Monitoring cotranslational protein folding in mammalian cells at codon resolution
Monitoring cotranslational protein folding in mammalian cells at codon resolution
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DOI：
10.1073/pnas.1208138109
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发表时间：
2012-07-31
影响因子：
11.1
通讯作者：
Qian, Shu-Bing
中科院分区：
文献类型：
--
作者：
Han, Yan;David, Alexandre;Qian, Shu-Bing
核糖体结合的新生链如何折叠以承担其功能三级结构仍然是生物学中的一个中心难题。与变性蛋白的重折叠相比，共翻译折叠由于新生链的载体性质、核糖体的频繁停顿和细胞拥挤而变得复杂。在这里，我们提出了一种称为折叠相关共翻译测序的策略，该策略能够在密码子分解延伸期间监测新生链的折叠能力。通过使用工程多结构域融合蛋白，我们证明了在完整结构域序列出现后立即进行有效的共翻译折叠。我们还应用折叠相关的共翻译测序来跟踪甲型流感病毒感染细胞中血凝素的共翻译折叠。与不同表位的顺序形成相反，血凝素的受体结合域遵循全局折叠路线，当完整序列可用时，同时显示两个表位。我们的研究结果提供了在哺乳动物细胞中共翻译发生的区域智能全局折叠的直接证据。
How the ribosome-bound nascent chain folds to assume its functional tertiary structure remains a central puzzle in biology. In contrast to refolding of a denatured protein, cotranslational folding is complicated by the vectorial nature of nascent chains, the frequent ribosome pausing, and the cellular crowdedness. Here, we present a strategy called folding-associated cotranslational sequencing that enables monitoring of the folding competency of nascent chains during elongation at codon resolution. By using an engineered multidomain fusion protein, we demonstrate an efficient cotranslational folding immediately after the emergence of the full domain sequence. We also apply folding-associated cotranslational sequencing to track cotranslational folding of hemagglutinin in influenza A virus-infected cells. In contrast to sequential formation of distinct epitopes, the receptor binding domain of hemagglutinin follows a global folding route by displaying two epitopes simultaneously when the full sequence is available. Our results provide direct evidence of domain-wise global folding that occurs cotranslationally in mammalian cells.