Ewbank J J, Creighton T E
European Molecular Biology Laboratory, Heidelberg, Germany.
Biochemistry. 1993 Apr 13;32(14):3694-707. doi: 10.1021/bi00065a023.
Specific three- and two-disulfide intermediates that accumulate transiently during reduction of the disulfide bonds of Ca(2+)-bound bovine alpha-lactalbumin have been trapped, isolated, and characterized. The three-disulfide intermediate was shown to lack the Cys6-120 disulfide bond, confirming the observations of others. The newly-recognized two-disulfide form has been shown to lack the Cys6-120 and Cys28-111 native disulfide bonds. The remaining native disulfide bonds in the two partially reduced derivatives of alpha-lactalbumin are stable only when the proteins are in a Ca(2+)-bound state. Otherwise, they adopt an equilibrium between molten globule and unfolded conformations, and rapid thiol-disulfide interchange occurs, at a rate as high as when the proteins are fully unfolded in 8 M urea, to generate distinct mixtures of rearranged products. Urea gradient electrophoresis, circular dichroism, fluorescence, and ANS binding have been combined to give a detailed structural picture of alpha-lactalbumin, its derivatives with native and with nonnative disulfide bonds, and the fully reduced protein. The native structure of alpha-lactalbumin appears to be split by selective disulfide bond cleavage into at least one subdomain, which retains the Ca(2+)-binding site. The alpha-lactalbumin molten globule state is shown largely to result from nonspecific hydrophobic collapse, to be devoid of cooperative or specific tertiary interactions, and not to be stabilized substantially by the native or rearranged disulfide bonds.
在还原与钙结合的牛α-乳白蛋白的二硫键过程中短暂积累的特定三硫键和二硫键中间体已被捕获、分离和表征。已证明三硫键中间体缺乏Cys6-120二硫键，这证实了其他人的观察结果。新发现的二硫键形式已被证明缺乏Cys6-120和Cys28-111天然二硫键。α-乳白蛋白的两种部分还原衍生物中剩余的天然二硫键仅在蛋白质处于钙结合状态时才稳定。否则，它们在熔融球蛋白和未折叠构象之间达到平衡，并且会发生快速的硫醇-二硫键交换，其速率与蛋白质在8M尿素中完全展开时一样高，从而产生重排产物的不同混合物。尿素梯度电泳、圆二色性、荧光和ANS结合已被结合起来，以给出α-乳白蛋白、其具有天然和非天然二硫键的衍生物以及完全还原的蛋白质的详细结构图像。α-乳白蛋白的天然结构似乎通过选择性二硫键断裂被分成至少一个亚结构域，该亚结构域保留了钙结合位点。α-乳白蛋白的熔融球蛋白状态在很大程度上被证明是由非特异性疏水塌陷导致的，缺乏协同或特异性三级相互作用，并且基本上不会被天然或重排的二硫键稳定。
