Department of Biology, University of Rochester, Rochester, NY 14627.
Mass Spectrometry Resource Laboratory, University of Rochester Medical Center, Rochester, NY 14627.
Proc Natl Acad Sci U S A. 2023 Aug 15;120(33):e2303167120. doi: 10.1073/pnas.2303167120. Epub 2023 Aug 8.
The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.
大多数蛋白质的折叠发生在其翻译过程中，而它们与 tRNA 结合的 C 末端嵌入核糖体中。新生蛋白质与核糖体的接近程度如何影响其折叠热力学仍然知之甚少。在这里，我们开发了一种基于质谱的方法，使用甲硫氨酸氧化作为折叠探针来确定新生多肽链的稳定性。该方法能够在复杂的蛋白质混合物和提取物中定量测量核糖体新生链的亚全局折叠稳定性。为了验证该方法，我们分析了三种模型蛋白（二氢叶酸还原酶、趋化蛋白 Y 和 DNA 聚合酶 IV）在可溶性和核糖体结合状态下的折叠热力学。数据表明，核糖体可以显著改变新生多肽的稳定性。核糖体诱导的稳定性调制在不同的折叠结构域之间差异很大，并且依赖于新生多肽中的局部电荷分布。结果表明，核糖体表面和新生多肽之间的静电相互作用是核糖体诱导稳定性调制的原因。该研究建立了一种强大的蛋白质组学方法，用于分析核糖体结合的新生多肽中的局部稳定性，并阐明了核糖体如何影响蛋白质折叠的热力学。
