Fig. 2

Fig. 2

a In vitro affinity titration fitted curves of recombinant purified GreenT-EC and two variants with higher (GreenT-EC.b) and lower (GreenT-EC.c) affinity. b Off-rate kinetics of the three variants of the sensor. The proteins were prepared in MOPS 30 mM, KCl 100 mM, Mg 2.5 mM, Ca²⁺ 100 mM and rapidly mixed with BAPTA 200 mM. c Fitted curves of pka measurements for the three sensors studied in this article. Measurements were obtained by preparing pH solutions in MOPS/MES containing 100 mM Ca²⁺. d On-rate kinetics of GreenT-EC (green), GreenT-EC.b (blue) and lower affinity GreenT-EC.c (yellow) were measured by rapidly mixing solutions of protein in MOPS 30 mM, KCl 100 mM, Mg 1 mM with MOPS 30 mM, KCl 100 mM, Mg²⁺ 1 mM containing increasing concentration of Ca²⁺. e A double exponential was used for the fittings for each variant and the obtained Kon values were plotted against the calcium concentration. K_obs1 corresponds to the first step of the rate, representing 25–30% of the total response. K_obs2 corresponds to the slower process and accounts for the higher percentage of the total response. In all cases, a decrease in the K_obs is observed at increasing concentrations of calcium. This suggests a mechanism by which there is an equilibrium between at least two species of the sensor, and only one can progress to a fluorescent species upon calcium binding. Technical replicates of proteins produced and purified on the same day were used for all fittings. Source data are provided in the Source Data file.