Mass photometry

MP experiments were performed using a OneMP mass photometer (Refeyn Ltd, Oxford, UK). Data acquisition was performed using AcquireMP (Refeyn Ltd. v2.3). MP movies were recorded at 1 kHz, with exposure times varying between 0.6 and 0.9 ms, adjusted to maximize camera counts while avoiding saturation. Microscope slides (70 × 26 mm²) were cleaned for 5 min in 50% (v/v) isopropanol (HPLC grade in Milli-Q H₂O) and pure Milli-Q H₂O, followed by drying with a pressurized air stream. Silicon gaskets to hold the sample drops were cleaned in the same manner and fixed to clean glass slides immediately prior to measurement. The instrument was calibrated using the NativeMark Protein Standard (Thermo Fisher Scientific) immediately prior to measurements. The concentration during measurement of Aq880, Aq880 mutants, or Hhal2243 during measurements was typically 100 nM. Each protein was measured in a new gasket well (i.e., each well was used once). To find focus, 18 µL of fresh buffer (10 mM Tris pH 8.0, 100 mM KCl, and 0.1 mM EDTA) adjusted to room temperature was pipetted into a well, the focal position was identified and locked using the autofocus function of the instrument. For each acquisition, 2 µL of diluted protein was added to the well and thoroughly mixed. For each sample, three individual measurements were performed. The data were analyzed using the DiscoverMP software.