STRUCTURAL CHARACTERIZATION OF THE DISULFIDE FOLDING INTERMEDIATES OF BOVINE ALPHA-LACTALBUMIN
STRUCTURAL CHARACTERIZATION OF THE DISULFIDE FOLDING INTERMEDIATES OF BOVINE ALPHA-LACTALBUMIN
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DOI：
10.1021/bi00065a023
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发表时间：
1993-04-13
期刊：
影响因子：
2.9
通讯作者：
CREIGHTON, TE
中科院分区：
文献类型：
--
作者：
EWBANK, JJ;CREIGHTON, TE
在Ca2+结合的牛α -乳清蛋白的二硫键还原过程中积累的特异性三硫和二硫中间体已经被捕获、分离和表征。3 -二硫中间体被证明缺少Cys6-120二硫键，证实了其他人的观察。新发现的二硫化物形式缺乏Cys6-120和Cys28-111天然二硫键。α -乳清蛋白的两个部分还原衍生物中剩余的天然二硫键只有在蛋白质处于Ca2+结合状态时才稳定。否则，它们采用熔融球状和未展开构象之间的平衡，迅速发生硫醇-二硫交换，其速率与蛋白质在8 M尿素中完全展开时一样高，以产生不同的重排产物混合物。尿素梯度电泳、圆二色性、荧光和ANS结合相结合，给出了α -乳白蛋白、其天然和非天然二硫键衍生物以及完全还原蛋白的详细结构图。α -乳清蛋白的天然结构似乎被选择性二硫键分裂成至少一个亚结构域，保留Ca2+结合位点。α -乳清蛋白熔融球态主要是由非特异性疏水崩溃造成的，缺乏合作或特定的三级相互作用，并且不被天然或重新排列的二硫键稳定。
Specific three- and two-disulfide intermediates that accumulate transiently during reduction of the disulfide bonds of Ca2+-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 Ca2+-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 Ca2+-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.