Figure 1.

Design and in vitro properties of G-CatchER⁺

(A) Design of Ca²⁺ binding sites using MUG (MUltiple Geometries) algorithm. EGFP was modified with a direct Ca²⁺ binding site. Both CatchER and G-CatchER⁺ were designed using these techniques (binding site residues are highlighted in pink, residues mutated from CatchER to G-CatchER⁺ are highlighted in red, and Ca²⁺ is represented with the silver ball).

(B) Mutations S175G, S30R, and Y39N were added to CatchER to improve its brightness and thermostability at 37°C. G-CatchER⁺ contains all three mutations. n = 10 for CatchER and G-CatchER⁺. Statistical significance was determined using unpaired student t test. p < 0.0001.

(C) Components of the Ca²⁺ signaling toolkit help shape the spatial-temporal signal. Calcium signaling is mediated by various pumps, channels, receptors, and CaBPs to control intracellular Ca²⁺ release from calcium storage organelles such as the ER/SR. The rapid action of Ca²⁺ release receptors and pumps lining the ER/SR membrane along with the fast conformational changes of some CaBPs rapidly convert the signal for action potential generation in muscle contraction and neuron activation.

(D) Absorbance spectra of 10 μM G-CatchER⁺ sample before titration with 10 μM EGTA (dashed line) and after titrating up to 10 mM Ca²⁺ (solid line). Arrows indicate the increase and decrease in the 488 nm and 395 nm excitation peaks with the addition of Ca²⁺.

(E) Fluorescence increase of G-CatchER⁺ in response to addition of Ca²⁺ excited at 395 nm (blue) and 488 nm (black), respectively, with emission monitored at 510 nm. Slit widths for excitation and emission were 0.25 mm. Inset, Binding curves were fit with a 1:1 binding equation to obtain the K_d.

(F) Comparison of Ca²⁺-associated kinetics of G-CatchER⁺ and G-CEPIA1er.

(G) Fluorescence decay curves of G-CatchER⁺ in both D₂O and H₂O when excited at 372 nm, with and without Ca²⁺.(H) Average lifetimes of G-CatchER⁺ in both D₂O and H₂O when excited at 372 nm, with and without Ca²⁺. Scale bars, 20 μm.

See also Figures S1 and S2 and Tables S1–S3.