Fig. 1

Design, construction and in vitro characterization of NERNST.

a Proposed mechanism of NERNST interaction and electron exchange with NADP(H). Molecular organization of the input (NTRC with NTR and Trx domains) and output (roGFP2) modules of the biosensor, separated by the spacer arm (top, left), and the intra-complex flow of electrons from NADPH to the vicinal dithiols of a second NERNST subunit, as proposed in ref. ²⁷ (top, right). Numbering of active site cysteines corresponds to that of NTRC²⁷. Changes resulting from the NADPH-dependent turnover of NERNST and emission spectra of the fully oxidized (above) and reduced (below) biosensor are shown for comparison (bottom). The color bar reflects the oxidation state (R values) in pseudocolors. This applies to all figures. b Redox state, as estimated by the R values, of NERNST equilibrated with various oxidants and reductants in 100 mM K₃PO₄ pH 7, 150 mM NaCl. DTT and H₂O₂ were used at 10 mM; NADPH, NADH and GSH at 0.5 mM. c The Cys-to-Ser mutant of NERNST (Mut) is not reduced by NADPH. Conditions were those of panel (b). Wt, wild-type biosensor. d Reduction of NERNST by different concentrations of NADPH. e Plot of OxD_NERNST (as determined from the R values) against the redox potential E_NADP(H) (E). The biosensor was equilibrated with different NADPH/NADP⁺ ratios at 0.5 mM or 1 mM total NADP(H) concentration. The upper abscissa represents the ratio of NADPH/NADP⁺. f Effect of pH on the ratiometric behavior of purified NERNST. The biosensor was incubated with 10 mM DTT, 10 mM H₂O₂ or an equimolar NADPH/NADP⁺ mixture of 1 mM NADP(H) in a buffer containing 0.2 M NaH₂PO₄ and 0.2 M Na₂HPO₄ adjusted to the indicated pH. SD values were within the size of the data points. Data shown in (b–f) are means ± SD of 3 independent determinations. ****P ≤ 0.0001; ns, non-significant. b One-way ANOVA followed by Tukey’s multiple comparisons test, c Two-way ANOVA followed by Bonferroni’s multiple comparisons test. Source data, including exact P values, are provided as a Source Data file.