Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT 06510, USA.
Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20264-9. doi: 10.1073/pnas.0911556106. Epub 2009 Nov 13.
The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide. In order-of-addition experiments, approximately twice as much Rubisco activity was recovered when nonnative substrate protein was added after ATP compared with it being added before ATP, associated with twice as much Rubisco protein recovered with the chaperonin. Furthermore, the rate of Rubisco binding to an ATP-exposed ring was twice that observed in the absence of nucleotide. Finally, when both ATP and Rubisco were added simultaneously to a GroEL ring, simulating the physiological situation, the rate of Rubisco binding corresponded to that observed when ATP had been added first. We conclude that the physiological order, ATP binding before polypeptide, enables more efficient capture of nonnative substrate proteins, and thus allows greater recovery of the native state for any given round of the chaperonin cycle.
GroEL/GroES 反应循环包括在 GroES 包封步骤之前，ATP 和多肽结合到开放的 GroEL 环的步骤，该步骤触发在隔离腔室中的有效折叠。尽管已经假设多肽首先结合，允许随后的 ATP 介导的 GroEL 顶端结构域运动对非天然多肽施加强制展开的力，但非天然多肽和 ATP 向开放的反式环的添加的生理顺序（通常是不对称的 GroEL/GroES/ADP 复合物）尚不清楚。在这里，我们使用荧光测量法表明，生理添加顺序相反，涉及快速结合 ATP，伴随着几乎同样快速的顶端结构域运动，随后是较慢的非天然多肽结合。在添加顺序实验中，与将非天然底物蛋白添加到 ATP 之前相比，当将其添加到 ATP 之后时，回收的 Rubisco 活性大约增加了一倍，与伴侣蛋白回收的 Rubisco 蛋白增加了一倍有关。此外，在不存在核苷酸的情况下，Rubisco 与暴露于 ATP 的环的结合速率是观察到的两倍。最后，当同时将 ATP 和 Rubisco 添加到 GroEL 环中，模拟生理情况时，Rubisco 结合的速率与首先添加 ATP 时观察到的速率相对应。我们得出结论，生理顺序，即多肽结合之前的 ATP 结合，能够更有效地捕获非天然底物蛋白，从而允许给定伴侣蛋白循环的轮次中更大程度地恢复天然状态。
