Phase transition pathways encode distinct physicochemical properties of biomolecular condensates

The same intrinsically disordered proteins (IDPs) can form biomolecular condensates through distinct thermodynamic phase transition processes, such as temperature-dependent and osmosis-dependent pathways. These distinct thermodynamic driving forces should, in principle, induce phase transition by modulating different features of the solvent environments, so that different driving forces should correspond with distinct sequence grammars for phase transition. However, whether the molecular driving force is pathway-dependent and how these different pathways can define the properties and functions of condensates are largely unknown. Here, by employing a diverse set of solid-state and solution-state NMR techniques, we uncover that different phase transition pathways of the monomer unit of an IDP define the types of molecular interactions driving phase transition and stabilizing dense phases. By establishing a complete chemical shift profile of the IDP unit, we identified a unique interaction mode that specifically initiates the upper critical solution temperature transition process, an anion-dependent cation-pi interaction, in which Asp acts as a bistable molecular switch regulating a stepwise Arg-Tyr interaction in a critical temperature-dependent manner. We further show that the pathway-dependent molecular interactions encode condensates formed by the same IDP to exhibit different physical and electrochemical properties, which in turn enable distinct functions of condensates. Our study shows that besides the defined sequence grammar of a given IDP, the molecular driving forces under specific transition processes are different and can determine the structure and properties of condensates, which emphasizes an overlooked role of transition pathway on encoding the functions of condensates.