Fig. 2

Reducing bPAC dark activity. A Model of bPAC structure (5MBK, 5M2A), a parallel homodimer (chain A and B colored in light and dark shades), marked with the “FAD” chromophore, ATP substrate, and residues of interest. Asterisks indicate residues from the other chain of the dimer. Green spheres represent the two catalytic Mg²⁺ ions. B Point mutations of key residues and their effect on dark and light cAMP concentration in whole oocytes as in Fig. 1A. All bPAC mutants are either tagged with Venus at the N-terminus (S27A, L123R, F198Y, F198W, H266W, and T267Y) or eYFP at the C-terminus (K197A, K197A/D201A, and R278A). C Normalized cAMP production at different time delays in the dark after 500 ms, 473 nm, 0.3 mW mm^-2 light stimulation fitted with a mono-exponential function (bPAC(wt) τ = 10.1 s, Venus-bPAC(F198Y) τ = 4.5 s. D Effect of membrane anchoring on cAMP concentration of whole oocytes in dark and light conditions as in Fig. 1A. E Cytoplasmic and membrane distribution of bPAC variants indicated by Venus fluorescence. Data in B, D, and E are individual values and mean ± SEM. n = 3–6. Control = non-injected oocytes. ***p < 0.0001, *p < 0.05, ns = not significant, Dunnett’s multiple comparisons vs control following one-way ANOVA (p < 0.0001)