Effects of WT and Tm-binding mutants on binding to actin–Tm and actin. Effects of WT and Tm-binding mutants on actin–Tm (thick lines) and actin (thin lines) TR-F were tested. Tm-binding mutants reverse charges (EASE; R215E/K218E) or introduce additional positive charges (RRKK; A216R/S217K) in the Tm-binding loop 215–218, RASK, of C0–C2. (A) WT and R215E/K218E (EASE in red) effects on IAEDANS-actin–Tm and IAEDANS-actin for C0–C2 from 0 to 20 µM. (B) Zooming in on the lower concentrations (0–5 µM) C0–C2 added in A. (C and D) The same conditions as A and B above but comparing WT and A216R/S217K (RRKK in green). For the Tm-binding mutant (charge reversal-EASE), apparent K_d changes trended toward significant (P = 0.11) for actin–Tm, but not for actin alone. For the positive Tm-binding mutant (additional positive charges-RRKK), apparent K_d changes were significant (P < 0.05) for binding to both actin and actin–Tm. Refer to Table S1 for statistical analysis of fitted binding properties for curves and Table 2 for comparisons of binding at specific C0–C2 concentrations. Data are provided as mean ± SE (n > 4).