FIGURE 3.

Fluorescence properties of YFP-CBD-CFP in vitro. (A) Emission spectra of solubilized YFP-CBD-CFP (excited at 434 nm) in the absence or presence of 1 μM free Ca²⁺. Ca²⁺ addition induced a decrease in the YFP/CFP ratio without altering the spectral properties of YFP-CBD-CFP. (B) Changes in the emission ratio versus Ca²⁺ concentration. Each point represents the average of eight experiments. Emission ratios were normalized to the value obtained in the absence of Ca²⁺ and fit to the Hill function (1/1 + ([Ca²⁺]/K_D)ⁿ), where K_D is the dissociation constant and n the Hill coefficient. The Ca²⁺ titration curve has a K_D of 140 nM and a Hill coefficient of 2.9. Addition of 1 mM Mg²⁺ (▪) induced a shift in the K_D value to 383 nM. (C) Emission spectra of YFP-CBD-YFP (excited at 434 nm) before and after an eightfold dilution. Both spectra were normalized to emission at 514 nm.