Figure 2. Metal binding properties of MagFRET-1.

(A) Normalized fluorescence emission spectra of MagFRET-1 at 0 and at 16 mM Mg²⁺ after excitation at 420 nm. (B, C) Emission ratio (Citrine to Cerulean) of MagFRET-1 as a function of the Mg²⁺ (B) or Ca²⁺ (C) concentration. Solid lines indicate a fit to a single (B) or a double (C) binding event, yielding a K _d of 0.15±0.02 mM for Mg²⁺ and K _d's of 10±4 µM and ∼35 mM for Ca²⁺, respectively. (D) Emission ratios of MagFRET-1 in absence of metal, in the presence of 10 µM Ba²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Fe³⁺, and in the presence of the same metals and 1 mM Mg²⁺. Measurements were performed in triplicate, error bars indicate SEM. All measurements were performed in 150 mM Hepes (pH 7.1), 100 mM NaCl and 10% (v/v) glycerol with 0.2 µM sensor protein.