Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen–deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unanticipated pathway through structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing carboxy-terminal sequences remain confined in the ribosome exit tunnel. Trigger factor binds partially folded states without disrupting their structure, and the nascent chain is poised to complete folding immediately upon emergence of the C terminus from the exit tunnel. By mapping interactions between the nascent chain and ribosomal proteins, we trace the path of the emerging polypeptide during synthesis. Our work reveals new mechanisms by which cellular factors shape the conformational search for the native state.

体内蛋白质折叠在核糖体合成过程中开始，并受与新生多肽结合的分子伴侣调节。蛋白质生物发生的这些特征如何影响新生蛋白质的成熟途径尚不完全清楚。在这里，我们使用氢 - 氘交换质谱法以肽分辨率定义大肠杆菌二氢叶酸还原酶的共翻译伴侣辅助折叠途径。新生的多肽沿着意想不到的途径折叠，穿过从变性物重新折叠过程中未填充的结构化中间体。与核糖体的结合允许这些中间体形成，否则不稳定的羧基末端序列仍然局限在核糖体出口隧道中。触发因子结合部分折叠状态而不破坏其结构，并且新生链准备在 C 末端从出口隧道中出现后立即完成折叠。通过绘制新生链和核糖体蛋白之间的相互作用，我们追踪了合成过程中出现的多肽的路径。我们的工作揭示了细胞因子塑造天然状态的构象寻找的新机制。