Ty c001

Thermal scans with measurement of the intrinsic fluorescence of the protein samples were carried out using a Tycho instrument (Nanotemper, Germany). 10 μL samples were loaded in capillaries (TY-C001, Nanotemper). The intrinsic protein fluorescence was measured at 350 nm and 330 nm, and the first derivative of the intensity ratio was calculated as a function of temperature. The temperature ramp rate was 30°C/min and data were acquired from 35 to 95 °C.

The stabilities of differnerent sNASPc constructs and mutants were evaluated through the inflection temperature (T_i). The proteins were diluted into 0.1 mg/mL with 1 X PBS buffer (10 mM Na₂HPO₄, 1.75 mM KH₂PO₄, 137 mM NaCl, 2.65 mM KCl, pH 7.4) and loaded onto the standard capillaries (Cat# TY-C001; NanoTemper Technologies). The samples were measured by using Tycho NT.6 (NanoTemper Technologies). When a 30°C/min thermal ramp from 35 to 95°C was applied, the changes of the intrinsic fluorescence of protein (from residues tryptophan and tyrosine), monitored at both 350 nm and 330 nm, were recorded. The initial ratio (350 nm/330 nm at 35°C) and the change in signal over the run (Δ ratio; change between 95 and 35°C) were also recorded. The inflection temperature (T_i) was evaluated using the Tycho NT.6 software version 1.2.2.859.

DSF was carried out using a Tycho instrument (Nanotemper, Germany). 10 μL samples were loaded in capillaries (TY-C001, Nanotemper). The intrinsic protein fluorescence was measured at 350 nm and 330 nm, and the first derivative of the intensity ratio was calculated as a function of temperature. The temperature ramp rate was 30°C/min and data were acquired from 35 to 95 °C.