Structural and Mechanstic Aspects of Cotranslational Protein Folding

共翻译蛋白质折叠的结构和机制

• 批准号: 1616459
• 负责人: Silvia Cavagnero
• 金额: $ 92.87万
• 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: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616459&HistoricalAwards=false
• 关键词: Structural; Mechanstic; Aspects; Cotranslational; Protein

TITLE: Structural and Mechanistic Aspects of Cotranslational Protein FoldingThis project will lead to a better understanding of how proteins attain their 3-dimensional shape in the cell, providing a gateway to a deeper understanding and control of crucial life-related processes. The shape of proteins is extraordinarily important because it determines biological activity. However, little is known about how proteins achieve their bioactive three-dimensional shape inside the living cell. This research will elucidate how proteins gain their precise fold from the earliest stages of their life, from the time they are made a by a complex machine known as the ribosome. The role of the ribosome and molecular helpers known as chaperones will be explored. Molecular movies, showing folding as a function of time, will be generated to watch the microscopic details of the folding process in real time. This research will foster the participation of several underrepresented graduate students, and will be a benchmark to learn advanced biophysical techniques as well as the mechanisms of protein folding in the cell. In addition, classroom demonstrations showing illustrative renderings of the protein folding and unfolding processes will be developed, providing unprecedented opportunities for active learning and for undergraduate student participation in the classroom. As part of the broader impacts of this project, campus pre-visits for minority and disadvantaged undergraduate students will also be organized, to encourage them to pursue graduate studies.Despite the extensive literature available on the folding of purified proteins upon dilution from denaturant and upon temperature-jump, there is very little information on how proteins fold in the cellular environment, where chaperones, molecular crowding and the ribosome can profoundly affect the sequence and nature of the events leading to bioactive proteins. This project addresses the earliest stages of folding in the cellular context, as proteins emerge from the ribosome. The role of nascent chain compaction and thermodynamic stability, together with the effect of the ribosomal surface and the rate of full-length protein release from the ribosome, will be addressed at the molecular level. Experiments will be carried out by a combination of molecular-biology and biophysical techniques, including fluorescence depolarization and pulsed proteolysis on wild-type and rationally designed variants of the E. coli globin apoHmpH and on other control nascent proteins. Data will be collected in the absence and presence of the DnaK/DnaJ/GrpE chaperones, to assess the role played by each of these folding facilitators. The specific interaction between the ribosomal surface and nascent proteins will be assessed as function of ionic strength. The rate of release of ribosome-bound nascent proteins will be followed in real time to gain high-resolution insights into the events accompanying protein birth. Overall, this project will lead to an improved understanding of intracellular protein folding, one of the most fascinating aspects of gene expression.

标题：共翻译蛋白质折叠的结构和机制方面该项目将有助于更好地理解蛋白质如何在细胞中获得其 3 维形状，为更深入地理解和控制与生命相关的关键过程提供了途径。蛋白质的形状非常重要，因为它决定了生物活性。然而，人们对蛋白质如何在活细胞内实现其生物活性三维形状知之甚少。这项研究将阐明蛋白质如何从其生命的最早阶段，即从被称为核糖体的复杂机器制造出来的那一刻起，获得精确的折叠。将探讨核糖体和分子伴侣（称为伴侣）的作用。将生成显示折叠随时间变化的分子电影，以实时观察折叠过程的微观细节。这项研究将促进几名代表性不足的研究生的参与，并将成为学习先进生物物理技术以及细胞中蛋白质折叠机制的基准。此外，还将开发展示蛋白质折叠和展开过程的说明性效果图的课堂演示，为主动学习和本科生参与课堂提供前所未有的机会。作为该项目更广泛影响的一部分，还将组织少数民族和弱势本科生参观校园，以鼓励他们继续研究生学习。温度跳跃时，关于蛋白质如何在细胞环境中折叠的信息非常少，其中分子伴侣、分子拥挤和核糖体可以深刻地影响导致生物活性蛋白质的事件的序列和性质。该项目解决了细胞中蛋白质从核糖体中出现时折叠的最早阶段。新生链压缩和热力学稳定性的作用，以及核糖体表面的影响和全长蛋白质从核糖体释放的速率，将在分子水平上得到解决。实验将结合分子生物学和生物物理技术进行，包括对野生型和合理设计的大肠杆菌球蛋白 apoHmpH 变体以及其他对照新生蛋白进行荧光去极化和脉冲蛋白水解。将在 DnaK/DnaJ/GrpE 伴侣不存在和存在的情况下收集数据，以评估每个折叠促进子所发挥的作用。核糖体表面和新生蛋白质之间的特异性相互作用将作为离子强度的函数进行评估。将实时跟踪核糖体结合的新生蛋白质的释放速率，以获得对伴随蛋白质诞生的事件的高分辨率洞察。总体而言，该项目将提高对细胞内蛋白质折叠的理解，这是基因表达最令人着迷的方面之一。

项目成果

Net Charge and Nonpolar Content Guide the Identification of Folded and Prion Proteins

净电荷和非极性含量指导折叠蛋白和朊病毒蛋白的识别

• DOI: 10.1021/acs.biochem.9b01114
• 发表时间: 2020-05
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Yaeger;Jennaro, Theodore S.;Mecha, Miranda;Becker, Jenna H.;Yang, Hanming;Winkler, Gordon L.;Cavagnero, Silvia
• 通讯作者: Cavagnero, Silvia

Kinetic trapping in protein folding

蛋白质折叠中的动力学捕获

• DOI: 10.1093/protein/gzz018
• 发表时间: 2019-02
• 期刊: Design and Selection
• 作者: Varela, Angela E;England, Kevin A;Cavagnero, Silvia;Daggett, Valerie
• 通讯作者: Daggett, Valerie

Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins

荧光各向异性衰减和微量粘度测定揭示核糖体结合新生蛋白的压缩

• DOI: 10.1021/acs.jpcb.1c04473
• 发表时间: 2021-06
• 期刊: The Journal of Physical Chemistry B
• 作者: Hutchinson, Rachel B.;Chen, Xi;Zhou, Ningkun;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: 2021-10-29
• 期刊: Communications Biology
• 影响因子: 5.9
• 作者: Guzman-Luna V;Fuchs AM;Allen AJ;Staikos A;Cavagnero S
• 通讯作者: Cavagnero S

Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis

共翻译事件和翻译后事件在蛋白质从头生物合成中的互补作用

• DOI: 10.1021/acs.jpcb.0c03039
• 发表时间: 2020-07
• 期刊: The Journal of Physical Chemistry B
• 作者: Addabbo, Rayna M.;Dalphin, Matthew D.;Mecha, Miranda F.;Liu, Yue;Staikos, Alexios;Guzman;Cavagnero, Silvia
• 通讯作者: Cavagnero, Silvia