Chang Jui-Yoa
Research Center for Protein Chemistry, Institute of Molecular Medicine, and Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030, USA.
Biochemistry. 2004 Apr 20;43(15):4522-9. doi: 10.1021/bi0360354.
The pathways of oxidative folding of disulfide proteins exhibit a high degree of diversity, which is illustrated by the varied extent of (a) the heterogeneity of folding intermediates, (b) the predominance of intermediates containing native disulfide bonds, and (c) the level of accumulation of fully oxidized scrambled isomers as intermediates. BPTI and hirudin exemplify two extreme cases of such divergent folding pathways. We previously proposed that the underlying cause of this diversity is associated with the degree of stability of protein subdomains. Here we present compelling evidence that substantiates this hypothesis by studying the folding pathway of alphaLA-IIA. alphaLA-IIA is a partially folded intermediate of alpha-lactalbumin (alphaLA). It comprises a structured beta-sheet (calcium-binding) domain linked by two native disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) and a disordered alpha-helical domain with four free cysteines (Cys(6), Cys(28), Cys(111), and Cys(120)). Purified alphaLA-IIA was allowed to refold without and with stabilization of its structured beta-sheet domain by calcium. In the absence of calcium, the folding pathway of alphaLA-IIA resembles that of hirudin, displaying a highly heterogeneous population of folding intermediates, including fully oxidized scrambled species. Upon stabilization of its beta-sheet domain by bound calcium, oxidative folding of alphaLA-IIA undergoes a pathway conspicuously similar to that of BPTI, exhibiting limited species of folding intermediates containing mostly native disulfide bonds.
二硫键蛋白的氧化折叠途径具有高度多样性，这体现在以下几个方面的不同程度上：(a) 折叠中间体的异质性；(b) 含有天然二硫键的中间体的优势；(c) 作为中间体的完全氧化的混乱异构体的积累水平。抑肽酶和水蛭素是这种不同折叠途径的两个极端例子。我们之前提出，这种多样性的根本原因与蛋白质亚结构域的稳定性程度有关。在这里，我们通过研究αLA-IIA的折叠途径，提供了有力证据来证实这一假设。αLA-IIA是α-乳白蛋白（αLA）的部分折叠中间体。它由一个通过两个天然二硫键（Cys(61)-Cys(77)和Cys(73)-Cys(91)）连接的结构化β-折叠（钙结合）结构域和一个具有四个游离半胱氨酸（Cys(6)、Cys(28)、Cys(111)和Cys(120)）的无序α-螺旋结构域组成。纯化后的αLA-IIA在没有钙和有钙稳定其结构化β-折叠结构域的情况下进行重折叠。在没有钙的情况下，αLA-IIA的折叠途径类似于水蛭素的折叠途径，显示出高度异质的折叠中间体群体，包括完全氧化的混乱物种。当通过结合的钙稳定其β-折叠结构域时，αLA-IIA的氧化折叠途径明显类似于抑肽酶的途径，表现出有限种类的主要含有天然二硫键的折叠中间体。
