Ikeguchi M, Fujino M, Kato M, Kuwajima K, Sugai S
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo, Japan.
Protein Sci. 1998 Jul;7(7):1564-74. doi: 10.1002/pro.5560070710.
The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of alpha-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact alpha-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of alpha-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of alpha-lactalbumin is discussed.
测定了α-乳白蛋白衍生物（其中6-120二硫键被选择性还原并进行了S-羧甲基化）折叠和解折叠速率常数对盐酸胍浓度的依赖性，并将其与完整二硫键的α-乳白蛋白进行了比较。基于我们之前提出的两种替代模型——中间态控制折叠模型和多途径折叠模型，分析了折叠和解折叠速率常数对浓度的依赖性。所有数据均支持多途径折叠模型。因此，在α-乳白蛋白折叠早期积累的熔球态不是一个必经的中间体。6-120二硫键的断裂导致解折叠加速而不改变重折叠速率，这表明由6-120二硫键封闭的环在过渡态时是展开的。从理论上表明，从具有螺旋伸展的无规卷曲链中去除交联时链熵的增加与从完全无规链中去除交联时的增加相当。因此，目前的结果与熔球态中间体中已知的结构并不矛盾。基于这一结果和迄今获得的其他知识，讨论了α-乳白蛋白折叠反应过渡态的结构。
