Figure 5
From: Structure determination and activity manipulation of the turfgrass ABA receptor FePYR1
ABA binding affinity of FePYR1 and its AtPYL9-like mutants. (A) ABA binding assay of the wild-type FePYR1. 5 μM fluorescently labeled FePYR1 protein was titrated from about 2000 μM to about 4 μM of ABA diluted at 2:3 gradient concentration. The Kd is fitted to 249 ± 14.1 μM. (B) ABA binding assay of the double mutant FePYR1-H73P/L185C. 5 μM of the mutant protein was fluorescently labeled and titrated from about 2000 μM of ABA diluted at 1:2 gradient concentration. The Kd is fitted to 11.6 ± 0.44 μM. (C,D) ABA binding assay of the triple mutants FePYR1-V182L/A179V/F125I and FePYR1-I75V/I127V/E131D. 5 μM fluorescently labeled mutant protein was titrated from about 2000 μM of ABA diluted at 1:3 (C) or 1:2 (D) gradient concentration, respectively. The Kd is fitted to 28 ± 2.3 μM and 3.86 ± 0.28 μM respectively. The analysis of thermophoresis is plotted by normalized fluorescence [‰] and random fluorescence intensity is ruled out when calculating the fit curve.
