Representative curves showing binding of S1-ADP to actin-tropomyosin-troponin in the absence of Ca2+ at 120 mM ionic strength. Binding curves for each mutant were repeated three to four times. (A) The binding of WT (circles) and mutant 45 (squares) with increasing S1-ADP concentrations; (B) WT (circles) and mutant 43/45 (squares); (C) WT (circles) and mutant 43/45/144 (squares). Insets show saturation: x axis, [S1-ADP]Free (0–1 μM); y axis, ActinBound/ActinTotal (0–1.1). Conditions are: 25°C, pH 7, 0.075 μM phalloidin-actin, 0.2 mg/mL BSA, 14 units/mL hexokinase, 1 mM glucose, 20 μM AP5A, 2 mM ADP, 20 mM MOPS, 5 mM MgCl2, 88 mM NaCl, 1 mM dithiothreitol, 1 mM EGTA. Actin/tropomyosin/troponin was 1:1:1. Light scattering was used to measure the binding with excitation at 340 nm and emission at 360 nm.