEDANS-actin–Tm and IAEDANS-actin-binding curves for C0–C2 mutants. TR-F measurements of the effects of mutant C0–C2 on the reduction (percent decrease) in lifetimes are plotted for increasing concentration without (solid red lines) and with PKA treatment (dotted red lines). For comparison, curves for WT C0–C2 are included in each graph (black lines). (A) C0–C1/actin–Tm. (B) C0–C1/actin. (C) L352P/actin–Tm. (D) L352P/actin. (E) E334K/actin–Tm. (F) E334K/actin. (G) R282W/actin–Tm. (H) R282W/actin. Arrows indicate the C0–C2 concentrations to be used in future screens (thick arrows, 1.25 µM for actin–Tm in A, C, E, and G; thin arrows, 2.5 µM for actin alone in B, D, F, and H). Data are provided as mean ± SE (n > 4).