Microcalorimetric investigation of the ATPase activity and the refolding activity of GroEL system
Microcalorimetric investigation of the ATPase activity and the refolding activity of GroEL system
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GroEL 系统 ATP 酶活性和重折叠活性的微量热研究
DOI：
10.1007/s10973-018-7443-0
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发表时间：
2019-02-01
影响因子：
4.4
通讯作者：
Gan, Min
中科院分区：
文献类型：
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作者：
Chen, Daixiong;Zhang, Ke;Gan, Min
GroEL是Hsp 60分子伴侣的成员之一，其ATP酶活性和复性机制已被广泛应用于分子生物学研究。在这里，一个C80等温量热仪被施加到了解GroEL辅助复性的分子机制。得到了不同反应体系的功率-时间曲线，并对积分热进行了计算和分析。热休克蛋白60的ATP酶活性在高温下非常稳定，并具有很高的温度依赖性。在ATP水解实验中，我们发现GroEL催化的ATP水解是一个放热反应，其中二价阳离子尤其是镁离子起着重要的作用。ATP浓度对ATP水解速率也有影响。此外，GroEL蛋白在ATP和辅助分子伴侣GroES的存在下可以辅助热变性木聚糖酶的复性，且复性过程为吸热反应，表明非天然蛋白质与GroEL蛋白疏水顶端结构域发生了以疏水相互作用为主的相互作用。本研究中给出的热力学参数通过微量热法解释了GroEL的工作机理，对进一步研究其他相互作用机理具有重要意义。
GroEL, a member of the Hsp60 class molecular chaperones, has been extensively studied by general molecular biological method not only on its ATPase activities but also on refolding mechanism. Here, a C80 isothermal calorimeter was applied to understand the molecular mechanism of GroEL-assisted refolding. The power–time curves were obtained from different reaction systems, and the integral heat was calculated and analyzed. The ATPase activity of HSP60 was proven to be very stable at high temperature and had a high temperature dependence. In ATP hydrolysis experiments, we found that ATP hydrolysis catalyzed by GroEL was an exothermic reaction, in which divalent cations especially magnesium cation played an important role. And the ATP concentration also influences the ATP hydrolysis rate. Moreover, GroEL can assist in refolding thermodenatured xylanase with the presence of ATP and co-chaperone GroES, and the refolding process was demonstrated to be endothermic reaction, indicating that non-native proteins interacted with hydrophobic apical domains of GroEL, mainly hydrophobic interaction. The thermodynamic parameters presented in this study illustrated the work mechanism of GroEL by microcalorimetry and have important implications for further study of other interaction mechanism.