Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy
Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy
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DOI：
10.1073/pnas.0903750106
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发表时间：
2009-12-29
影响因子：
11.1
通讯作者：
Christodoulou, John
中科院分区：
文献类型：
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作者：
Cabrita, Lisa D.;Hsu, Shang-Te Danny;Christodoulou, John
多肽链如何获得其独特的三维结构是生物学中的一个基本问题。在核糖体上的合成过程中，一个新生的链(NC)以矢量的方式出现，并开始以共翻译的方式折叠。细胞的复杂环境，再加上在合成过程中逐渐出现的核糖体拴系NC，对其折叠格局施加了不同于单独蛋白质的构象限制，当在溶液中变性后被刺激折叠时。为了开始研究细胞内发生的共翻译折叠，我们在体内生产了高度选择性的、同位素标记的NC与同位素沉默的核糖体结合。然后，我们应用核磁共振波谱在残基特定的水平上研究NCS的构象，该构象由对应于给定蛋白质的多肽链的不同分数长度组成。这种结合的方法提供了一种强大的方法，当NC从核糖体中出现时，可以生成NC折叠的一系列快照。将这一策略应用于Ig样结构域的渐进合成的核磁共振分析中，揭示了部分折叠的核糖体结合物种的存在，该物种可能代表在共翻译折叠过程中填充的中间物种。
The means by which a polypeptide chain acquires its unique 3-D structure is a fundamental question in biology. During its synthesis on the ribosome, a nascent chain (NC) emerges vectorially and will begin to fold in a cotranslational fashion. The complex environment of the cell, coupled with the gradual emergence of the ribosome-tethered NC during its synthesis, imposes conformational restraints on its folding landscape that differ from those placed on an isolated protein when stimulated to fold following denaturation in solution. To begin to examine cotranslational folding as it would occur within a cell, we produce highly selective, isotopically labeled NCs bound to isotopically silent ribosomes in vivo. We then apply NMR spectroscopy to study, at a residue specific level, the conformation of NCs consisting of different fractional lengths of the polypeptide chain corresponding to a given protein. This combined approach provides a powerful means of generating a series of snapshots of the folding of the NC as it emerges from the ribosome. Application of this strategy to the NMR analysis of the progressive synthesis of an Ig-like domain reveals the existence of a partially folded ribosome-bound species that is likely to represent an intermediate species populated during the cotranslational folding process.