Biophysical Aspects of Co- and Post-Translational Protein Folding

共翻译和翻译后蛋白质折叠的生物物理方面

• 批准号: 2124672
• 负责人: Silvia Cavagnero
• 金额: $ 82.98万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2124672&HistoricalAwards=false
• 关键词: Biophysical; Aspects; Co; Post; Translational

Proteins perform biological functions that are crucial to enable, support and protect life. Protein shape is extraordinarily important because it determines biological activity. The goal of this project is to reveal how proteins achieve their complex and highly organized three-dimensional shape in the context of living cells. This process is commonly referred to as protein folding. This is an intricate process, and it occurs at the level of individual molecules in the complex cellular environment. Unfortunately, very little is still known about how proteins fold in the biological milieu. As part of this project, the high-resolution structure of the ribosome, the protein-making machinery, as it binds and generates proteins will be mapped. With the help of sophisticated lasers, magnets and electron beams, how the nascent protein interacts with the ribosome and how the ribosome in turn helps the protein fold is expected to be revealed. This process is similar to picture-taking at the molecular level. The process of protein making in real time and watching 3D-protein-shape formation as it happens will be followed. These studies are important because they will show how proteins avoid misfolding, which could compromise their biological function. This project will promote participation of underrepresented graduate and undergraduate students and will be a benchmark to learn advanced biological techniques and mechanisms. Classroom demonstrations involving colorful renderings of protein folding/unfolding will be developed, providing unprecedented opportunities for active learning.This project focuses on the earliest stages of protein folding as nascent chains emerge out of the ribosome in the absence and presence of molecular chaperones. Very little is still known about how proteins fold in the cellular environment. Importantly, translation through the ribosome is often required to generate fully folded and bioactive proteins within the in vivo milieu. Yet, the role of the ribosome in the protein-making process is still extremely poorly understood. In this research, single-domain proteins of variable size originating from codon-optimized genes in E. coli will be studied. The project involves the determination of 3D structure of ribosome-bound nascent proteins by single-particle cryo-electron microscopy, as well as the elucidation of nascent-protein dynamics by time-resolved fluorescence anisotropy. In addition, nascent-protein ribosome interactions will be studied by chemical crosslinking. Through the proposed studies, insights into how interactions with the ribosome and molecular chaperones ensure that nascent proteins attain their native state devoid of competing aggregation will be gained. The principal investigator and her research group has determined that the last stages of translation and the events accompanying full-length ribosome-bound nascent-chain (RNC) release from the ribosome are crucial to kinetically channel proteins to their native state, away from aggregation. Next, these stages at much higher resolution will be characterized to fully understand the function of ribosome and molecular chaperones in the cellular context. These investigations are significant for a better understanding of biological phenomena as well as for the future custom-design of proteins able to more robustly and efficiently withstand severe environmental perturbations.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

蛋白质所发挥的生物学功能对于生命的存在、支持和保护至关重要。蛋白质形状非常重要，因为它决定生物活性。该项目的目标是揭示蛋白质如何在活细胞中实现复杂且高度组织的三维形状。这个过程通常被称为蛋白质折叠。这是一个复杂的过程，并且发生在复杂细胞环境中的单个分子水平上。不幸的是，人们对蛋白质在生物环境中如何折叠仍然知之甚少。作为该项目的一部分，将绘制核糖体（蛋白质制造机器）结合和生成蛋白质时的高分辨率结构。在复杂的激光、磁铁和电子束的帮助下，新生蛋白质如何与核糖体相互作用以及核糖体如何帮助蛋白质折叠有望被揭示。这个过程类似于分子水平上的拍照。我们将跟踪实时蛋白质制造过程并观察 3D 蛋白质形状的形成过程。这些研究很重要，因为它们将展示蛋白质如何避免错误折叠，从而损害其生物功能。该项目将促进代表性不足的研究生和本科生的参与，并将成为学习先进生物技术和机制的基准。将开发涉及蛋白质折叠/展开的彩色渲染的课堂演示，为主动学习提供前所未有的机会。该项目重点关注蛋白质折叠的最早阶段，即在分子伴侣不存在或存在的情况下，新生链从核糖体中出现。关于蛋白质在细胞环境中如何折叠仍然知之甚少。重要的是，通常需要通过核糖体翻译才能在体内环境中产生完全折叠的生物活性蛋白质。然而，人们对核糖体在蛋白质制造过程中的作用仍然知之甚少。在这项研究中，将研究源自大肠杆菌中密码子优化基因的可变大小的单域蛋白质。该项目涉及通过单粒子冷冻电子显微镜测定核糖体结合的新生蛋白的 3D 结构，以及通过时间分辨荧光各向异性阐明新生蛋白动力学。此外，新生蛋白质核糖体相互作用将通过化学交联进行研究。通过拟议的研究，将深入了解与核糖体和分子伴侣的相互作用如何确保新生蛋白质达到其天然状态，而无需竞争聚集。首席研究员和她的研究小组已经确定，翻译的最后阶段以及伴随全长核糖体结合的新生链 (RNC) 从核糖体释放的事件对于动态地将蛋白质引导至其天然状态、远离聚集至关重要。接下来，将以更高的分辨率表征这些阶段，以充分了解核糖体和分子伴侣在细胞环境中的功能。这些研究对于更好地理解生物现象以及未来定制设计能够更稳健、更有效地承受严重环境扰动的蛋白质具有重要意义。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准。

项目成果

Critical Beginnings: Selective Tuning of Solubility and Structural Accuracy of Newly Synthesized Proteins by the Hsp70 Chaperone System

关键起点：Hsp70 分子伴侣系统选择性调节新合成蛋白质的溶解度和结构准确性

• DOI: 10.1021/acs.jpcb.2c08485
• 发表时间: 2023-05
• 期刊: The Journal of Physical Chemistry B
• 作者: Addabbo, Rayna M.;Hutchinson, Rachel B.;Allaman, Heather J.;Dalphin, Matthew D.;Mecha, Miranda F.;Liu, Yue;Staikos, Alexios;Cavagnero, Silvia
• 通讯作者: Cavagnero, Silvia

Mapping Protein–Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome–Nascent Globin Complex

绘制出生时蛋白质与蛋白质相互作用图谱：核糖体与新生球蛋白复合物的单粒子冷冻电镜分析

• DOI: 10.1021/acscentsci.3c00777
• 发表时间: 2024-02
• 期刊: ACS Central Science
• 影响因子: 18.2
• 作者: Masse, Meranda M.;Hutchinson, Rachel B.;Morgan, Christopher E.;Allaman, Heather J.;Guan, Hongqing;Yu, Edward W.;Cavagnero, Silvia
• 通讯作者: Cavagnero, Silvia

Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome

Hsp70 伴侣结合位点在大肠杆菌蛋白质组中的分布和溶剂暴露

• DOI: 10.1002/prot.26456
• 发表时间: 2023-01
• 期刊: and Bioinformatics
• 作者: Chen, Xi;Hutchinson, Rachel B.;Cavagnero, Silvia
• 通讯作者: Cavagnero, Silvia

An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel

本质上无序的新生蛋白与出口隧道附近核糖体表面的特定区域相互作用

• DOI: 10.1038/s42003-021-02752-4
• 发表时间: 2021-10
• 期刊: Communications Biology
• 影响因子: 5.9
• 作者: Guzman;Fuchs, Andrew M.;Allen, Anna J.;Staikos, Alexios;Cavagnero, Silvia
• 通讯作者: Cavagnero, Silvia

Protein folding in vitro and in the cell: From a solitary journey to a team effort

体外和细胞内的蛋白质折叠：从单独的旅程到团队的努力

• DOI: 10.1016/j.bpc.2022.106821
• 发表时间: 2022-08
• 期刊: Biophysical Chemistry
• 影响因子: 3.8
• 作者: Mecha, Miranda F.;Hutchinson, Rachel B.;Lee, Jung Ho;Cavagnero, Silvia
• 通讯作者: Cavagnero, Silvia