S100A4 single-chain EF-hand mutant proteins only weakly promote myosin-IIA disassembly. (A) Fluorescence anisotropy measurements of sc-S100A4–5M1 (solid gray line), sc-S100A4–10M1 (solid black line), sc-S100A4–5M2 (dashed gray line), and sc-S100A4–10M2 (dashed black line) demonstrated that sc-S100A4-M1 and -M2 EF-hand mutants have a 30–60-fold reduced affinity for FITC-MIIA1908–1923. Values represent the mean ± standard deviation from two or three independent experiments. (B) Myosin-IIA disassembly assays. Myosin-IIA disassembly in the absence of any added S100A4 (light gray bar). Ca2+-dependent myosin-IIA disassembly in the presence of wt-S100A4 (black bars) and EF-hand mutant proteins sc-S100A4–5M1 (white bars), sc-S100A4–5M2 (white-hatched bars), sc-S100A4–10M1 (dark gray bars), sc-S100A4–10M2 (dark gray-hatched bars), and wt-S100A4-E33A/E74A (white-lined bars). Values represent the mean ± standard error of the mean for three independent experiments.