150 µM of non-tagged RhlR protein purified in the presence of mBTL was first buffer-exchanged into labeling buffer (50 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol, 50 µM mBTL) using Zeba Spin desalting columns. RhlR was then mixed with a 1.5-fold molar excess of ATTO488-maleimid and incubated on ice for 2 h in the dark. Conjugated protein was separated from unreacted dye by gel filtration using a NAP-5 desalting column (Cytiva) and buffer exchanged to RhlR buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 50 µM mBTL). The purified protein was then aliquoted and flash frozen in liquid nitrogen for storage at −80 °C until further use.
Before every measurement, a single aliquot of RhlR and PqsE was thawed and briefly centrifuged (10 min, 17.000 × g). PqsE was diluted 1:1 into assay buffer (50 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 50 µM mBTL, 0.05% Tween) starting from 10 µM and mixed with an equal volume of RhlR-ATTO488 (final concentration: 20 nM). Samples were then incubated for 10 min at RT, loaded into standard-treated capillaries and measured using a Monolith NT.155 (NanoTemper Technologies GmbH) at 25 °C. For the evaluation, samples with irregular MST traces (notable aggregates) were omitted.
Before every measurement, a single aliquot of RhlR and PqsE was thawed and briefly centrifuged (10 min, 17.000 × g). PqsE was diluted 1:1 into assay buffer (50 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 50 µM mBTL, 0.05% Tween) starting from 10 µM and mixed with an equal volume of RhlR-ATTO488 (final concentration: 20 nM). Samples were then incubated for 10 min at RT, loaded into standard-treated capillaries and measured using a Monolith NT.155 (NanoTemper Technologies GmbH) at 25 °C. For the evaluation, samples with irregular MST traces (notable aggregates) were omitted.