Department of Chemistry, Yale University, New Haven, Connecticut, USA.
Protein Sci. 2023 Jul;32(7):e4698. doi: 10.1002/pro.4698.
Because steric crowding is most effective when the crowding agent is similar in size to the molecule that it acts upon and the average macromolecule inside cells is much larger than a small protein or peptide, steric crowding is not predicted to affect their folding inside cells. On the other hand, chemical interactions should perturb in-cell structure and stability because they arise from interactions between the surface of the small protein or peptide and its environment. Indeed, previous in vitro measurements of the λ-repressor fragment, λ , in crowding matrices comprised of Ficoll or protein crowders support these predictions. Here, we directly quantify the in-cell stability of λ and distinguish the contribution of steric crowding and chemical interactions to its stability. Using a FRET-labeled λ construct, we find that the fragment is stabilized by 5°C in-cells compared to in vitro. We demonstrate that this stabilization cannot be explained by steric crowding because, as anticipated, Ficoll has no effect on λ stability. We find that the in-cell stabilization arises from chemical interactions, mimicked in vitro by mammalian protein extraction reagent (M-PER™). Comparison between FRET values in-cell and in Ficoll confirms that U-2 OS cytosolic crowding is reproduced at macromolecule concentrations of 15% w/v. Our measurements validate the cytomimetic of 15% Ficoll and 20% M-PER™ that we previously developed for protein and RNA folding studies. However, because the in-cell stability of λ is reproduced by 20% v/v M-PER™ alone, we predict that this simplified mixture could be a useful tool to predict the in-cell behaviors of other small proteins and peptides.
由于空间位阻在与被作用分子大小相似的拥挤剂作用时最有效，并且细胞内的平均大分子远远大于小蛋白质或肽，因此预计空间位阻不会影响它们在细胞内的折叠。另一方面，化学相互作用应该会扰乱细胞内结构和稳定性，因为它们源于小蛋白质或肽的表面与其环境之间的相互作用。事实上，先前在拥挤基质中对 λ 抑制因子片段 λ 的体外测量支持了这些预测。在这里，我们直接量化了 λ 在细胞内的稳定性，并区分了空间位阻和化学相互作用对其稳定性的贡献。使用 FRET 标记的 λ 构建体，我们发现与体外相比，该片段在细胞内稳定 5°C。我们证明这种稳定性不能用空间位阻来解释，因为正如预期的那样，Ficoll 对 λ 的稳定性没有影响。我们发现这种细胞内稳定性来自化学相互作用，在体外通过哺乳动物蛋白提取试剂 (M-PER™) 模拟。细胞内和 Ficoll 中的 FRET 值比较证实，U-2 OS 细胞质拥挤在大分子浓度为 15%w/v 时得以重现。我们的测量结果验证了我们之前为蛋白质和 RNA 折叠研究开发的 15%Ficoll 和 20%M-PER™ 的细胞模拟物。然而，由于 λ 的细胞内稳定性仅由 20%v/v 的 M-PER™ 单独重现，我们预测这种简化的混合物可以成为预测其他小蛋白质和肽在细胞内行为的有用工具。
