The N-terminal His-tag of the regulatory C-terminus of KEA3 or a mutant carrying a G65>A point mutation was labelled with the RED-tris-NTA kit (NanoTemper Technologies GmbH, Munich, Germany). After 10 minutes incubation on ice, 50 nM of protein sample in PBS together with the indicated concentrations of nucleotides were loaded into standard Monolith NT.115 capillaries (NanoTemper Technologies GmbH, Munich, Germany). TRIC measurements were performed using the Monolith NT.115 instrument (NanoTemper Technologies GmbH, Munich, Germany) at ambient temperature of 23 °C. Instrument parameters were adjusted to 80 % LED and 60 % MST power. TRIC as differences in normalized fluorescence was calculated between the initial fluorescence and 1.5 s after the onset of the laser. 10 mM nucleotide stocks were prepared freshly in PBS pH 8.0 directly before the experiment and the pH was checked.