Kinetics of the redox reactions in STEAP1 and STEAP2
Data files
Nov 08, 2023 version files 4.25 MB
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Figure2-FigureSupplement1.xlsx
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Figure2.xlsx
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Figure3-FigureSupplement1.xlsx
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Figure3-FigureSupplement2.xlsx
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Figure3.xlsx
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Figure4-FigureSupplement1.xlsx
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Figure5.xlsx
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README.md
Abstract
The data repository includes the kinetic data of the redox reactions in STEAP1 and STEAP2, which are presented in "Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2" by Kehan Chen, Lie Wang, Jiemin Shen, Ah-lim Tsai, Ming Zhou and Gang Wu. The data include: 1) the reduction of ferric STEAP1 by reduced FADH2 and ferrous STEAP2; 2) the reduction of ferric STEAP1 by cytochrome b5 reductase; 3) the reduction of ferric STEAP2 with NADPH; and 4) the oxidation of ferrous STEAP1 and STEAP2 by ferric.NTA.
README: Kinetics of the redox reactions in STEAP1 and STEAP2
https://doi.org/10.5061/dryad.00000008r
The kinetics data are stored in Excel files and the filenames correspond to the figures in "Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2" by Kehan Chen, Lie Wang, Jiemin Shen, Ah-lim Tsai, Ming Zhou and Gang Wu, https://doi.org/10.7554/eLife.88299.1. In each data file, the title of each sheet indicates the panel in the figure and the reaction.
The structures of different types of kinetic data are described as follows.
- Time-dependent spectral change (Figure2, Figure2-FigureSupplement1, Figure3, Figure3-FigureSupplement1, Figure4-FigureSupplement1) The first column is the wavelength and the spectra are listed continuously from column 2. The spectrum acquired at the first time point is always in column 2 and marked "spectrum#1" in the top, and the temporal sequence of the spectra are marked in the top of each column. The acquisition time points of the spectra are listed in the two columns following the last spectrum.
- Time courses of the Soret absorbance of ferrous heme (Figure3-FigureSupplement1, Figure5) The data is presented in two columns, the left one is the time points (in second) and the right is the absorbance at 427 nm (A427).
- Rate-dependence on the concentration of reduced FADH2 or ferric.NTA (Figure2, Figure2-FigureSupplement1, Figure5) The left column is the concentration of reduced FAD (FADH2) (in uM) or ferric.NTA (Fe3+.NTA) (in uM), followed by the columns of the rate constants, kobs (in s-1) of the fast phase, kobs_1 and the slow phase, kobs_2.
- Bio-layer Interferometry (Figure3-FigureSupplement2). The profiles of association and dissociation are labeled with the concentration cytochrome b5 reductase (b5R) in the top and the response (Red) is listed versus the concentration of b5R (in uM).
The data sheets in each file are listed below.
1. Figure 2
a. "PanelA STEAP1+FADH2": the spectral change during the reaction of STEAP1 with reduced FAD (FADH2); Panel A in Figure 2.
b. "PanelBkobs(STEAP1+FADH2)vsFADH2": the dependence of the rate constants, kobs in the reaction of STEAP1 with FADH2, on the concentration of FADH2; Panel B in Figure 2.
c. "PanelC STEAP1-STEAP2+NADPH": the spectral change in the reaction of the mixture of STEAP1 and STEAP2 with NADPH; Panel C in Figure 2.
2. Figure 2-FigureSupplement1
"PanelC L230GSTEAP1+FADH2": the spectral change in the reaction of STEAP1 Leu230Gly mutant with reduced FAD (FADH2) and the dependence of rate constants, kobs, on the concentration of FADH2; Panel C in Figure 2-figuresupplement1.
3. Figure 3
a. "PanelA STEAP1-b5R+NADH": the spectral change in the reaction of STEAP1 plus cytochrome b5 reductase (b5R) with NADH; Panel A in Figure 3.
b. "PanelB L230GSTEAP1-b5R+NADH": the spectral change in the reaction of STEAP1 Leu230Gly mutant plus b5R with NADH; Panel B in Figure 3.
4. Figure 3-FigureSupplement1
a. "PanelA": the time courses of A427 in the reaction of STEAP1 only with NADPH and STEAP1 plus b5R with NADH; Panel A in Figure 3-
figuresupplement1.
b. "PanelB" the spectral change in the reaction of STEAP1 plus FAD with NADPH; Panel B-figuresupplement1.
5. Figure 3-FigureSupplement2
"Sheet1": (1) the bio-layer interferometry profiles in the binding of b5R to STEAP1; Panel A in Figure 3-figuresupplement2; (2) the dependence of
response on the concentration of b5R; Panel B in Figure 3-figuresupplement2.
6. Figure 4-FigureSupplement1
a. "PanelA STEAP2+NADPH": the spectral change in the reaction of STEAP2 plus FAD with NADPH; Panel A in Figure 4-figuresupplement1.
b. "PanelB STEAP2+FADH2": the spectral change in the reaction of STEAP2 with reduced FAD (FADH2); Panel B in Figure 4-figuresupplement1.
7. Figure 5
a. "PanelA&B STEAP1+Fe.NTA": (1) the time courses of A427 in the reaction of ferrous STEAP1 with Fe3+.NTA; Panel A in Figure 5; (2) the
dependence of the rate constants, kobs, on the concentration of Fe3+.NTA; Panel B in Figure 5.
b. "PanelC&D STEAP2+Fe.NTA": (1) the time courses of A427 in the reaction of ferrous STEAP2 with Fe3+.NTA; Panel C in Figure 5; (2) the
dependence of the rate constants, kobs, on the concentration of Fe3+.NTA; Panel D in Figure 5.
Methods
All the data was collected under anaerobic conditions. The time-dependent optical changes in the absorbance of the heme in STEAP1 and STEAP2 were monitored using either a diode-array UV-Vis spectrophotometer or a stopped-flow machine with a rapid-scan accessory. In each spectrum, the background absorbance is subtracted. The absorbance at 800 nm is taken as the background in the spectra acquired on the spectrophotometer and the absorbance at 703–708 nm is taken as the background in the data obtained using the stopped-flow rapid-scan method. The time courses of the Soret wavelength of the heme were either extracted from the data of the time-dependent spectral change or acquired on the stopped-flow machine operated in the single wavelength mode.
For more experimental details, please refer to the Method section in "Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2" by Kehan Chen, Lie Wang, Jiemin Shen, Ah-lim Tsai, Ming Zhou and Gang Wu.