Extended Data Fig. 3: The kinetics of the H2O2 response of HyPerFLEX depend on the protonation state of Cys121, with Phe130 being essential for its functioning.
From: A color-tailored fluorogenic sensor for hydrogen peroxide
a, HyPerFLEX is a dimer, as determined by size exclusion chromatography. The elution profile and calibration curve are shown, with HyPerFLEX represented by the red point. b, The KD of fluorogen binding to HyPerFLEX is in the low micromolar range for all fluorogens, regardless of oxidation state. For each fluorogen, intensity at the excitation peak was normalized to the maximum and minimum intensity. KD and Hill coefficient (h) values were obtained by fitting the data to a “specific binding with Hill slope’ model. The mean +/- SEM of at least n = 3 independent experiments is shown. The 95% confidence interval is indicated with dashed lines. c, Anti-HyPer7 western blot for HyPerFLEX and its mutants, along with the corresponding post-transfer Ponceau stain. d, The F130A mutation does not affect the secondary structure of HyPerFLEX or structural changes upon oxidation. CD spectra of reduced and oxidized HyPerFLEX WT and F130A are shown. The data are the mean of n = 3 independent experiments. e, The F130A mutant displays a similar KD for WT HyPerFLEX for HBR-3,5-DOM. The intensity at the excitation peak was normalized to the maximum and minimum intensity. KD and Hill coefficient (h) values were obtained by fitting the data to a “specific binding with Hill slope’ model. The mean +/- SEM of n = 2 independent experiments is shown. The 95% confidence interval is indicated with dashed lines. f, Diagnostic residual plots from fitting the reaction curves of HyPerFLEX with Η2Ο2 to one-phase or two-phase exponential models (Fig. 3e, f). g, kobs values vs Η2Ο2 concentration obtained from fitting response curves of the HyPerFLEX C261S mutant to at least a 10x higher Η2Ο2 concentration. The mean of n = 3 independent experiments +/- SEM, is shown. h, Diagnostic residual plots from fitting the reaction curves of HyPerFLEX C261S mutant exposed to a 10x lower molar excess of Η2Ο2 to a one-phase exponential model (Fig. 2f).
