Effect of the structure of the denatured state of lysozyme on the aggregation reaction at the early stages of folding from the reduced form
Effect of the structure of the denatured state of lysozyme on the aggregation reaction at the early stages of folding from the reduced form
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DOI：
10.1016/j.jmb.2005.01.022
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发表时间：
2005-03-18
影响因子：
5.6
通讯作者：
Ueda, T
中科院分区：
文献类型：
--
作者：
Ohkuri, T;Shioi, S;Ueda, T
我们以前证明，在变性条件下存在于鸡溶菌酶中的疏水簇被破坏的Trp62突变为甘氨酸（W62G）。为了考察W62G溶菌酶变性态结构对折叠的影响，我们对还原态W62G溶菌酶折叠的早期事件进行了详细的分析。从使用含有一对半胱氨酸残基（1SS）的变体的二硫键交换测量中发现，在W62GISS溶菌酶中二硫键的形成并不伴随着氨基酸残基之间的显著相互作用，这表明疏水核心的破坏导致了还原溶菌酶在折叠过程的早期阶段的随机折叠。对还原的W62 G溶菌酶的氧化复性的分析以及对还原的和羧酰胺甲基化的W62 G溶菌酶的聚集程度的测量表明，在还原的W62 G溶菌酶中聚集体的形成比在还原的野生型溶菌酶中更显著。此外，一个滞后期被检测到在氧化复性还原W62G溶菌酶，根据观察的恢复活动。折叠过程的模拟表明，中间体存在于折叠的早期阶段的减少W62G溶菌酶。这些结果表明，中间体的存在来自于在还原W62G溶菌酶的折叠过程中的早期阶段的随机折叠，由于变性状态的结构的破坏。因此，折叠似乎已被这些过程，然后导致显着的减少溶菌酶聚集动力学延迟。此外，从淀粉样蛋白聚集的还原溶菌酶的分析，这表明，在变性状态下的残留结构的破坏W62G突变阻止淀粉样蛋白纤维的形成溶菌酶。(C)2005爱思唯尔有限公司保留所有权利。
We previously demonstrated that the hydrophobic clusters present in hen lysozyme under denaturing conditions were disrupted by the mutation of Trp62 to Gly (W62G). In order to examine the effects of the structure of the denatured state of W62G lysozyme on folding, we analyzed the early events in the folding of reduced W62G lysozyme in detail. From the exchange measurements of disulfide bonds using the variants containing a pair of cysteine residues (1SS), it was found that the formation of disulfide bond in the W62GISS lysozyme was not accompanied by a prominent interaction between amino acid residues, indicating that the disruption of the hydrophobic core led to the random folding at the early stages in the process of folding of the reduced lysozyme.On the other hand, analyses of the oxidative-renaturation of reduced W62G lysozymes, as well as measurements of the extent of aggregation of the reduced and carboxy amido methylated W62G lysozyme, indicated that the formation of an aggregate is more prominent in the reduced W62G lysozyme than in the reduced wild-type lysozyme. Moreover, a lag phase was detected in the oxidative-renaturation of reduced W62G lysozyme, as based on observations of the recovery of activity. The simulation of the folding process indicated that intermediates were present at the early stages in the folding of the reduced W62G lysozyme. These results suggest that the presence of the intermediates was derived from the random folding at the early stages in the folding process of reduced W62G lysozyme due to the disruption of the structure of the denatured state. Folding thus appears to have been kinetically delayed by these processes, which then led to the significant aggregation of reduced lysozyme. Moreover, from the analysis of amyloid aggregation of the reduced lysozymes, it was suggested that the disruption of the residual structure in denatured state by W62G mutation deterred the formation of the amyloid fibrils of lysozyme. (C) 2005 Elsevier Ltd. All rights reserved.