Weak, specific chemical interactions dictate barnase stability in diverse cellular environments.

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Protein Science Pub Date : 2025-05-01 DOI:10.1002/pro.70128
Ume Tahir, Caitlin M Davis
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引用次数: 0

Abstract

It is well-established that in vitro measurements do not reflect protein behaviors in-cell, where macromolecular crowding and chemical interactions modulate protein stability and kinetics. Recent work suggests that peptides and small proteins experience the cellular environment differently from larger proteins, as their small sizes leave them primarily susceptible to chemical interactions. Here, we investigate this principle in diverse cellular environments, different intracellular compartments and host organisms. Our small protein folding model is barnase, a bacterial ribonuclease that has been extensively characterized in vitro. Using fast relaxation imaging, we find that FRET-labeled barnase is stabilized in the cytoplasm and destabilized in the nucleus of U2-OS cells. These trends could not be reproduced in vitro by Ficoll and M-PER™, which mimic macromolecular crowding and non-specific chemical interactions, respectively. Instead, in-cell trends were best replicated by cytoplasmic and nuclear lysates, indicating that weak specific interactions with proteins in either compartment are responsible for the in-cell observations. Interestingly, in the cytoplasm barnase's unfolded state is unstable and prone to aggregation, while in the nucleus a stable unfolded state exists prior to aggregation. In the more biologically relevant environment of bacterial cells, barnase folding resembled that in the nucleus, but with no aggregation at higher temperatures. These findings show that protein interactions are evolved for their native environment, which highlights the importance of studying and designing proteins in situ.

弱的,特定的化学相互作用决定了在不同的细胞环境中藤本酶的稳定性。
这是公认的体外测量不能反映蛋白质在细胞内的行为,其中大分子拥挤和化学相互作用调节蛋白质的稳定性和动力学。最近的研究表明,肽和小蛋白质与大蛋白质经历的细胞环境不同,因为它们的小尺寸使它们主要容易受到化学相互作用的影响。在这里,我们在不同的细胞环境、不同的细胞内区室和宿主生物中研究了这一原理。我们的小蛋白质折叠模型是藤蔓酶,一种细菌核糖核酸酶,已在体外广泛表征。通过快速弛缓成像,我们发现在U2-OS细胞中,fret标记的藤蔓酶在细胞质中稳定,而在细胞核中不稳定。这些趋势不能被Ficoll和M-PER™在体外重现,它们分别模拟大分子拥挤和非特异性化学相互作用。相反,细胞质和核裂解物可以最好地复制细胞内趋势,这表明细胞内观察与任何一个区室中蛋白质的弱特异性相互作用有关。有趣的是,在细胞质中,藤蔓酶的未折叠状态是不稳定的,易于聚集,而在细胞核中,在聚集之前存在一个稳定的未折叠状态。在细菌细胞的生物相关环境中,藤蔓酶折叠类似于细胞核中的折叠,但在更高的温度下没有聚集。这些发现表明,蛋白质相互作用是为其天然环境而进化的,这突出了原位研究和设计蛋白质的重要性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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