Fluorescence Polarization (FP) Binding Assays.

Protein aliquots were thawed and mixed 1:1 with glycerol free buffer (300 mM NaCl, 50 mM HEPES, pH 7.5) to obtain stock solutions at 5% glycerol. The proteins were then concentrated using Amicon centrifugal filter units with suitable MW cut-offs to the concentrations needed for the experiments. A 1:1 dilution series spanning a total of 16 different concentrations was set up for each protein in assay buffer (300 mM NaCl, 50 mM HEPES, 5% glycerol, pH 7.5). In wells of a 384-well solid black polystyrene microplate (Corning), 5 μL of a 1 μM FITC-SgbA(leader) or fluorescein-AciA(leader) stock solution was mixed with 45 μL of the protein solutions. For each dilution series, a reference containing 5 μL of fluorescein-labeled peptide mixed with 45 μL assay buffer was also set up, to ensure that the highest protein dilutions have the same FP values as the buffer control. All assays were set up in triplicates.

After mixing, samples were equilibrated for 30 min at RT until parallel and perpendicular fluorescence intensities were measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm with a bandwidth of 20 nm using a Synergy H4 Hybrid Reader (BioTek). To calculate the FP, the perpendicular fluorescence intensity was first subtracted from the parallel one and the result was then divided through the sum of both fluorescence intensities (all collected data is shown in Supporting Information Tables 4a–m). For completed binding curves, the K_d values were determined in OriginPro2017 (OriginLab) by using a non-linear dose-response fit after plotting the normalized FP (i.e. the measured FP minus the mean FP of the buffer references) against the log of the protein concentrations.