Studies on the Critical Structure of Protein Folding by Means of Site-Directed Amino Acid Replacements.

研究蛋白质折叠的过渡态对于阐明蛋白质折叠的机制具有重要意义。在这里，我们使用葡萄球菌核酸酶（SNase）作为模型蛋白分析过渡状态下的关键结构，并建立了一个有效的表达系统，利用位点定向氨基酸替换进行这种分析。得到了以下结果：(1)通过停流CD测量，研究了尿素浓度跳跃诱导的野生型SNase的展开和再折叠。具有相当数量的肽二级结构的中间体在重折叠的早期阶段积累，但它也被证明比以前在其他蛋白质中观察到的类似中间体明显不稳定。(2)定量分析了SNase的特定配体（Ca^<2+>和pdTp）对动力学折叠的影响，从而深入了解了过渡态的结构。将本研究结果与以往研究的另外两种Ca^<2+>-结合蛋白（α -乳清蛋白和小白蛋白）的结果进行比较，可以得出过渡态的关键结构反映了蛋白质结构折叠的层阶性。(3)利用lac-tac串联启动子、核糖体结合位点和大肠杆菌蛋白酶III信号序列构建了SNase在大肠杆菌中的表达分泌质粒，该质粒可用于SNase突变体的构建。(4)制备了甘氨酸取代Pro117的SNase突变体P117G，研究了其展开和再折叠动力学，研究了脯氨酸顺反异构对SNase动力学的影响。脯氨酸到甘氨酸的突变简化了展开和再折叠的动力学，并已被证明对折叠过渡状态的分析有用。

Investigations of the transition state of folding have primary importance in elucidating the mechanism of protein folding. Here, we have used staphylococcal nuclease (SNase) as a model protein for analysis of the critical structure in the transition state and established an effective expression system to utilize site-directed amino acid replacements for such an analysis. The following results were obtained. (1) Unfolding and refolding of wild-type SNase induced by concentration jumps of urea have been studied by stopped-flow CD measurements in the peptide region. An intermediate that has an appreciable amount of peptide secondary structure accumulates at an early stage of the refolding, but it has also been shown to be significantly less stable than similar intermediates previously observed in other proteins. (2) Effects of specific ligands (Ca^<2+> and pdTp) for SNase on the kinetic folding have been analyzed quantitatively to obtain an insight into the structure of the transition state. Comparison of the present results with those on the two other Ca^<2+>-binding proteins (alpha-lactalbumin and parvalbumin) in previous studies has led to the conclusion that the critical structure in the transition state reflects the hierarchical nature of protein structure folding. (3) An expression-secretion plasmid for SNase in E. Coli, which is useful for making SNase mutants, was constructed by use of a lac-tac tandem promoter followed by the ribosome-binding site and the signal sequence of E. Coli protease III. (4) A mutant SNase (P117G) in which Pro117 was replaced by glycine was made, and its kinetic unfolding and refolding were investigated to study the effect of proline cis-trans isomerism on the kinetics. The proline to glycine mutation simplifies the kinetics of both unfolding and refolding and has been shown to be useful for analysis of the transition state of folding.