Figure 1.

In vitro properties of the purified NCaMP7 indicator. (a) A scheme of the original library for optimization of linkers in the NCaMP7 indicator and a cartoon representation of its crystal structure (PDB ID—6XW2). (b) Absorbance spectra for NCaMP7 in Ca²⁺-bound (10 mM Ca²⁺) and Ca²⁺-free (10 mM EDTA) state at pH 7.2. (c) Excitation and emission spectra for NCaMP7 in Ca²⁺-bound (10 mM Ca²⁺) and Ca²⁺-free (10 mM EDTA) states, pH 7.2. (d) Fluorescence intensity for NCaMP7 in Ca²⁺-bound (10 µM Ca²⁺) and Ca²⁺-free (10 µM EDTA) states as a function of pH. Error bars represent the standard deviation. (e) Ca²⁺ titration curves for NCaMP7 in the absence and in the presence of 1 mM MgCl₂, pH 7.2. Error bars represent the standard deviation. (f) Calcium-association and -dissociation kinetics for the NCaMP7 and control GCaMP6s indicators investigated using stopped-flow fluorimetry. Calcium-association and -dissociation kinetics curves were acquired at 300 nM final and at 1000 nM starting Ca²⁺-free concentrations, respectively. (d–f) Three replicates were averaged for analysis.