Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate.
Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate.
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DOI：
10.1021/bi00067a021
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发表时间：
1993-04
期刊：
影响因子：
2.9
通讯作者：
D. Palleros;L. Shi;K. L. Reid;A. Fink
中科院分区：
文献类型：
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作者：
D. Palleros;L. Shi;K. L. Reid;A. Fink
采用圆二色法、荧光法、粒径排除高效液相色谱法和动态光散射法研究了盐酸胍（Gdn-HCl）对热休克蛋白DnaK的变性作用。DnaK的展开分为两个独立的步骤。在Mg/ADP或Mg/ATP存在的情况下，第一次转变（0.5 M）的中点（Cm）转移到更高的变性剂浓度（0.8 M），而第二次转变（Cm = 1.6 M）不受核苷酸的影响。随着Gdn的增加而不断膨胀的中间状态。观察HCl浓度；讨论了其与熔融球的关系。此外，发现分子体积与222 nm处的椭圆率直接相关，无论构象状态（天然、中间、未折叠）如何；讨论了这些发现的意义。DnaK的展开最好用展开的层次模型来解释。
The denaturation of the heat shock protein DnaK induced by guanidine hydrochloride (Gdn-HCl) was investigated by circular dichroism, fluorescence, size-exclusion HPLC, and dynamic light scattering. DnaK unfolding takes place in two discrete steps. The midpoint (Cm) of the first transition (0.5 M) was shifted to higher denaturant concentrations (0.8 M) in the presence of Mg/ADP or Mg/ATP, whereas the second transition (Cm = 1.6 M) was unaffected by nucleotides. An intermediate state which continuously expands with increasing Gdn.HCl concentration was observed; its relation to molten globules is discussed. In addition, a direct correlation between molecular volume and ellipticity at 222 nm was found, regardless of the conformational state (native, intermediate, unfolded); the implications of these findings are discussed. The unfolding of DnaK is best explained by a hierarchical model of unfolding.