Hydrogen peroxide induces the dissociation of GroEL into monomers that can facilitate the reactivation of oxidatively inactivated rhodanese.
Hydrogen peroxide induces the dissociation of GroEL into monomers that can facilitate the reactivation of oxidatively inactivated rhodanese.
复制标题
DOI：
10.1016/j.biocel.2003.08.012
复制
发表时间：
2004-03
期刊：
影响因子：
--
通讯作者：
G. Melkani;C. McNamara;G. Zardeneta;J. A. Mendoza
中科院分区：
文献类型：
--
作者：
G. Melkani;C. McNamara;G. Zardeneta;J. A. Mendoza
虽然，已经有一些关于氧化剂对其他分子伴侣的结构和功能的影响的研究报道，但迄今为止还没有关于伴侣蛋白GroEL的报道。通过监测过氧化氢(H_2O_2)对GroEL蛋白结构和复性活性的影响，研究了GroEL在氧化应激下的功能。利用荧光光谱和光散射技术，我们观察到GroEL暴露的疏水位置增加，三、四元结构发生变化。差示沉淀法、凝胶电泳法和圆二色谱分析表明，经H_2O_2处理的GroEL发生了不可逆解离成单体，部分二级结构丧失。相对于其他蛋白质，GroEL被发现具有高度的抗氧化性损伤。有趣的是，在这些条件下产生的GroEL单体可以促进H_2O_2灭活的罗丹明重新激活，但不能促进尿素变性的罗丹明重新激活。在没有GROES或∼的情况下，用天然或氧化的GroEL均可获得84%的活性罗丹明的回收率。相比之下，尿素变性的GroEL、BSA和不含蛋白质的复性混合物分别恢复了72%、50%和49%的罗丹尼酶活性。以前的研究表明，GroEL单体可以重新激活罗丹明。在这里，我们证明了氧化单体GroEL可以重新激活氧化的若丹明，这表明GroEL在氧化应激过程中保留了保护蛋白质的能力。
Although, several studies have been reported on the effects of oxidants on the structure and function of other molecular chaperones, no reports have been made so far for the chaperonin GroEL. The ability of GroEL to function under oxidative stress was investigated in this report by monitoring the effects of hydrogen peroxide (H2O2) on the structure and refolding activity of this protein. Using fluorescence spectroscopy and light scattering, we observed that GroEL showed increases in exposed hydrophobic sites and changes in tertiary and quaternary structure. Differential sedimentation, gel electrophoresis, and circular dichroism showed that H2O2treated GroEL underwent irreversible dissociation into monomers with partial loss of secondary structure. Relative to other proteins, GroEL was found to be highly resistant to oxidative damage. Interestingly, GroEL monomers produced under these conditions can facilitate the reactivation of H2O2-inactivated rhodanese but not urea-denatured rhodanese. Recovery of ∼84% active rhodanese was obtained with either native or oxidized GroEL in the absence of GroES or ATP. In comparison, urea-denatured GroEL, BSA and the refolding mixture in the absence of proteins resulted in the recovery of 72, 50, and 49% rhodanese activity, respectively. Previous studies have shown that GroEL monomers can reactivate rhodanese. Here, we show that oxidized monomeric GroEL can reactivate oxidized rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.