Tm in 1.5M GdmCl, pH7.0, 4μM protein

Stability/Folding

Melting Temperature (Tm)

°C (Celsius)

Circular Dichroism (CD) , Thermal Denaturation

0.1 M sodium cacodylate–HCl (pH 7.0); 1.5 M GdmCl

7.0

4 μM

223 nm

By CD: Thermal unfolding transitions of 1, 4, and 10 μM protein in 0.1 M Na cacodylate–HCl (pH 7.0) and 1.5 M GdmCl were followed by CD at 223 nm with a 1-nm bandwidth in 10-mm cells using a Jasco J-600A spectropolarimeter equipped with a PTC-348 WI Peltier device. The data were analyzed using nonlinear regression and the program Grafit (Erithacus Software, Staines, UK), and the heat capacity change ΔCp was held constant at 4000 J mol− 1 K− 1 . The reversibility of thermal unfolding was examined by heating protein samples for 5 min at a temperature that was 10 °C above its TM value and cooled to 20 °C. Subsequent thermal unfolding transitions were identical with those obtained without preheating.
The precisions of Tm and ΔHvH are ±0.2° and ±5 kJ mol−1, respectively.

By DSC (for protein concentrations > 60 μM): For the wild-type Gβ1 protein and Gβ1-M2, DSC measurements were performed with a VP-DSC instrument (MicroCal, Northampton, MA, USA) at concentrations between 62 and 139 μM in 0.1 M sodium cacodylate–HCl (pH 7.0) and 1.5 M GdmCl at a scan rate of 1.5 K min− 1 (cell volume, 0.523 ml). The measured excess molar heat capacity CEp(T) was analyzed by a nonlinear least-squares method according to a non-two-state model after correc- tion for a progressive baseline (assumption of 0 ΔCp). For the analysis, the Origin software provided by MicroCal was used.