盐酸胍诱导的DnaK三态变性。可扩展中间体的证据。

Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate.

作者信息

Palleros D R, Shi L, Reid K L, Fink A L

机构信息

Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.

出版信息

Biochemistry. 1993 Apr 27;32(16):4314-21. doi: 10.1021/bi00067a021.

DOI:10.1021/bi00067a021

PMID:8476860

Abstract

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.

摘要

通过圆二色性、荧光、尺寸排阻高效液相色谱和动态光散射研究了盐酸胍（Gdn-HCl）诱导的热休克蛋白DnaK的变性。DnaK的去折叠发生在两个离散步骤中。在Mg/ADP或Mg/ATP存在下，第一个转变的中点（Cm）（0.5 M）向更高的变性剂浓度（0.8 M）移动，而第二个转变（Cm = 1.6 M）不受核苷酸影响。观察到一种随着Gdn.HCl浓度增加而持续扩展的中间状态；讨论了其与熔球态的关系。此外，发现无论构象状态（天然、中间、未折叠）如何，分子体积与222 nm处的椭圆率之间都存在直接相关性；讨论了这些发现的意义。DnaK的去折叠最好用分级去折叠模型来解释。