. (A) Schematic illustration of a hybrid-type ATP sensor composed of the ε subunit of FoF1-ATP synthase having a cysteine point mutation at which a small-molecule fluorophore is conjugated. (B) Results of HyFInD screening. Bars indicate fluorescence changes (ΔF/F0) of fluorescence conjugates labeled with OG (upper left), Alexa488 (upper right), Cy3 (bottom left), and SiR650 (bottom right) in response to 2 mM ATP. The numbers 1–134 on the horizontal axis represent the positions in the amino-acid sequence at which cysteine point mutation is introduced for fluorophore labeling. The fluorescent conjugates indicated by red bars were tested in the second screening. (C) Normalized excitation (blue) and emission spectra (red) of ATPOS with (solid line) or without 1 mM ATP (dashed line). (D) Dose-response curves of ATPOS to ATP, ADP, AMP, adenosine, CTP, GTP, and UTP (n = 3). Plots were fitted with the Hill equation. (E) The pH dependence of ATPOS. Fluorescence intensities normalized to the fluorescence intensity at pH 7.4 without ATP (F/F0) are plotted as a function of pH (n = 3). Data are presented as mean ± SEM. In some figures, error bars representing SEM are smaller than the symbols indicating the mean.