Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants
Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants
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DOI：
10.1021/bi983037t
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发表时间：
1999-05-18
期刊：
影响因子：
2.9
通讯作者：
Dobson, CM
中科院分区：
文献类型：
--
作者：
Canet, D;Sunde, M;Dobson, CM
采用停流荧光和氢交换脉冲标记结合质谱技术研究了人溶菌酶和两种淀粉样蛋白变体Ile 56 Thr和Asp 67 His的去折叠和重折叠特性。每种蛋白质在5.4 M盐酸胍（GuHCl）中的解折叠被很好地描述为双态过程，但是Ile 56 Thr变体和Asp 67 His变体在5.4 M GuHCl中的解折叠速率约为1.5。分别是野生型的30倍和160倍。在0.54 M盐酸胍在pH 5.0的所有三种蛋白质的重折叠收益通过持久的中间体，揭示了在荧光实验中的多步动力学和通过检测的定义明确的群体在淬灭流氢交换实验。这些发现与人溶菌酶重折叠的主要机制一致，其中一个结构域（α-结构域）在两个不同的步骤中形成，然后在两个结构域组装形成天然结构之前折叠另一个结构域（β-结构域）。Asp 67 His变体的重折叠动力学与野生型蛋白的重折叠动力学非常相似，这与β结构域的外环中该突变的位置一致，该突变仅在重折叠过程的末端获得天然结构。相比之下，Ile 56 Thr突变位于β结构域的碱基，并参与结构域界面。α-结构域的重折叠不受这种取代的影响，但后者具有显著减缓β-结构域的折叠和天然结构的最终组装的效果。这些研究表明，溶菌酶变体的淀粉样变性性质是由于天然折叠相对于部分折叠的中间体的稳定性降低而引起的。这种不稳定性的起源在两种变体中是不同的，在一种情况下主要是由折叠速率的降低引起的，而在另一种情况下是由展开速率的增加引起的。在这两种情况下，这导致可溶性部分折叠种类的低群体，其可以以缓慢和受控的方式聚集以形成淀粉样蛋白原纤维。
The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the a-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.