Small Molecules Influence the Physical Microenvironment of Biomolecular Condensates

Biomolecular condensates exhibit distinct microenvironments that arise from interactions between proteins, RNA, and solutions. In aqueous solutions, these membraneless structures constantly encounter small molecules that could affect the structure and properties of the condensates. However, the effects of organic small molecules in water solutions on the microenvironments of condensates remain poorly understood. In this study, we used various organic solutes as an example to explore how small molecules could influence the physicochemical properties in the microenvironment of protein condensates. Particularly, we quantitatively studied micropolarity and microviscosity using a combination of techniques, including fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, and passive rheology. Unexpectedly, our results revealed that the microenvironment was not correlated with the polarity of organic solutes; instead, the correlation was observed on the interaction strength between water and small molecules. We found that solutes with stronger interaction with water and weaker interaction with proteins increase the micropolarity and decrease the microviscosity of condensates. Furthermore, we demonstrated that the modulation of the micropolarity of condensates could impact the miscibility of multicomponent condensates. Finally, we showed that organic solutes could influence the micropolarity of condensates and the partitioning of products in condensates, thus affecting the rate and equilibrium of the chemical reactions. In summary, our work provides a quantitative analysis of how the microenvironment of biomolecular condensates is impacted by organic solutes. Since protein condensates coexist with various types of metabolites in the aqueous cellular milieu, results from this work offer insights into how organic metabolites could regulate the microenvironment and behaviors of biological condensates.