Extended Data Fig. 3: Engineering and characterization of ChemoX-CaM calcium sensors.
From: A general method for the development of multicolor biosensors with large dynamic ranges
a. Fluorescence intensity (FI) emission spectra of eGFP-CaM/M13-HT7 labeled with SiR in presence of different concentrations of free Ca2+. Shown are the means of 3 technical replicates. b. FRET/eGFP ratios of calcium sensors differing in the number of interface mutations (Supplementary Table S5) in presence (39 μM) or absence (0 μM) of free Ca2+. Sensors were labeled with SiR. The variant corresponding to the final calcium sensor ChemoG-CaM is marked with an asterisk. Shown are the means ±s.d. n = 4 technical replicates. c. Maximal FRET/eGFP ratio changes (MaxΔR/R0) of calcium sensors differing in the number of interface mutations (Supplementary Table S5). Sensors were labeled with SiR. The variant corresponding to the final calcium sensor ChemoG-CaM is marked in red and with an asterisk. Shown are the means ±s.d. n = 4 technical replicates. d, g, j. Ca2+ titrations of SiR-labeled calcium sensors differing in the number of interface mutations (d, Supplementary Table S5), ChemoG-CaM labeled with different fluorophores (g, Supplementary Table S6) or ChemoX-CaM calcium sensors labeled with SiR (j, Supplementary Table S5). Shown are the means ±s.d. n = 3 technical replicates. e, f. Fluorescence intensity (FI) emission spectra of ChemoG-CaM labeled with TMR (e) or CPY (f) in presence (39 μM) or absence (0 μM) of free Ca2+. Shown are the means of 3 technical replicates. h, i. Fluorescence intensity (FI) emission spectra of ChemoB-CaM (h) or ChemoR-CaM (i) labeled with SiR in presence (39 μM) or absence (0 μM) of free Ca2+. Shown are the means of 3 technical replicates. k. pH sensitivity of the FRET/eGFP ratio of ChemoG-CaMSiR in presence (2 mM CaCl2, +Ca2+) or absence (2 mM EGTA, -Ca2+) of free Ca2+. Shown are the means ±s.d of three technical replicates.
