Macromolecular crowding in vitro and in cells

体外和细胞内的大分子拥挤

• 批准号: 1909664
• 负责人: Gary Pielak
• 金额: $ 90万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1909664&HistoricalAwards=false
• 关键词: Macromolecular; crowding; vitro; cells

Protein-protein interactions regulate all cellular functions, yet they are almost always studied in water rather than in the complex and crowded environment that exists inside cells. The results of this project will fill a key gap that prevents a complete description of metabolism. The goal of moving quantitative biophysics from simple solutions to crowded environments, including inside living cells, is a major challenge with important outcomes. The knowledge gained will add to both the fundamental understanding of biology and inform efforts to produce and stabilize protein-based reagents. The work will also facilitate the training of undergraduate and graduate students in the practice of cutting-edge research. All these efforts are key to building the US bioeconomy.The overall goal of the project is to gain broadly-applicable knowledge about protein-protein interactions under physiologically-relevant conditions. The focus is on homodimeric interactions in vitro, in Escherichia coli cells and in zebrafish (Danio rerio) oocytes. The overarching hypothesis is that electrostatic interactions between cosolutes and protein complexes dominate the effect of hard-core excluded volume under physiologically-relevant conditions. More specifically, electrostatic attractions between cosolutes and protein complexes are expected to decrease stability of the complexes, whereas electrostatic repulsions are expected to increase stability. The supported research will also deliver to modelers and theoreticians the information required to produce accurate simulations of cellular processes. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.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.

蛋白质之间的相互作用调节着所有的细胞功能，但它们几乎总是在水中进行研究，而不是在细胞内部复杂而拥挤的环境中进行研究。这个项目的结果将填补一个关键的空白，阻止新陈代谢的完整描述。将定量生物物理学从简单的解决方案转移到拥挤的环境，包括活细胞内部，是一项具有重要成果的重大挑战。所获得的知识将增加对生物学的基本理解，并为生产和稳定基于蛋白质的试剂提供信息。这项工作还将有助于培养本科生和研究生从事前沿研究的实践。所有这些努力都是建立美国生物经济的关键。该项目的总体目标是获得在生理相关条件下蛋白质-蛋白质相互作用的广泛应用知识。重点是在体外，在大肠杆菌细胞和斑马鱼（Danio rerio）卵母细胞的同二聚体相互作用。总体假设是，在生理相关条件下，co溶质和蛋白质复合物之间的静电相互作用主导了硬核排除体积的影响。更具体地说，co溶质和蛋白质复合物之间的静电吸引预计会降低复合物的稳定性，而静电排斥预计会增加稳定性。这项支持的研究还将为建模者和理论家提供精确模拟细胞过程所需的信息。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理集群的支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Buffers, Especially the Good Kind

• DOI: 10.1021/acs.biochem.1c00200
• 发表时间: 2021-05-03
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Pielak, Gary J.

Positively Charged Tags Impede Protein Mobility in Cells as Quantified by 19F NMR.

• DOI: 10.1021/acs.jpcb.9b02162
• 发表时间: 2019-05
• 期刊: The journal of physical chemistry. B
• 作者: Yansheng Ye;Qiong Wu;Wenwen Zheng;Bin Jiang;G. Pielak;Maili Liu;Conggang Li

Dynamical spectroscopy and microscopy of proteins in cells

• DOI: 10.1016/j.sbi.2021.02.001
• 发表时间: 2021-10-01
• 期刊: CURRENT OPINION IN STRUCTURAL BIOLOGY
• 影响因子: 6.8
• 作者: Gruebele, Martin;Pielak, Gary J.
• 通讯作者: Pielak, Gary J.

Controlling and quantifying protein concentration in Escherichia coli

• DOI: 10.1002/pro.3637
• 发表时间: 2019-07-01
• 期刊: PROTEIN SCIENCE
• 影响因子: 8
• 作者: Speer, Shannon L.;Guseman, Alex J.;Pielak, Gary J.
• 通讯作者: Pielak, Gary J.

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis.

• DOI: 10.1016/j.bpj.2020.03.031
• 发表时间: 2020-04
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Samantha S. Stadmiller;Jhoan S. Aguilar;C. Waudby;G. Pielak