Reconstitution of Pure Chaperonin Hetero-Oligomer Preparations in Vitro by Temperature Modulation.
Reconstitution of Pure Chaperonin Hetero-Oligomer Preparations in Vitro by Temperature Modulation.
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DOI：
10.3389/fmolb.2018.00005
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发表时间：
2018
影响因子：
5
通讯作者：
Weiss CE
中科院分区：
文献类型：
--
作者：
Vitlin Gruber A;Vugman M;Azem A;Weiss CE
伴侣蛋白是一种大的、必需的寡聚体，可以促进叶绿体、线粒体和真细菌中的蛋白质折叠。植物叶绿体伴侣蛋白由多个具有独特性质的同源亚基组成。我们之前在体外鉴定了均一的、重组的、叶绿体伴侣蛋白低聚体，每个寡聚体由三个高度同源的海藻β亚基中的一个组成。在目前的工作中，我们描述了来自同一物种的阿尔法型亚基，并研究了它们与β亚型的相互作用。两个阿尔法亚基都不能单独形成高阶低聚物。当与β亚基结合时，只有α2亚基能够形成稳定的功能杂寡体，能够与天然叶绿体共伴侣蛋白复性变性蛋白。由于β寡聚体能够在没有α的情况下进行齐聚，我们寻找了在不污染αβ同源低聚体的情况下生产β杂化低聚体的条件。我们发现β-2亚基在低温下不能齐聚，并利用这一性质获得了均一的功能性α-2-β-2低聚物的制备。这项研究的结果强调了温度和浓度等反应条件对叶绿体伴侣蛋白低聚体体外重组的重要性。
Chaperonins are large, essential, oligomers that facilitate protein folding in chloroplasts, mitochondria, and eubacteria. Plant chloroplast chaperonins are comprised of multiple homologous subunits that exhibit unique properties. We previously characterized homogeneous, reconstituted, chloroplast-chaperonin oligomers in vitro, each composed of one of three highly homologous beta subunits from A. thaliana. In the current work, we describe alpha-type subunits from the same species and investigate their interaction with β subtypes. Neither alpha subunit was capable of forming higher-order oligomers on its own. When combined with β subunits in the presence of Mg-ATP, only the α2 subunit was able to form stable functional hetero-oligomers, which were capable of refolding denatured protein with native chloroplast co-chaperonins. Since β oligomers were able to oligomerize in the absence of α, we sought conditions under which αβ hetero-oligomers could be produced without contamination of β homo-oligomers. We found that β2 subunits are unable to oligomerize at low temperatures and used this property to obtain homogenous preparations of functional α2β2 hetero-oligomers. The results of this study highlight the importance of reaction conditions such as temperature and concentration for the reconstitution of chloroplast chaperonin oligomers in vitro.
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