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. 2016 Aug 22;11(10):2854–2864. doi: 10.1021/acschembio.6b00453

Figure 4.

Figure 4

(A) Sensor mechanism of BLZinCh-3 (CER = Cerulean, CIT = Citrine). (B) Bioluminescence emission spectra (normalized to emission at 455 nm) of BLZinCh-3 in Zn2+-depleted (blue) and Zn2+-saturated state (red). The measurement was performed using 0.1 nM protein and 3000-fold diluted furimazine in 150 mM HEPES (pH 7.1), 100 mM NaCl, 10% (v/v) glycerol, 5 μM DTT, 1 mM TCEP, and 1 mg mL–1 BSA, at 20 °C. (C) Bioluminescence emission ratio (emission 500–545 nm/emission 400–455 nm) of BLZinCh-3 in the presence of a range of free Zn2+ concentrations buffered using 1 mM HEDTA, 1 mM DHPTA, 5 mM EGTA, or 1 mM EGTA. Measurements were performed using 0.2 nM protein and 3200-fold diluted furimazine in 150 mM HEPES (pH 7.1), 100 mM NaCl, 10% (v/v) glycerol, 5 μM DTT, 1 mM TCEP, and 1 mg mL–1 BSA, at 23 °C. Data points represent the average of two measurements, and the solid line depicts a fit of the data assuming a single binding event using eq 1, from which the Kd of 15.6 ± 1.0 pM was determined.