Figure 1.
A raw DSC heating scan of MalZ in a 20 mM sodium phosphate buffer that contained 50 mM NaCl and 50 mM imidazole, at pH 7.4.
The scan rate was 1.0 K/min, and the protein concentration was 10 µM. The profile shows an endothermic unfolding peak at 327 K, followed by a typical exothermal trace caused by protein aggregation above 337 K. The DSC scan shown is that before baseline subtraction.
Figure 2.
The CD (A) and UV-absorption (B) spectra of native (solid line) and thermally denatured (dashed line) MalZ at 20°C and 58°C, respectively.
The solution condition was in 20 mM sodium phosphate buffer, containing 50 mM NaCl and 50 mM imidazole, at pH 7.4. The protein concentration used was 2.5 µM for CD measurement and 0.5 µM for UV-absorption measurements.
Figure 3.
Temperature dependence of the molar excess heat capacity () (A) and the normalized turbidity at 340 nm (B) at different concentrations of MalZ.
(A) 1.25 µM (short dashed line (black)), 2.5 µM (dash dotted line (blue)), 5 µM (dotted line (red)) and 10 µM (dashed line (magenta)); and (B) 0.38 µM (short dashed line (black)), 0.75 µM (dash dotted line (blue)), 1 µM (dotted line (red)) and 1.25 µM (dashed line (magenta)). The scan rate was 1.0 K/min.
Figure 4.
Temperature dependence of (A), the CD ellipticity at 230 nm (B) and the UV absorption at 340 nm (C) of MalZ at different scan rates.
The scan rate were 0.5 K/min (short dashed line), 0.75 K/min (solid line), 1.0 K/min (dash dotted line) and 1.5 K/min (dashed line). The protein concentration was 1.25 µM for DSC measurements, 2.5 µM for CD measurement, and 0.75 µM for UV absorption measurements.
Table 1.
Tmax and T1/2 values of the MalZ thermal denaturation at different scan rates.
Figure 5.
Theoretical fitting of the DSC profiles (A), the thermal unfolding profiles obtained by CD ellipticity at 230 nm (B), and the turbidity transition profiles obtained by UV absorption at 340 nm (C) of MalZ at different scan rates.
The scan rates were 0.5 K/min (▾), 0.75 K/min (▴), 1.0 K/min (•) and 1.5 K/min (▪). The continuous lines indicate the theoretical fitting curves based on Eq. (4) (A) and Eq. (5) (B) and (C). The experimental data were the same as those in Fig. 4, and the representative data points are shown by the symbols.
Table 2.
Arrhenius-equation parameters obtained by the non-linear least-squares fitting of the DSC data (Fig. 4(A)) to Eq. (4), *.
Table 3.
Arrhenius-equation parameters obtained by the non-linear least-squares fitting of the far-UV CD data (Fig. 4(B) and the UV-absorption data (Fig. 4(C)) to Eq. (5), *.
Figure 6.
Dependence of 1/T on for MalZ (Eq. (6)).
The symbols are the same as in Fig. 5 and indicate different scan rates.
Table 4.
Comparison of parameters of the one-step irreversible thermal denaturation of different proteins.