Schmidt M, Bücheler U, Kaluza B, Buchner J
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
J Biol Chem. 1994 Nov 11;269(45):27964-72.
The protein-protein interactions during GroE-mediated protein refolding are of crucial importance for understanding how the assisted refolding of non-native proteins is achieved. Since GroEL seems to be a rather promiscuous polypeptide-binding protein it is not surprising that conditions for efficient dissociation from GroEL are promiscuous as well. To understand assisted protein refolding it is necessary to elucidate the underlying principles of the different partial steps of the functional cycle. Here we show a correlation between the overall stability of the complex between GroEL and ligand protein and the conditions for functional release from the chaperonin. As a model system, differently denatured species of an antibody Fab fragment were used. While weakly bound Fab fragments are functionally released in the absence of GroES, stably associated non-native forms of the same protein are dependent on the presence of the co-chaperonin for optimal GroE-mediated reactivation, suggesting that complex stability determines the release requirement. However, the observed overall stability of the complex between GroEL and substrate protein may be regarded as the net product of constant binding and rebinding of the ligand protein, once associated with GroEL, as shown by competition experiments.
在GroE介导的蛋白质重折叠过程中，蛋白质 - 蛋白质相互作用对于理解非天然蛋白质的辅助重折叠是如何实现的至关重要。由于GroEL似乎是一种相当混杂的多肽结合蛋白，因此从GroEL上有效解离的条件同样混杂也就不足为奇了。为了理解辅助蛋白质重折叠，有必要阐明功能循环中不同部分步骤的潜在原理。在这里，我们展示了GroEL与配体蛋白之间复合物的整体稳定性与从伴侣蛋白上进行功能释放的条件之间的相关性。作为一个模型系统，我们使用了抗体Fab片段的不同变性形式。虽然弱结合的Fab片段在没有GroES的情况下会进行功能释放，但同一蛋白质的稳定结合的非天然形式则依赖于共伴侣蛋白的存在才能实现最佳的GroE介导的再活化，这表明复合物稳定性决定了释放需求。然而，如竞争实验所示，一旦与GroEL结合，GroEL与底物蛋白之间复合物的观察到的整体稳定性可能被视为配体蛋白持续结合和解离的净产物。
