Chang Jui-Yoa
Research Center for Protein Chemistry, Institute of Molecular Medicine and the Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030, USA.
J Biol Chem. 2002 Jan 4;277(1):120-6. doi: 10.1074/jbc.M108057200. Epub 2001 Sep 17.
The technique of disulfide scrambling permits reversible conversion of the native and denatured (scrambled) proteins via shuffling and reshuffling of disulfide bonds. Under strong denaturing conditions (e.g. 6 m guanidinium chloride) and in the presence of a thiol initiator, alpha-lactalbumin (alphaLA) denatures by shuffling its four native disulfide bonds and converts to an assembly of 45 species of scrambled isomers. Among them, two predominant isomers, designated as X-alphaLA-a and X-alphaLA-d, account for about 50% of the total denatured structure of alphaLA. X-alphaLA-a and X-alphaLA-d, which adopt the disulfide patterns of (1-2,3-4,5-6,7-8) and (1-2,3-6,4-5,7-8), respectively, represent the most unfolded structures among the 104 possible scrambled isomers (Chang, J.-Y., and Li, L. (2001) J. Biol. Chem. 276, 9705-9712). In this study, X-alphaLA-a and X-alphaLA-d were purified and allowed to refold through disulfide scrambling to form the native alphaLA. Folding intermediates were trapped kinetically by acid quenching and analyzed quantitatively by reversed phase high pressure liquid chromatography. The results revealed two major on-pathway productive intermediates, two major off-pathway kinetic traps, and at least 30 additional minor transient intermediates. Of the two major on-pathway intermediates, one takes on a native-like alpha-helical domain, and the other comprises a structured beta-sheet, calcium binding domain. The two major kinetic traps are apparently stabilized by locally formed non-native-like structures. Overall, the folding mechanism of alphaLA is essentially congruent with the model of "folding funnel" furnished with a rather intricate energy landscape.
二硫键重排技术可通过二硫键的重排和再重排实现天然蛋白质和变性（重排）蛋白质之间的可逆转换。在强变性条件下（如6 M盐酸胍）并存在硫醇引发剂时，α-乳白蛋白（αLA）会通过重排其4个天然二硫键而变性，转化为45种重排异构体的集合。其中，两种主要异构体，分别命名为X-αLA-a和X-αLA-d，约占αLA总变性结构的50%。X-αLA-a和X-αLA-d分别采用(1-2,3-4,5-6,7-8)和(1-2,3-6,4-5,7-8)的二硫键模式，代表了104种可能的重排异构体中最展开的结构（Chang, J.-Y., and Li, L. (2001) J. Biol. Chem. 276, 9705-9712）。在本研究中，X-αLA-a和X-αLA-d被纯化，并通过二硫键重排进行复性以形成天然αLA。通过酸淬灭动力学捕获折叠中间体，并通过反相高压液相色谱进行定量分析。结果揭示了两种主要的沿途径生产性中间体、两种主要的偏离途径动力学陷阱以及至少30种额外的次要瞬时中间体。在两种主要的沿途径中间体中，一种具有类似天然的α-螺旋结构域，另一种包含结构化的β-折叠钙结合结构域。两种主要的动力学陷阱显然是由局部形成的非天然样结构稳定的。总体而言，αLA的折叠机制与具有相当复杂能量景观的“折叠漏斗”模型基本一致。
