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Studies of Protein Folding with Gene Manipulation and X-ray Solution Scattering -The Case of Staphylococcal Nuclease-

通过基因操作和 X 射线溶液散射研究蛋白质折叠 - 以葡萄球菌核酸酶为例 -

基本信息

批准号：
02680217
负责人：
KATAOKA Mikio
金额：
$ 1.47万
依托单位：
Osaka University (1992)Tohoku University (1990-1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

The structural information of unfolded state is essential for the better understanding of protein folding. We have revealed that X-ray solution scattering is useful and unique method to derive the compactness and the globularity of a protein molecule, which are estimated from the radius of gyration and the integral intensity of Kratky plot. The compactness and the globularity are the important factors to describe the extent of folding.Urea denaturation of Staphylococcal nuclease has been studied by X-ray solution scattering. The denaturation curve obtained from the compactness shows a good agreement with those obtained from CD and fluorescence in the case of wild type nuclease, while the denaturation curve of the mutant belonging to the class m- is different between X-ray and CD or fluorescence. This result strongly suggests that the acquisition of the compactness occurs earlier stage than the formation of the secondary structure.Staphylococcal nuclease fragment which lacks 13 amino acid residues from its C-terminus is unfolded under a physiological condition, while it is folded upon the addition of the inhibitor, pdTp. The fragment is a good model system for the folding study. The mutant fragments which have amino acid substitutions belonging to class m- are more compact than wild type fragment, while the mutant fragments with amino acid substitution of class m+ are much larger than wild type. The m- fragment takes a different inner structure from native protein. The m+ fragment takes a chain-like conformation. These results indicate that the effect of amino acid substitution is appeared in the unfolded state as well as the folded state.X-ray solution scattering is successfully applied to the studies of various conformational states of the other proteins.

未折叠状态的结构信息对于更好地理解蛋白质折叠至关重要。我们发现，X 射线溶液散射是推导蛋白质分子的致密性和球状性的有用且独特的方法，这些紧密性和球状性是根据回转半径和 Kratky 图的积分强度来估计的。致密性和球状性是描述折叠程度的重要因素。利用X射线溶液散射研究了葡萄球菌核酸酶的尿素变性。在野生型核酸酶的情况下，从紧密性获得的变性曲线与从CD和荧光获得的变性曲线显示出良好的一致性，而属于m-类的突变体的变性曲线在X射线和CD或荧光之间是不同的。这一结果强烈表明，致密性的获得发生在二级结构形成的更早阶段。 C 末端缺少 13 个氨基酸残基的葡萄球菌核酸酶片段在生理条件下会展开，而在添加抑制剂 pdTp 时会折叠。该片段是折叠研究的良好模型系统。具有m-类氨基酸取代的突变体片段比野生型片段更紧凑，而具有m+类氨基酸取代的突变体片段比野生型大得多。 m-片段采用与天然蛋白质不同的内部结构。 m+片段呈链状构象。这些结果表明，氨基酸取代的效应在未折叠状态和折叠状态下均出现。X射线溶液散射已成功应用于其他蛋白质各种构象状态的研究。