Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

生物体利用称为渗透剂的有机分子来适应环境条件。体外研究表明渗透剂可以热稳定蛋白质，但其机制存在争议，并且缺乏细胞环境内的系统研究。我们通过原位渗透剂和整个蛋白质组分析了大肠杆菌和人类蛋白质的热稳定性。利用结构蛋白质组学，我们探讨了渗透剂对蛋白质热稳定性、结构和聚集的影响，揭示了常见机制以及渗透剂和蛋白质特异性的影响。所有测试的渗透剂（三甲胺N-氧化物、甜菜碱、甘油、脯氨酸、海藻糖和葡萄糖）主要通过优先排斥机制稳定许多蛋白质，并导致大多数蛋白质聚集的温度向上变化。人类蛋白质组的热分析提供了原位内在无序的证据，但也确定了预测无序区域的潜在结构。我们的分析提供了对复杂生物基质内渗透剂功能的机制洞察，并揭示了本质无序区域的原位流行情况。