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Fig. 3. Coronin 1B preferentially binds to ATP/ADP-P_i–F-actin and mutation of R30 abolishes binding. (A) Representative Coomassie-Blue-stained gel showing the near-saturation binding of coronin 1B to ATP/ADP-P_i–F-actin (1 µM total actin). Upper band, coronin 1B; lower band, actin. Numbers below bands indicate protein concentrations in µM as determined by densitometry. (B) Representative Coomassie-Blue-stained gel showing F-actin binding of coronin 1B at indicated KCl concentrations (1.1 µM total actin, 1.3 µM coronin 1B). (C,D) Representative immunoblot showing F-actin bound coronin 1B in the pellets of high-speed co-sedimentation experiments (top panel); Coomassie-Blue-stained gel show the corresponding actin pellets (bottom panel). Identical amounts of coronin 1B (0.1 µM) were used and concentrations of total ADP- and ATP-G-actin used in each lane are indicated above the blots. In this experiment, the ATP G-actin used was recharged from ADP–G-actin as described previously (Pollard, 1986). (E) Equilibrium binding of coronin 1B to ADP or ATP/ADP-P_i–F-actin filaments. Bound coronin 1B was calculated from the depletion of coronin 1B from the supernatant fraction of experiments similar to those shown in C and D, and quantified by densitometry. Three different protein preparations were used to generate the data points, which are presented as means ± standard errors of the mean (±s.e.m.). (F) Representative Coomassie-Blue-stained gels comparing F-actin-binding capability of wild-type coronin 1B and R30D mutant. (G) Equilibrium binding of wild-type coronin 1B or R30D mutant to ATP/ADP-P_i–F-actin (1.5 µM F-actin in pellets).

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