Melting Temperature (Tm)
Circular Dichroism (CD) , Thermal Denaturation
20-100 °C with 1 °C/min increments
Thermal denaturation curves were obtained in SB250buffer using 25-50μM Ni-affinity purified protein in a 2.0 mm path-length cuvette. Samples were heated and cooled between 20°C and100C with a 2°C step size and 2 min sample equilibration time ateach temperature. At each temperature, ellipticity was monitored at222 nm using 60 s total data collection time, and 50 s integration time. Thermal melt fitting was performed as described assuming atwo-state model and a ΔCp value of 896 cal/mol K was set for all variants (14 cal/mol K per residue).