Fig. 2: Enzymatic activity of PPIases and their effect on SthK.
From: Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel
a Quenching kinetics from the Tl+ flux assay for WT SthK + 100 µM cAMP after incubation for 12 ms (black) and 2.5 s (teal) and after 12 ms in the presence of 1 µM CypD (purple) or 1 µM SlyD (orange). b Rate constants (Eqs. (2) and (3)) obtained from kinetics as shown in (a) for WT SthK in the presence of 100 µM cAMP and 1 µM CypD (purple, n = 3 independent experiments) or 1 µM SlyD (orange, n = 6 independent experiments). Dashed line represents the activation of SthK in the absence of PPIases, taken from Fig. 1f. c Quenching kinetics from the Tl+ flux assay performed in the presence of different concentrations of CypD (0 M CypD—black, 10−10 M—yellow, 10−7 M—cyan, 10−5 M—pink). Kinetics were obtained after 12 ms incubation with 100 µM cAMP. d Normalized rate constants (Eqs. (2) and (3)) from kinetics as shown in (c) for CypD (purple, n = 4 independent experiments) and SlyD (orange, n = 4 independent experiments). Data were analyzed using Eq. (4) yielding EC50 values of ~30 nM. e Normalized rate of Tl+ flux for SthK after activation by 100 µM cAMP for 12 ms in the presence of 1 µM CypD and increasing concentrations of CsA (n = 3 independent experiments). Data were fitted to Eq. (5) giving IC50 = 4 ± 1.6 µM, s = 0.9 ± 0.2. f Catalytic activities kcat/KM of SlyD and CypD from a peptide-based isomerization assay. Values, plotted as bars, are obtained from fits of data points as shown in Supplementary Fig. 3b for the substrates Abz-ALPF-pNa (filled bars) and Abz-AEPF-pNa (hatched bars). The assays were performed in 20 mM Hepes, 100 mM KCl, pH 7.4 (gray) and 10 mM Hepes, 140 mM KNO3, pH 7.4 (navy). Numerical values together with the S.E. of fits are also in Supplementary Table 1. Symbols in (b, d, e) are mean ± S.D., in (f) fitted values ± S.E. of fits. Source data are provided as a Source Data file.
