Analysis of Early Secodary Structure in Globular-Protein Folding.

球状蛋白质折叠的早期二级结构分析。

• 批准号: 60580217
• 负责人: KUWAJIMA Kunihiro
• 金额: $ 1.02万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60580217/
• 关键词: Folding Reaction; CD Spectrum; Stopped-Flow; Lysozyme; <alpha> -Lactalbumin; Parvalbumin; Cytochrome c; β-ラクトグロブリン

In order to investigate whether the presence of a transient intermediate that has folded secondary structure is a general phenomenon in globular-protein folding or not, kinetic refolding reactions of various proteins have been studied by stopped-flow circular dichroism (CD). Refolding reactions were induced by concentration jumps of guanidine hydrochloride from the unfolded to the native conditions, and resulting CD changes in peptide and side-chain regions were monitored. In all the proteins examined, i.e., lysozyme, <alpha> -lactalbumin, parvalbumin, ferricytochrome c and <beta> -lactoglobulin, there was rapid formation of secondary structure, within the dead time of the stopped-flow apparatus, before the formation of specific tertiary structure. Therefore, there is a transient intermediate formed early in the folding. In lysozyme and <alpha> -lactalbumin, their transient intermediates are similar to each other as expected from their structural homology and also essentially identical to the equilibrium unfolding intermediate of <alpha> -lactalbumin. In parvalbumin and cytochrome c, the transient intermediates have <alpha> -helical structure as expected from their structural patterns in the native state. A <beta> -structural protein, <beta> -lactoglobulin also shows a transient accumulation of the intermediate that involves <beta> -structure, comparable to the structure in the native protein, but also contains an excess of <alpha> -helix. From these results, it is concluded that the protein folding occurs in a hierarchical mechanism in which the framework of secondary structure is restored at an early stage of the reaction.

为了研究具有折叠二级结构的瞬态中间体的存在是否为球形蛋白质折叠的普遍现象，本文采用停流圆二色性（CD）研究了各种蛋白质的动力学再折叠反应。利用盐酸胍的浓度从未展开状态跳跃到自然状态，诱导了再折叠反应，并监测了肽区和侧链区CD的变化。所有检测的蛋白，即溶菌酶、< α > -乳清蛋白、小白蛋白、铁细胞色素c和< β > -乳球蛋白，在停流仪死亡时间内，在形成特定的三级结构之前，二级结构迅速形成。因此，在褶皱早期形成了一个瞬态中间体。在溶菌酶和< α > -乳清蛋白中，它们的瞬态中间体如结构同源性所预期的那样彼此相似，并且与< α > -乳清蛋白的平衡展开中间体基本相同。在小白蛋白和细胞色素c中，瞬态中间体具有< α > -螺旋结构，这与它们在天然状态下的结构模式一致。一种< β > -结构蛋白，< β > -乳球蛋白也表现出涉及< β > -结构的中间体的短暂积累，与天然蛋白中的结构相当，但也含有过量的< α > -螺旋。从这些结果可以得出结论，蛋白质折叠发生在一个层次机制中，二级结构的框架在反应的早期阶段被恢复。

项目成果

桑島邦博,春島嘉章,須貝新太郎: Int.J.Peptide Protein Res.27. 18-27 (1986)

Kunihiro Kuwashima、Yoshiaki Harushima、Shintaro Sugai：Int.J.Peptide Protein Res.27（1986）。

Kuwajima, K., Harushima, Y. and Sugai, S.: "Influence of <Ca^(2+)> Binding on the Structure and Stability of Bovine <alpha> -Lactalbumin Studied by Circular Dichroism and Nuclear Magnetic Resonance Spectra." Int. J. Peptide Protein Res.27. 18-27 (1986)

Kuwajima, K.、Harushima, Y. 和 Sugai, S.：“通过圆二色性和核磁共振光谱研究 <Ca^(2)> 结合对牛 <α> -乳白蛋白结构和稳定性的影响”。

池口雅道,桑島邦博,須貝新太郎: J.Biochem.99. 1191-1201 (1986)

池口正通、桑岛邦宏、菅井慎太郎：J.Biochem.99（1986）。

三谷尚洋,春島嘉章,桑島邦博,池口雅道,須貝新太郎: J.Biol.Chem.261. 8824-8829 (1986)

Naohiro Mitani、Yoshiaki Harushima、Kunihiro Kuwashima、Masamichi Ikeguchi、Shintaro Sugai：J.Biol.Chem.261 8824-8829（1986）。

浜野真城,新田勝利,桑島邦博,須貝新太郎: J.Biochem.100. 1617-1622 (1986)

Mashiro Hamano、Katsutoshi Nitta、Kunihiro Kuwashima、Shintaro Sugai：J.Biochem.1617-1622（1986）。