Sensor properties of the different MagFRET variants.

1<p>Mutations introduced in the first or second 12-residue metal binding loops of HsCen3 are indicated in bold and are underlined.</p>2<p>The dissociation constant (<i>K</i>_d) for each variant's Mg²⁺ and first Ca²⁺ binding event is indicated, together with the standard error (SE).</p>3<p>A binding event's dynamic range (D.R.) is defined as the difference in emission ratio between the unbound and fully metal bound form divided by the emission ratio in the unbound form, multiplied by 100%.</p

Metal binding properties of MagFRET-1.

<p>(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 <i>K</i>_d of 0.15±0.02 mM for Mg²⁺ and <i>K</i>_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.</p

Design of the genetically encoded magnesium FRET sensor MagFRET.

<p>(A) Crystal structure (PDB code 2GGM) of HsCen2 in the calcium-bound, compact state. The typical helix-loop-helix structure can be observed, with EF-hands indicated by Roman numerals. The dotted lines indicate the N-terminal truncated part of the domain used in the sensor. In HsCen3, the high-affinity Mg²⁺/Ca²⁺ binding site is in loop I, and a much weaker Ca²⁺-binding site is found in loop II. (B) Schematic representation of MagFRET, where the N-terminal truncation of HsCen3 is flanked by Cerulean and Citrine. In absence of Mg²⁺, the HsCen3 domain is in a molten globule-like state, with little tertiary structure and a relatively large average distance between the fluorescent domains. Mg²⁺-binding induces a compact, well-defined tertiary structure, resulting in increased energy transfer between Cerulean and Citrine.</p

<i>In situ</i> characterization of MagFRET-1 in HEK293 cells.

<p>(A–D) Confocal fluorescence microscopy images showing HEK293 cells expressing MagFRET-1 (A, B) and MagFRET-1-NLS (C, D) showing Cerulean (A,C) or Citrine emission (B, D). (E, F) Investigation of MagFRET-1's <i>in situ</i> Ca²⁺ sensitivity. (E) Emission ratio over time of intact HEK293 cells expressing MagFRET-1 measured by widefield fluorescence microscopy. At t = 120 s, 50 µM of PAR-1 agonist peptide was added to activate Ca²⁺ signaling. (F) To confirm Ca²⁺ signaling took place in stimulated cells, the fluorescence intensity of intact HEK293 cells loaded with Ca²⁺-dye Oregon Green–BAPTA was followed. At t = 120 s, 50 µM of PAR-1 agonist peptide was added to activate Ca²⁺ signaling, and at t = 240 s, 20 µM A23187 was added. In E and F, each trace represents the response of an individual cell, with ratio (E) or intensity (F) normalized to the value at t = 0 s. (G, H) Response of MagFRET-1 expressed in permeabilized HEK293 cells to changes in [Mg²⁺]. MagFRET-1 emission ratio was followed over time as the concentration of MgCl₂ (G) or EDTA (H) was increased, as indicated on the panels. (I, J) Response of negative control construct Cerulean-linker-Citrine expressed in permeabilized HEK293 cells to changes in [Mg²⁺]. To maintain an isotonic solution, the increase in Cl⁻ concentration due to addition of MgCl₂ was compensated for by reducing the KCl concentration in the buffer. Prior to imaging, cells were permeabilized using 10 µg/mL digitonin. Traces in G to J represent averages of at least 9 cells, error bars indicate SEM, ratios were normalized to the emission ratio at t = 0.</p