Ohmae E, Kurumiya T, Makino S, Gekko K
Department of Materials Science, Faculty of Science, Hiroshima University.
J Biochem. 1996 Nov;120(5):946-53. doi: 10.1093/oxfordjournals.jbchem.a021511.
The acid and thermal unfolding of Escherichia coli dihydrofolate reductase (DHFR) were studied by means of circular dichroism (CD) and fluorescence spectroscopy. There existed at least one intermediate around pH 4 in the acid unfolding process at 15 degrees C, in which the tertiary structure was disrupted before unfolding of the secondary structure. The fluorescence energy transfer from intrinsic tryptophan residues to 1-anilinonaphthalene-8-sulfonate suggested the disruption of the tertiary structure around some tryptophan residues of the intermediate. The thermal unfolding process at pH 7.0 also involved at least one intermediate having a disrupted tertiary structure and a folded secondary structure. The three-state thermodynamic analysis showed that the intermediate in thermal unfolding was less stable by 1.8 kcal/mol than the native state. The similarity of the far-ultraviolet CD spectra of acid and thermally unfolded forms suggests that both types of unfolding produce the same structure, which may be a molten globule intermediate such as that in the folding kinetics of DHFR. The acid and thermal unfolding were depressed in the presence of KCl due to stabilization of the native form.
通过圆二色性（CD）和荧光光谱法研究了大肠杆菌二氢叶酸还原酶（DHFR）的酸变性和热变性。在15℃下的酸变性过程中，pH 4左右至少存在一种中间体，其中三级结构在二级结构展开之前就已被破坏。从内在色氨酸残基到1-苯胺基萘-8-磺酸盐的荧光能量转移表明中间体的一些色氨酸残基周围的三级结构被破坏。pH 7.0时的热变性过程也涉及至少一种具有被破坏的三级结构和折叠的二级结构的中间体。三态热力学分析表明，热变性中的中间体比天然状态稳定性低1.8千卡/摩尔。酸变性和热变性形式的远紫外CD光谱相似，表明两种类型的变性产生相同的结构，这可能是一种熔球中间体，如DHFR折叠动力学中的中间体。由于天然形式的稳定化，在KCl存在下酸变性和热变性受到抑制。
