Fig. 2: Effect of QhpG on QhpD-catalyzed thioether bond formation in QhpC and inter-protein interactions among QhpCDG proteins.

a MALDI-TOF mass spectra are shown for IAA-treated products (crosslinked QhpC) of the QhpD-catalyzed thioether bond formation in the presence of an equimolar amount of QhpG (top panel) and in its absence before (middle) and after (bottom) the QhpD reaction. Inset: Calculated m/z values of 1–4 acetamidated (AA) peptides (monoprotonated form). Intensity is expressed in arbitrary units (arb. units) for all mass spectra. Mobility shift assays for interactions between QhpG and the QhpCD binary complex (b) and between QhpG and QhpC (d). Indicated amounts (pmol) of respective proteins were applied in each lane. In b, d, the experiments repeated twice independently gave similar results. c BLI assays for interactions between QhpG and linear (left) and crosslinked (right) QhpC immobilized on the biosensor tip. The analyte solution (4 μl) contained QhpG at 1.0 μM (magenta), 0.50 μM (blue), 0.25 μM (green), and 0.13 μM (orange) for linear QhpC (left) and at 62.5 nM (magenta) and 31.3 nM (blue) for crosslinked QhpC (right). Binding-induced changes in wavelength (nm) of the transmitted light were recorded for measuring time (s). Thin black curves represent the theoretical fitting of the calculated data (Supplementary Table 1).