Fig. 5.

Effects of RLC phosphorylation on ATPase activity and motility. (A) Steady-state ATPase of unphosphorylated (blue) or phosphorylated (red) Myo2 in the absence or presence of tropomyosin (Tpm). Circles and squares represent independent preparations of N-FLAG-Myo2-C-Biotin. Diamonds represent N-FLAG-Myo2. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl₂, 1 mM ATP, and 2 mM DTT. Tpm was added at a 2:1 Actin-Tpm molar ratio. (B) In vitro motility speeds of unphosphorylated or phosphorylated Myo2, in the absence or presence of Tpm. Speeds represent motility data from two experimental repeats conducted in parallel with two independent preparations of N-FLAG-Myo2-C-Biotin (n, number of moving filaments). To ensure that all heads were available for interaction with actin, Myo2 was attached to neutravidin-coated coverslips by a biotin tag at its C terminus. Conditions were as follows: 30 °C, 0.5% methylcellulose, 25 mM imidazole, pH 7.4, 50 mM KCl, 4 mM MgCl₂, 1 mM EGTA, 1 mM ATP, and 10 mM DTT. When indicated, Tpm was added to a final concentration of 2 μM.