m for GB1-M2 at 0.25μM protein
Slope of the Denaturant Unfolding Curve Cooperativity Value (m)
Fluorescence Intensity , Guanidinium Denaturation
0.1 M sodium cacodylate–HCl (pH 7.0); 1.5 M GdmCl
The parameters were derived from the GdmCl-induced unfolding transitions shown in Figure S1) (see details under [GdmCl]m measurements ). The cooperativity value m represents the dependence of ΔGD(25°C) on the concentration of GdmCl.
[GdmCl]m of GB1-M2 at 0.25 μM protein details: For the GdmCl-induced unfolding transitions of Gβ1- M2, 0.25, 1, and 10 μM protein samples were incubated for 2 h in 0.1 M Na cacodylate–HCl (pH 7.0) and various concentrations of GdmCl. The emission at 342 nm (bandwidth, 5 nm), after excitation at 280 nm (bandwidth, 3 nm), was measured at 25 °C in 10-mm cells with a Hitachi F-4010 fluorescence spectrophotometer. The experimental data were analyzed according to a two- state model and by assuming a linear dependence of fluorescence emission on the concentration of GdmCl.
Tm in 1.5M GdmCl, pH7.0, 4μM protein details: 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.