Microscale Thermophoresis (MST)

Purified RTA was labeled according to the manufacturer’s instructions with a Monolith NT RED NHS NT647 labeling kit (NanoTemper Technologies) using red fluorescent dye NT-647 N-hydroxysuccinimide (amine-reactive). The fluorescent dye to protein ratios were determined using photometry at 650 and 280 nm. Labeling reagents were removed by buffer-exchange column chromatography into MST reaction buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 0.05% Tween-20. The RTA protein samples were titrated with a serial twofold dilution with 1 mg/mL of bovine albumin. The concentration of labeled proteins was adjusted to approximately 5–20 nM. Twofold dilution series of up to 16 unlabeled P1-P2 protein concentrations were prepared in a final volume of 20 μL for the MST measurements starting from 50 to 100 μM. Thermophoresis was measured using a Monolith NT.115 pico instrument (NanoTemper) at an ambient temperature of 25 °C and 5/30/5 sec. laser off/on/off times, respectively. The instrument parameters were adjusted to 90% LED power and 20% MST power. The data were analyzed using NanoTemper’s NT analysis software (version 1.5.41) using the “Thermophoresis and T Jump” resulting signal. The change in thermophoresis was expressed as the change in the normalized fluorescence (ΔFnorm), which is defined as F_hot/F_cold (F-values correspond to average fluorescence values between the defined areas marked by the red and blue cursors, respectively). Titration of the non-fluorescent ligand resulted in a gradual change in thermophoresis, which was plotted as ΔFnorm to yield a binding curve, which was fitted to derive the binding constants.