FIGURE 1.

FRET-based Ca²⁺-CaM biosensor. A, the domain structure of Ca²⁺-CaM biosensor. Two versions of the biosensor were developed: BsCaM-2 with a high Ca²⁺-CaM binding affinity versus BsCaM-45 with a lower Ca²⁺-CaM binding affinity similar to that of CaMKII. B, diagram showing the Ca²⁺-CaM-dependent FRET changes of the biosensors. C and D, titration of BsCaM-2 (C) and BsCaM-45 (D) in the presence of 50 μm Ca²⁺ with increasing concentrations of CaM. The data were fitted to a standard single-site binding model. The K_d values were obtained as 2.5 nm (BsCaM-2) and 46.3 nm (BsCaM-45), respectively. R is the ratio of F₄₈₀ to F₅₃₀. R_min (the minimal ratio of biosensor in absence of Ca²⁺-CaM) and R_max (the maximal ratio of biosensor saturated with Ca²⁺-CaM) are shown in the insets.