Thermal activation of the co-chaperonins GroES and gp31 probed by mass spectrometry
Thermal activation of the co-chaperonins GroES and gp31 probed by mass spectrometry
复制标题
DOI：
10.1002/rcm.3782
复制
发表时间：
2008-11-01
影响因子：
2
通讯作者：
Heeren, Ron M. A.
中科院分区：
文献类型：
--
作者：
Geels, Rimco B. J.;Calmat, Stephane;Heeren, Ron M. A.
许多具有生物活性的蛋白质被组装成蛋白质复合物。因此，理解这种复合物的（分解）组装是主要的兴趣。在这里，我们使用质谱监测的七聚体共伴侣GroES和gp 31的热激活诱导的解体。我们使用本地电喷雾电离质谱（ESI-MS）的傅立叶变换离子回旋共振（FT-ICR）质谱仪监测化学计量的伴侣蛋白。一个热控制的电喷雾装置来分析构象和化学计量的变化，在不同的温度下的伴侣蛋白。天然ESI-MS数据与从荧光光谱获得的数据一致，因为测得的复合物的热解离温度非常一致。此外，我们观察到GroES和gp 31的热变性。通过所有低聚物形式的中间步骤进行，没有证据表明存在瞬时稳定的未折叠七聚体。我们还评估了在气相中使用红外多光子解离（IRMPD）的热活化的伴侣蛋白的热解离。使用气相活化，未检测到较小的（2-4）寡聚体，仅低至五聚体，此后复合物似乎完全解离。这些结果清楚地表明，GroES和gp 31的构象变化，由于在溶液中和在气相中加热是显着不同的。版权所有（c）2008约翰威利父子有限公司。
Many biological active proteins are assembled in protein complexes. Understanding the (dis)assembly of such complexes is therefore of major interest. Here we use mass spectrometry to monitor the disassembly induced by thermal activation of the heptameric co-chaperonins GroES and gp31. We use native electrospray ionization mass spectrometry (ESI-MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to monitor the stoichiometry of the chaperonins. A thermally controlled electrospray setup was employed to analyze conformational and stoichiometric changes of the chaperonins at varying temperature. The native ESI-MS data agreed welt with data obtained from fluorescence spectroscopy as the measured thermal dissociation temperatures of the complexes were in good agreement. Furthermore, we observed that thermal denaturing of GroES and gp31. proceeds via intermediate steps of all oligomeric forms, with no evidence of a transiently stable unfolded heptamer. We also evaluated the thermal dissociation of the chaperonins in the gas phase using infrared multiphoton dissociation (IRMPD) for thermal activation. Using gas-phase activation the smaller (2-4) oligomers; were not detected, only down to the pentamer, whereafter the complex seemed to dissociate completely. These results demonstrate clearly that conformational changes of GroES and gp31 due to heating in solution and in the gas phase are significantly different. Copyright (c) 2008 John Wiley & Sons, Ltd.