FIGURE 4.

Mutation of aspartates 447 and 498 alters Ca²⁺ dependency of FRET signals. (A) Changes in FRET value versus free Ca²⁺ concentrations for WT and mutants D447V and D498I. Emission ratios were normalized to the value obtained in the absence of Ca²⁺ and fitted to a single Hill function for wild-type and to the sum of two Hill functions for mutants D447V and D498I. K_D values are WT, 140 nM; D447V, 1.2 and 2.5 μM; and D498I, 1.2 and 130 μM. Each point is the mean of eight experiments for WT, five experiments for D497V, and six experiments for D498I. (B) The graph shows the total change in FRET signal (ΔFRET) for the WT and mutants D447V and D498I. Values are WT = 0.109 ± 0.005 (n = 8), D447V = 0.091 ± 0.003 (n = 5), and D498I = 0.075 ± 0.005 (n = 5). ΔF is the difference between the YFP/CFP value in 0 Ca²⁺ and YFP/CFP value in presence of high [Ca²⁺].