Figure 1: In vitro characterization of calredoxin.
(a) Kd estimation for Ca2+-binding by MST measurements. Fluorescently labelled recombinant WT CRX (CRX) was incubated with defined concentrations of free Ca2+ (filled circles) or Mg2+ (open circles) and the ratio of detected fluorescence before and after the thermophoretic movement was plotted against the corresponding cation concentration. Data for CRX incubated with Ca2+ were fitted according to the law of mass action (black line) and gave a Kd of 88.2 nM±16.5 nM. Each data point represents the mean value of at least three experiments (±s.d.). (b) CRX shows Ca2+-dependent redox activity. 10 μM recombinant WT CRX (closed circles) was reduced by E. coli NTR and NADPH in defined Ca2+ concentrations for 10 min at RT. 200 μM DTNB were added as substrate for reduction by CRX and the increase in absorption at 412 nm was recorded to calculate the redox activity (slope 0–80 s after addition of DTNB). Data were normalized on the highest activity measured for each protein purification and fitted by Michaelis–Menten kinetics (Kd: 281.1±153.8 nM). Error bars represent s.d. of three independent measurements. Assay modified after ref. 62. (c) Oxidation–reduction titration of WT CRX. The disulfide/dithiol redox state at each Eh value was monitored using the monobromobimane fluorescence method. The line represents a fit of the data to a two-electron Nernst curve and yielded an Eh of −288.2±5.3 mV. Data were acquired in two independent experiments.
