Minuth T, Frey G, Lindner P, Rachel R, Stetter K O, Jaenicke R
Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Germany.
Eur J Biochem. 1998 Dec 1;258(2):837-45. doi: 10.1046/j.1432-1327.1998.2580837.x.
The archaeon Pyrodictium occultum is one of the most thermophilic organisms presently known. Previous experiments provided support for the significant contribution of a high-molecular-mass protein complex to the extreme thermotolerance of P. occultum. This protein complex, the 'thermosome', is composed of two subunits, alpha and beta, which form a hexadecameric double ring complex. In order to obtain the thermosome in amounts sufficient for structural and functional investigations, we produced the two subunits jointly and separately in Escherichia coli BL21(DE3). In all three cases, we isolated soluble, high-molecular-mass double-ring complexes from E. coli BL21(DE3). On electron micrographs, the recombinant complexes were indistinguishable from each other and from the natural thermosome. To characterize the quaternary structure of the recombinant particles, we used native gel electrophoresis, analytical gel filtration, and analytical ultracentrifugation. Spectral analysis, using absorption, fluorescence emission and far-UV circular dichroism spectroscopy were applied to compare the three recombinant protein complexes with the natural thermosome from P. occultum. All three recombinant complex species exhibit ATPase activity. Furthermore, we could demonstrate that the recombinant complexes slow down the aggregation of citrate synthase, alcohol dehydrogenase, and insulin. Thus, we conclude that the recombinant protein complexes exhibit a chaperone-like activity, interacting with non-native proteins; they do so at temperatures far below the lower physiological limit of growth.
隐秘嗜热栖热菌是目前已知的最嗜热的生物之一。先前的实验支持了一种高分子质量蛋白质复合物对隐秘嗜热栖热菌的极端耐热性有重大贡献。这种蛋白质复合物，即“热体”，由α和β两个亚基组成，形成一个十六聚体双环复合物。为了获得足以进行结构和功能研究的热体，我们在大肠杆菌BL21(DE3)中联合和分别生产了这两个亚基。在所有三种情况下，我们都从大肠杆菌BL21(DE3)中分离出了可溶性的、高分子质量的双环复合物。在电子显微镜照片上，重组复合物彼此之间以及与天然热体没有区别。为了表征重组颗粒的四级结构，我们使用了非变性凝胶电泳、分析性凝胶过滤和分析性超速离心。利用吸收光谱、荧光发射光谱和远紫外圆二色光谱进行光谱分析，以比较这三种重组蛋白复合物与来自隐秘嗜热栖热菌的天然热体。所有三种重组复合物都表现出ATP酶活性。此外，我们能够证明重组复合物减缓了柠檬酸合酶、乙醇脱氢酶和胰岛素的聚集。因此，我们得出结论，重组蛋白复合物表现出类似伴侣蛋白的活性，与非天然蛋白质相互作用；它们在远低于生长的生理下限温度下就能做到这一点。
