Microscale thermophoresis (MST) binding assay

The binding affinities of Csx1–Crn2 and its variants with cA₄ were measured by the MST technology-based binding assay as previously described (33). In preparation for the experiment, the purified recombinant proteins of Csx1–Crn2^H495A and Csx1–Crn2^{182–563/H495A} were dialyzed into MST buffer containing 20 mM HEPES at pH 7.8 and 100 mM NaCl, whereas the protein of Csx1–Crn2^1–444 was maintained in the MST buffer contained 500 mM NaCl. Then, each protein was mixed and incubated with the fluorescent dye RED-NHS (MO-L011, NanoTemper Technologies) at room temperature for 30 min in darkness. After the reaction, the labeled proteins were purified through column-B, and stored in phosphate-buffered saline buffer (pH 7.4) supplemented with 0.05% Tween-20 (PBST). Then, the labeled proteins (50 nM) were incubated with 16 different serial concentrations of cA₄ at room temperature for 15 min in PBST. Subsequently, each sample was loaded into a capillary (MO-K022, NanoTemper Technologies), and the MST experiment was conducted on a Monolith NT.115 instrument, using 60–80% light-emitting diode power and medium MST power settings at 25°C. All experiments were conducted with a minimum of three replicates, and data were analyzed using MO.Affinity Analysis v.2.3 software (NanoTemper Technologies, München, Germany). Figures were generated using the GraphPad Prism 6.0 software.