FIGURE 1.

Schematic of sensor function and in vitro characterization. A, mechanism for the sensor response to Zn²⁺. In the absence of Zn²⁺, the sensing domain is unstructured, whereas in the presence of Zn²⁺, it becomes structured leading to a change in the distance and orientation between CFP and YFP. B, emission spectrum of the His₄ sensor as a function of Zn²⁺ upon excitation of CFP at 420 nm. The arrows indicate the decrease in CFP fluorescence intensity and an increase in YFP fluorescence intensity with increasing Zn²⁺ concentrations. The Zn²⁺ concentrations were as follows: 0 μm (long dash), 70 μm (short dash), 180 μm (medium dash), solid line (1000 μm). C and D, Zn²⁺ binding curves for the Cys₂His₂ (●), His₄ (■), and Ala₄ (♦) sensors. The FRET ratio change (R − R_min) is plotted as a function of Zn²⁺ for each sensor. The lines represent fits to a single site binding equation and yield the following dissociation constants: K′_d = 1.7 ± 0.2 μm (●) and 160 ± 4 μm (■). The FRET ratio (R) was calculated as Em₅₂₉/Em₄₇₅ upon 420-nm excitation.