Luo J, Iwakura M, Matthews C R
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1995 Aug 22;34(33):10669-75. doi: 10.1021/bi00033a043.
The reversible temperature-induced unfolding of a cysteine-free mutant (C85S/C152E, des-Cys) of dihydrofolate reductase from Escherichia coli has been studied by absorbance and by both far- and near-ultraviolet circular dichroism spectroscopies. The non-coincidence of all three transition curves demonstrated the existence of a highly populated partially-folded form near 39 degrees C at pH 7.8. This intermediate retains substantial secondary structure and partially excludes one or more of the five tryptophans from solvent; however, the intermediate has lost specific tertiary packing around its aromatic residues. Increases in enthalpy, entropy, and heat capacity are observed for both the native/intermediate and intermediate/unfolded transitions; the majority of the changes in these parameters occurs in the first transition. These results suggest that the thermal unfolding reaction of des-Cys dihydrofolate reductase involves a stable intermediate whose properties resemble those of a molten globule.
通过吸光度以及远紫外和近紫外圆二色光谱法，对来自大肠杆菌的二氢叶酸还原酶的无半胱氨酸突变体（C85S/C152E，去半胱氨酸）的可逆温度诱导解折叠进行了研究。所有三条转变曲线的不一致表明，在pH 7.8、接近39℃时存在一种高度富集的部分折叠形式。该中间体保留了大量二级结构，并且部分地将五个色氨酸中的一个或多个排除在溶剂之外；然而，该中间体在其芳香族残基周围失去了特定的三级堆积。对于天然态/中间体和中间体/解折叠态的转变，均观察到焓、熵和热容增加；这些参数的大部分变化发生在第一次转变中。这些结果表明，去半胱氨酸二氢叶酸还原酶的热解折叠反应涉及一个稳定的中间体，其性质类似于熔球态。
