Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis
Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis
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DOI：
10.1021/acs.jpcb.0c03039
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发表时间：
2020-07-30
影响因子：
3.3
通讯作者：
Cavagnero, Silvia
中科院分区：
文献类型：
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作者：
Addabbo, Rayna M.;Dalphin, Matthew D.;Cavagnero, Silvia
在细胞中，共翻译和翻译后蛋白质折叠和聚集之间的关系知之甚少。在这里，我们采用了组合的荧光各向异性衰减在频域，荧光检测的溶解度测定，和NMR光谱，以探索在生物相关的背景下的蛋白质折叠的核糖体的作用。首先，我们发现核糖体的主要功能是促进共翻译起始蛋白的溶解性，从而支持共翻译折叠，即使在没有分子伴侣的情况下。然而，在这些条件下，只有一部分可溶性表达的蛋白质折叠并在溶液中自由翻滚。因此，单独的核糖体不足以保证脱辅基肌红蛋白（apoMb）模型蛋白的天然状态的定量形成。生物合成后，新生的链编码apoMb出现从核糖体出口隧道，并经历一个关键的不可逆的翻译后动力学进一步折叠和聚集之间的分配。突变分析结合蛋白质表达动力学和NMR表明，新生蛋白质可以达到其天然状态，只有当从核糖体释放后立即形成的可溶性和不溶性产物的相对速率是倾斜的，有利于可溶性物种。最后，上述立即翻译后动力学分配的结果对氨基酸序列扰动比对突变不敏感的天然折叠敏感得多。因此，从核糖体释放的初生蛋白构象的动力学通道可能是进化压力的主要决定因素。
The relation between co- and post-translational protein folding and aggregation in the cell is poorly understood. Here, we employ a combination of fluorescence anisotropy decays in the frequency domain, fluorescence-detected solubility assays, and NMR spectroscopy to explore the role of the ribosome in protein folding within a biologically relevant context. First, we find that a primary function of the ribosome is to promote cotranslational nascent-protein solubility, thus supporting cotranslational folding even in the absence of molecular chaperones. Under these conditions, however, only a fraction of the soluble expressed protein is folded and freely tumbling in solution. Hence, the ribosome alone is insufficient to guarantee quantitative formation of the native state of the apomyoglobin (apoMb) model protein. Right after biosynthesis, nascent chains encoding apoMb emerge from the ribosomal exit tunnel and undergo a crucial irreversible post-translational kinetic partitioning between further folding and aggregation. Mutational analysis in combination with protein-expression kinetics and NMR show that nascent proteins can attain their native state only when the relative rates of soluble and insoluble product formation immediately upon release from the ribosome are tilted in favor of soluble species. Finally, the outcome of the above immediately post-translational kinetic partitioning is much more sensitive to amino acid sequence perturbations than the native fold, which is rather mutation-insensitive. Hence, kinetic channeling of nascent-protein conformation upon release from the ribosome may be a major determinant of evolutionary pressure.