Fig. 1: NAPstars respond specifically to changes in the NADP redox state.

a Diagram showing the development of selected NAD and NADP sensors including NAPstars. b AlphaFold2 prediction of NAPstar structure. Graphs showing the normalised logarithm of the cpT-Sapphire/mCherry fluorescence ratio at c different NADPH/NADP⁺ and d NADH/NAD⁺ ratios. NADPH and NADH concentration was titrated against a fixed background of 150 µM NADP⁺ and 500 µM NAD⁺ respectively. In c and d, the dashed lines show a fitted sigmoidal function that was used to determine K_d(NAD(P)H). e Table summarising the determined K_d(NADPH) and K_d(NADH) values of all NAPstars. Graphs showing the normalised logarithm of the cpT-Sapphire/mCherry fluorescence ratio at f different NADPH/NADP⁺ and g NADH/NAD⁺ ratios. NADP⁺ and NAD⁺ concentration was titrated against a fixed concentration of NADPH and NADH respectively that for each probe corresponded approximately to the determined K_d(NADPH) and K_d(NADH) values. This experimental regime, by definition, only allows a maximum of approximately 50% NADPH binding and explains the difference in shape of the titration curve between panels c and f. h Table summarising the determined K_{r(NADPH/NADP+)} for all NAPstars. For panels c, d, f, g, n = 3 technical replicates. Data are presented as mean ± s.d. normalised to the lowest data point.