Melting temperatures and thermodynamic parameters for urea-induced unfolding equilibrium of Pim-1 wild type and mutants measured by far-UV CD spectroscopy.

The temperature-induced changes were followed by monitoring the ellipticity at 209 nm. The Tm values were calculated by taking the first derivative of the ellipticity at 209 nm with respect to temperature. Tm1 and Tm2 refer to the first and second transition observed for wild type and Y53H, respectively. For urea-induced unfolding equilibrium the data were obtained at 10°C in 20 mM Tris/HCl, pH 7.5, containing 0.2 M NaCl and 200 µM DTT by measuring circular dichroism ellipticity at 222 nm [Θ222]. ΔGH2O and m values were obtained from Eqn. 3; [Urea]0.5 was calculated from Eqn. 4. Data are reported as the mean ± SE of the fit.本研究通过监测209 nm处的椭圆率，追踪温度诱导的蛋白质结构变化。解链温度（Tm）值通过对209 nm处的椭圆率关于温度求取一阶导数计算得到。Tm1与Tm2分别对应野生型（wild type）和Y53H所观测到的第一、第二去折叠转变过程。针对尿素诱导的去折叠平衡实验，数据于10℃下在含0.2 M氯化钠、200 µM二硫苏糖醇（dithiothreitol, DTT）的20 mM三羟甲基氨基甲烷盐酸盐（Tris/HCl）缓冲液（pH 7.5）中，通过测定222 nm处的圆二色性椭圆率[Θ222]获取。ΔGH2O与m值通过公式3（Eqn. 3）计算得到；[Urea]0.5则通过公式4（Eqn. 4）计算获得。所有数据均以拟合结果的平均值±标准误（standard error, SE）形式报告。