Fig. 3: Phosphorylation of C-terminal peptides display different kinetics for individual phosphorylation sites.

Phosphorylation reaction of Rho Cterm, catalyzed by GRK1 (a), β₁AR Cterm, catalyzed by GRK2 (b), and β₂AR Cterm, catalyzed by GRK2 (c). Peak integrals of individual sites are plotted over time (left). Peak integrals were normalized to their individual 100% phosphorylation level according to Theillet et al.⁵¹ Data points were fitted to first-order kinetics (solid lines). Data which could not be fitted with classical first-order kinetics were subjected to analysis based on formulas 1.1–1.5 (Supplementary Fig. 1). Fits are depicted with dashed lines (see Fig. 4 and Supplementary Fig. 1 for details). Respective rate constants are summarized in Supplementary Table 5. On the top right of a–c, 2D [¹⁵N,¹H]- HMQC spectra displaying phosphorylated residues at individual timepoints are depicted. The last spectrum of each time-series displays the respective peak assignments. Sequence of C-terminal constructs is shown on the bottom right of a–c. Possible phosphorylation sites are marked in red (serines) or blue (threonines). Phosphorylation occurring in this assay are highlighted in yellow.