Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry
Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry
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DOI：
10.1073/pnas.240326597
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发表时间：
2000-12-19
影响因子：
11.1
通讯作者：
Robinson, CV
中科院分区：
文献类型：
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作者：
Fändrich, M;Tito, MA;Robinson, CV
我们已经分析了一个新描述的古细菌GimC/前折叠蛋白同源物，称为MtGimC，通过使用纳米流电喷雾耦合飞行时间MS。从嗜热甲烷杆菌的复合物的分子量对应于两个α亚基和四个β亚基的明确定义的六聚体。在气相内的复合物的解离揭示了两个中心亚基，两个α，和四个外围β亚基的四元排列。通过构建一个热控制的纳米流装置，我们已经监测了MS的复合物的热稳定性。这些实验的结果表明，在低盐条件下，在升高的温度下，MtGimC六聚体的显着比例保持完整。这一发现得到了CD光谱数据的支持，该数据表明，在生理盐浓度下，该复合物在65 ℃以上的温度下保持稳定。开发了质谱方法以真实的实时监测MtGimC六聚体从其组分亚基的组装。通过使用这种方法，在整个实验的时间过程中记录的质谱显示不存在任何显著填充的中间体，表明组装过程是高度协同的。总之，这些数据表明，该复合物是稳定的高温下，是适合于其超嗜热宿主，并证明，组装途径导致专门的六聚体，这可能是一个结构单元在体内。
We have analyzed a newly described archaeal GimC/prefoldin homologue, termed MtGimC, by using nanoflow electrospray coupled with time-of-flight MS. The molecular weight of the complex from Methanobacterium thermoautotrophicum corresponds to a well-defined hexamer of two alpha subunits and four beta subunits. Dissociation of the complex within the gas phase reveals a quaternary arrangement of two central subunits, both alpha, and four peripheral beta subunits. By constructing a thermally controlled nanoflow device, we have monitored the thermal stability of the complex by MS. The results of these experiments demonstrate that a significant proportion of the MtGimC hexamer remains intact under low-salt conditions at elevated temperatures. This finding is supported by data from CD spectroscopy, which show that at physiological salt concentrations, the complex remains stable at temperatures above 65 degreesC. Mass spectrometric methods were developed to monitor in real time the assembly of the MtGimC hexamer from its component subunits. By using this methodology, the mass spectra recorded throughout the time course of the experiment showed the absence of any significantly populated intermediates, demonstrating that the assembly process is highly cooperative. Taken together, these data show that the complex is stable under the elevated temperatures that are appropriate for its hyperthermophile host and demonstrate that the assembly pathway leads exclusively to the hexamer, which is likely to be a structural unit in vivo.