Lifetime changes of five fluorescent dyes attached to actin at Cys-374 upon binding to C0–C2. Either 1 or 5 µM of fluorescently labeled actin was mixed with 0, 5, or 20 µM unlabeled C0–C2. White bars indicate actin alone, gray/black bars indicate unphosphorylated C0–C2, and pink/red bars indicate phosphorylated C0–C2. (A) Fluorescence lifetime. (B) Relative reduction (% decrease) in lifetime compared with actin alone (see Materials and methods). Note that the actin alone (white bars) in B display only error bars, as the percent decrease in lifetime for actin alone relative to itself is 0. Significant differences were observed (P < 0.000002) for all probes comparing actin alone to actin plus C0–C2 (white bars versus gray, and white bars versus black) with two exceptions that trend to, but do not reach, significance in A or B: 1 µM IANBD-actin with 5 µM C0–C2 (P = 0.06) and 5 µM IANBD-actin with 5 µM C0–C2 (P = 0.16). For comparison of unphosphorylated versus phosphorylated C0–C2 (gray versus pink and black versus red), P < 0.05 for all probes and C0–C2 concentrations with four exceptions in A or B: 1 µM IANBD–actin 5 µM C0–C2 P = 0.86, 5 µM IANBD–actin 5 and 20 µM C0–C2 P = 0.94 and 0.40, and 5 µM IAEDANS–actin 20 µM C0–C2 P = 0.24. Data are provided as mean ± SE (n > 4).