Biochemical properties of mutant cofilins
| Parameter | Cofilin | Cofilin-M2 | Cofilin-M3 |
| Stability: ΔG (kcal mol−1) | 9.10 | 7.91 | 10.32 |
| Mg-ATP-actin monomer affinity, Kd (μM) | 0.08 | 0.11 | 0.17 |
| ADP-actin filament apparent affinity, Ka (μM−1) | 0.15 | 0.012 | 0.08 |
| ADP-actin filament affinity, Kd (μM) | 0.33 | 1.7 | 0.89 |
| ADP-actin filament binding cooperativity, ω | 19.9 | 48.7 | 14.2 |
| ADP-actin filament association rate constant, k+ (µM−1 s−1) | 12.5 | 1.4 | 3.8 |
| ADP-actin filament dissociation rate constant, k- (s−1) | 200 | 4600 | 140 |
| Ratio of k-/k+ (µM) | 16 | 3300 | 37 |
| Optimal severing concentration (nM) | 10 | 500 | 100 |
| Maximum serving rate (events per 1,000 subunits s−1) | 0.032 | 0.009 | 0.012 |
| Parameter | Cofilin | Cofilin-M2 | Cofilin-M3 |
| Stability: ΔG (kcal mol−1) | 9.10 | 7.91 | 10.32 |
| Mg-ATP-actin monomer affinity, Kd (μM) | 0.08 | 0.11 | 0.17 |
| ADP-actin filament apparent affinity, Ka (μM−1) | 0.15 | 0.012 | 0.08 |
| ADP-actin filament affinity, Kd (μM) | 0.33 | 1.7 | 0.89 |
| ADP-actin filament binding cooperativity, ω | 19.9 | 48.7 | 14.2 |
| ADP-actin filament association rate constant, k+ (µM−1 s−1) | 12.5 | 1.4 | 3.8 |
| ADP-actin filament dissociation rate constant, k- (s−1) | 200 | 4600 | 140 |
| Ratio of k-/k+ (µM) | 16 | 3300 | 37 |
| Optimal severing concentration (nM) | 10 | 500 | 100 |
| Maximum serving rate (events per 1,000 subunits s−1) | 0.032 | 0.009 | 0.012 |
Stability was determined from the dependence of the intrinsic fluorescence on urea concentration (Fig. S2 B). Affinity for ATP-actin monomers was measured from the effect on nucleotide exchange (Fig. 1 C and S2 C). Association equilibrium constants Ka and cooperativity ω were calculated from best fits to equilibrium binding data of Fig. 1 D. Apparent dissociation equilibrium constants Kd were calculated as Kd = 1/(ω*Ka). Association k+ and dissociation k- rate constants for cofilin binding pyrenyl-actin filaments were calculated from kinetic curves of Figs. 1, E–E’, and S2 D. Severing rates were measured by TIRF microscopy (Figs. 1 F and S2 E).