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

蛋白质折叠过程中的结构信息是理解蛋白质折叠过程的基础。我们揭示了X射线溶液散射是一种有用的和独特的方法来推导蛋白质分子的紧凑性和球形度，这是由旋转半径和Kratky图的积分强度估计。用X射线溶液散射法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，从紧凑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。葡萄球菌核酸酶C端缺失13个氨基酸残基的片段在生理条件下是解折叠的，而在加入抑制剂pdTp时则是折叠的。碎片是研究褶皱作用的一个很好的模型系统。具有属于m-类的氨基酸取代的突变体片段比野生型片段更紧凑，而具有m+类的氨基酸取代的突变体片段比野生型片段大得多。m片段具有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，氨基酸取代效应不仅存在于折叠态，而且也存在于非折叠态。X射线溶液散射法成功地应用于其它蛋白质的各种构象状态的研究。

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.