Mechanistic Studies of the Folding of Human Lysozyme and the Origin of Amyloidogenic Behavior in Its Disease-Related Variants
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- 作者：Denis Canet ; Margaret Sunde ; Alexander M. Last ; Andrew Miranker ; Andrew Spencer ; Carol V. Robinson ; and Christopher M. Dobson
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：May 18, 1999
- 年：1999
- 卷：38
- 期：20
- 页码：6419 - 6427
- 全文大小：119K
- 年卷期：v.38,no.20(May 18, 1999)
- ISSN：1520-4995
文摘
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 pulselabeling 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 andthe Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wildtype. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistentintermediates, 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 witha predominant mechanism for refolding of human lysozyme in which one of the structural domains (the
-domain) is formed in two distinct steps and is followed by the folding of the other domain (the 
-domain)prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67Hisvariant are closely similar to those of the wild-type protein, consistent with the location of this mutationin an outer loop of the -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 -domain and is involved in the domaininterface. The refolding of the -domain is unaffected by this substitution, but the latter has the effect ofdramatically slowing the folding of the -domain and the final assembly of the native structure. Thesestudies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in thestability of the native fold relative to partially folded intermediates. The origin of this instability is differentin the two variants, being caused in one case primarily by a reduction in the folding rate and in the otherby an increase in the unfolding rate. In both cases this results in a low population of soluble partiallyfolded species that can aggregate in a slow and controlled manner to form amyloid fibrils.
-domain) is formed in two distinct steps and is followed by the folding of the other domain (the -domain)prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67Hisvariant are closely similar to those of the wild-type protein, consistent with the location of this mutationin an outer loop of the -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 -domain and is involved in the domaininterface. The refolding of the -domain is unaffected by this substitution, but the latter has the effect ofdramatically slowing the folding of the -domain and the final assembly of the native structure. Thesestudies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in thestability of the native fold relative to partially folded intermediates. The origin of this instability is differentin the two variants, being caused in one case primarily by a reduction in the folding rate and in the otherby an increase in the unfolding rate. In both cases this results in a low population of soluble partiallyfolded species that can aggregate in a slow and controlled manner to form amyloid fibrils.