化学相互作用调节细胞中的6-85稳定性.
Edward Knab1, Caitlin M Davis1
1Department of Chemistry, Yale University, New Haven, Connecticut, USA.
Protein science : a publication of the Protein Society
|June 14, 2023
概括
细胞环境通过化学相互作用稳定小蛋白质,而不是硬质拥挤. 这项研究量化了细胞内蛋白质的稳定性,发现化学相互作用是关键,并提出了一种简化的体外方法来预测蛋白质的行为.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 细胞生物物理学 细胞生物物理学
背景情况:
- 固体拥挤对蛋白质折叠的影响取决于大小.
- 由于巨分子大小和化学相互作用,细胞环境对蛋白质稳定性提出了独特的挑战.
- 以前的体外研究表明化学相互作用,而不是固体拥挤,影响细胞内蛋白质的稳定性.
研究的目的:
- 直接量化兰巴抑制剂片段 (λ6-85) 的细胞内稳定性.
- 区分固体拥挤和化学相互作用对细胞环境中的蛋白质稳定性的贡献.
- 验证和完善用于预测细胞内蛋白质行为的体外模型.
主要方法:
- 使用一个标记为FRET的λ6-85结构进行稳定性测量.
- 细胞内稳定性与体外稳定性相比,在Ficoll (形) 和哺乳动物蛋白质提取试剂 (M-PERTM,化学相互作用) 的存在下.
- 通过使用FRET值,评估了宏分子度对细胞拥挤的影响.
主要成果:
- 与体外条件相比,λ6-85片段在细胞内表现出5°C的稳定性.
- 作为一种固体挤压器的 Ficoll 没有影响 λ6-85 的稳定性,这证实了固体挤压的作用很小.
- 细胞内稳定归因于化学相互作用,M-PERTM在体外有效模仿.
- 在U-2 OS细胞中的细胞质挤压被细胞模拟复制为15% (w/v) 大分子度.
- 20% (v/v) 的简化的体外混合物M-PERTM单独准确地重现了λ6-85.5的细胞内稳定性.
结论:
- 化学相互作用,而不是固态拥挤,是细胞环境中小蛋白质稳定的主要驱动因素.
- 该研究验证了用于蛋白质和RNA折叠研究的现有体外细胞相对应混合物.
- 一个基于M-PERTM的简化体外系统显示出预测其他小蛋白和的细胞内行为的前景.
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