Ficoll, an inert macromolecule, is a common in vitro crowder, but by itself it does not reproduce in-cell stability or kinetic trends for protein folding. Lysis buffer, which contains ions, glycerol as a simple kosmotrope, and mimics small crowders with hydrophilic/hydrophobic patches, can reproduce sticking trends observed in cells but not the crowding. We previously suggested that the proper combination of Ficoll and lysis buffer could reproduce the opposite in-cell folding stability trend of two proteins: variable major protein-like sequence expressed (VlsE) is destabilized in eukaryotic cells and phosphoglycerate kinase (PGK) is stabilized. Here, to discover a well-characterized solvation environment that mimics in-cell stabilities for these two very differently behaved proteins, we conduct a two-dimensional scan of Ficoll (0-250 mg/ml) and lysis buffer (0-75%) mixtures. Contrary to our previous expectation, we show that mixtures of Ficoll and lysis buffer have a significant nonadditive effect on the folding stability. Lysis buffer enhances the stabilizing effect of Ficoll on PGK and inhibits the stabilizing effect of Ficoll on VlsE. We demonstrate that a combination of 150 mg/ml Ficoll and 60% lysis buffer can be used as an in vitro mimic to account for both crowding and non-steric effects on PGK and VlsE stability and folding kinetics in the cell. Our results also suggest that this mixture is close to the point where phase separation will occur. The simple mixture proposed here, based on commercially available reagents, could be a useful tool to study a variety of cytoplasmic protein interactions, such as folding, binding and assembly, and enzymatic reactions. SIGNIFICANCE STATEMENT: The complexity of the in-cell environment is difficult to reproduce in the test tube. Here we validate a mimic of cellular crowding and sticking interactions in a test tube using two proteins that are differently impacted by the cell: one is stabilized and the other is destabilized. This mimic is a starting point to reproduce cellular effects on a variety of protein and biomolecular interactions, such as folding and binding.

中文翻译：

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用于蛋白折叠研究的细胞内溶剂化的体外模拟物。

Ficoll是一种惰性大分子，是一种常见的体外拥挤剂，但其本身无法再现细胞内稳定性或蛋白质折叠的动力学趋势。裂解缓冲液包含离子，甘油作为简单的共溶物，并模仿具有亲水/疏水补丁的小拥挤物，可以重现在细胞中观察到的粘附趋势，但不能重现拥挤的趋势。我们以前认为，Ficoll和裂解缓冲液的正确组合可以重现两种蛋白质的相反的细胞内折叠稳定性趋势：真核细胞中不稳定的主要蛋白样序列表达（VlsE）不稳定，磷酸甘油酸激酶（PGK）稳定。在这里，要发现一个表征良好的溶剂化环境，该环境模拟这两种行为迥异的蛋白质的细胞内稳定性，我们对Ficoll（0-250 mg / ml）和裂解缓冲液（0-75％）混合物进行二维扫描。与我们先前的预期相反，我们显示Ficoll和裂解缓冲液的混合物对折叠稳定性具有显着的非累加作用。裂解缓冲液可增强Ficoll对PGK的稳定作用，并抑制Ficoll对VlsE的稳定作用。我们证明了150 mg / ml Ficoll和60％裂解缓冲液的组合可以用作体外模拟物，以解决PGK和VlsE稳定性以及细胞中折叠动力学的拥挤和非空间效应。我们的结果还表明，该混合物接近发生相分离的点。本文提出的基于市售试剂的简单混合物可能是研究多种细胞质蛋白相互作用（例如折叠，结合和组装，以及酶促反应。意义声明：难以在试管中再现细胞内环境的复杂性。在这里，我们使用两种受细胞影响不同的蛋白质，验证了试管中细胞拥挤和黏附相互作用的模拟情况：一种是稳定的，另一种是不稳定的。这种模拟是在多种蛋白质和生物分子相互作用（例如折叠和结合）上复制细胞效应的起点。