Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding.
Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding.
复制标题
在不允许的大分子拥挤条件下需要 GroEL/GroES 依赖性蛋白质折叠。
DOI：
10.1021/bi015925l
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Martin,Jörg
中科院分区：
文献类型：
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作者：
Martin,Jörg
大分子拥挤是影响细胞蛋白质折叠效率的关键参数。在这里，我们表明，蛋白质二氢叶酸还原酶，烯醇化酶，和绿色荧光蛋白，可以折叠在稀释的缓冲液自发，失去这种能力在拥挤的环境。相反，它们作为可溶性的、蛋白酶敏感的非原生物种积累。它们的折叠变得依赖于完整的GroEL/GroES伴侣蛋白系统，并且不受陷阱GroEL的影响，这表明折叠必须发生在伴侣蛋白腔中，并将天然样蛋白释放到本体溶液中。此外，我们证明，在伴侣蛋白腔中的有效折叠需要ATP水解，因为在ADP存在下与底物蛋白形成三元GroEL/GroES复合物仅导致非常低效的再激活。然而，在仅发生单轮ATP水解的条件下，使用ADP-氟铝酸复合物或单环GroEL和GroES的蛋白质重折叠反应会产生大量重折叠酶。因此，初始三元复合物形成的模式似乎是关键的后续生产性释放的基板进入腔在某些拥挤的条件下，只有当触发ATP水解是有效的。我们的数据表明，严格的拥挤条件下，可以赋予折叠蛋白质的伴侣蛋白的援助更强的依赖性。
Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP−fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.