Molecular Aggregation Behavior and Microscopic Heterogeneity in Binary Osmolyte–Water Solutions

Osmolytes, small organic compounds, play a key role in modulating the protein stability in aqueous solutions, but the operating mechanism of the osmolyte remains inconclusive. Here, we attempt to clarify the mode of osmolyte action by quantitatively estimating the microheterogeneity of osmolyte–water mixtures with the aid of molecular dynamics simulation, graph theoretical analysis, and spatial distribution measurement in the four osmolyte solutions of trimethylamine-N-oxide (TMAO), tetramethylurea (TMU), dimethyl sulfoxide, and urea. TMAO, acting as a protecting osmolyte, tends to remain isolated with no formation of osmolyte aggregates while preferentially interacting with water, but there is a strong aggregation propensity in the denaturant TMU solution, characterized by favored hydrophobic interactions between TMU molecules. Taken together, the mechanism of osmolyte action on protein stability is proposed as a comprehensive one that encompasses the direct interactions between osmolytes and proteins and indirect interactions through the regulation of water properties in the osmolyte–water mixtures.渗透调节剂（osmolytes）作为一类小分子有机化合物，在水溶液中对蛋白质稳定性的调控发挥关键作用，但其具体作用机制仍未明确。为此，本研究借助分子动力学模拟、图论分析与空间分布测量技术，对三甲胺-N-氧化物（trimethylamine-N-oxide, TMAO）、四甲基脲（tetramethylurea, TMU）、二甲基亚砜以及尿素四种渗透调节剂的水溶液体系进行研究，通过定量估算其混合体系的微观异质性，以期阐明渗透调节剂的作用模式。作为保护性渗透调节剂的TMAO倾向于保持孤立状态，不会形成渗透调节剂聚集体，且优先与水分子相互作用；而作为变性剂的TMU溶液则表现出强烈的聚集倾向，其特征为TMU分子间存在优势疏水相互作用。综上，本研究提出渗透调节剂对蛋白质稳定性的作用机制为综合机制，既包含渗透调节剂与蛋白质之间的直接相互作用，也涵盖通过调控渗透调节剂-水混合体系中水的性质而产生的间接相互作用。