Thermal Shift Assay

Thermal shift assays (TSAs) were performed using a CFX96 Touch Real-Time PCR detection system (Bio-Rad). Protein samples were diluted to a final concentration of 15 μM and mixed with 4× SYPRO Orange Protein Gel Stain (diluted from a 5000× stock as supplied by Sigma-Aldrich) to detect protein unfolding. The temperature (T) was increased from 20 to 80 °C in steps of 1 °C, and samples were incubated for 1 min at each temperature before detection of the fluorescence signal. Six-fold measurements were performed. Data were analyzed using OriginPro version 9.1 (OriginLab) and fit using eq 1.

where T_m is the melting temperature in degrees Celsius, A₁ and A₂ are the initial and final values of fluorescence, respectively, and dT describes the steepness of the change in fluorescence upon unfolding and is thus a measure of the degree of cooperativity.