Chen Xiuqi, Kaiser Christian M
CMDB Graduate Program, Johns Hopkins University, Baltimore, MD, United States.
Department of Biology, Johns Hopkins University, Baltimore, MD, United States.
bioRxiv. 2023 Sep 1:2023.09.01.555749. doi: 10.1101/2023.09.01.555749.
Natural proteins have evolved to fold robustly along specific pathways. Folding begins during synthesis, guided by interactions of the nascent protein with the ribosome and molecular chaperones. However, the timing and progression of co-translational folding remain largely elusive, in part because the process is difficult to measure in the natural environment of the cytosol. We developed a high-throughput method to quantify co-translational folding in live cells that we term Arrest Peptide profiling (AP profiling). We employed AP profiling to delineate co-translational folding for a set of GTPase domains with very similar structures, defining how topology shapes folding pathways. Genetic ablation of major nascent chain-binding chaperones resulted in localized folding changes that suggest how functional redundancies among chaperones are achieved by distinct interactions with the nascent protein. Collectively, our studies provide a window into cellular folding pathways of complex proteins and pave the way for systematic studies on nascent protein folding at unprecedented resolution and throughput.
天然蛋白质已经进化到能够沿着特定途径稳健折叠。折叠在合成过程中开始，由新生蛋白质与核糖体和分子伴侣的相互作用引导。然而，共翻译折叠的时间和进程在很大程度上仍然难以捉摸，部分原因是该过程在细胞质的自然环境中难以测量。我们开发了一种高通量方法来量化活细胞中的共翻译折叠，我们将其称为滞留肽分析（AP分析）。我们采用AP分析来描绘一组结构非常相似的GTPase结构域的共翻译折叠，确定拓扑结构如何塑造折叠途径。主要新生链结合伴侣的基因缺失导致局部折叠变化，这表明伴侣之间的功能冗余是如何通过与新生蛋白质的不同相互作用实现的。总的来说，我们的研究为复杂蛋白质的细胞折叠途径提供了一个窗口，并以前所未有的分辨率和通量为新生蛋白质折叠的系统研究铺平了道路。
