Lilie H, Buchner J
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8100-4. doi: 10.1073/pnas.92.18.8100.
The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.
GroE蛋白是参与蛋白质折叠的分子伴侣。它们促进折叠的一般机制仍然是个谜。一个核心的未解决问题是与GroEL结合的非天然蛋白质的构象。关于蛋白质能与GroEL相互作用的折叠阶段已经提出了几种观点。此外，非天然蛋白质与GroEL的结合可能导致其去折叠。我们通过使用Fab抗体片段的折叠中间体作为分子探针来确定GroEL的结合特性，解决了这些理解GroE介导的折叠循环的基本问题。我们表明，除了与早期折叠中间体结合外，GroEL还能够识别并与晚期四级结构折叠中间体（Dc）相互作用，而不会使其明显去折叠。因此，结合的前提不是非天然蛋白质的特定折叠阶段。相反，非天然蛋白质的一般表面性质似乎对结合至关重要。此外，高度结构化的中间体在与GroEL结合时不一定会发生去折叠。在GroEL和ATP存在下Dc的折叠涉及结合和释放的循环。因为在这个系统中不涉及偏离途径的反应或动力学陷阱，所以可以对观察到的再激活动力学进行定量分析。我们的结果表明，在ATP存在下Dc与GroEL的缔合反应相当缓慢，而在没有ATP的情况下缔合效率要高几个数量级。因此，ATP似乎是通过抑制重新缔合而不是促进结合底物的释放来发挥作用的。
