Fig. 2

Hydrodynamic and spectroscopic features of rThBgl. a Analytical SEC experiments with purified rThBgl. The protein elution profiles were used to estimate the R _s. The detailed procedure is described in the “Methods” section. Inset Estimation of the rThBgl Stokes radii as a function of the values of −(logK_av)^1/2 using standard proteins. b Sedimentation velocity AUC experiments of rThBgl using a concentration range of 100–800 μg mL⁻¹ in buffer C. The figure presents the c(S) distribution of the experiment at 800 μg mL⁻¹. Even at high concentrations, all sedimentation profiles exhibited only one species. Inset: Dependence of rThBgl s_20,w (S) as a function of protein concentration. We calculated an \(S_{20,w}^{0}\) (S) of 4.53 ± 0.03 S. The results in A and B together indicate that rThBgl is a monomer in solution (Table 1). c rThBgl thermal-induced unfolding measurements followed by CD. The unfolding experiments were measured at 220 nm from 20 to 90 °C with a 1-mm-path length cell using 8 µM rThBgl in buffer A. rThBgl had a Tm of 49 ± 1 °C. The typical α-helix-rich CD spectrum of rThBgl is presented in the inset of panel c. d Fluorescence emission spectra excited at 280 nm of folded and denatured states of rThBgl. To ensure complete protein denaturation, rThBgl was incubated with a freshly prepared solution of GdnHCl at a final concentration of 6 M for 1 h before the fluorescence experiments. The intrinsic fluorescence emission spectra were collected from 300 to 500 nm and revealed a clear shift of 334–354 nm between the folded and denatured rThBgl samples, respectively