Macromolecular crowding modulates phase behaviour and structural maturation of β-lactoglobulin condensates

Liquid-liquid phase separation (LLPS) under crowded conditions promotes the formation of protein-rich condensates that can undergo structural and physicochemical transformations over time. Understanding how these dynamic assemblies evolve toward more ordered states is essential for elucidating the mechanisms governing condensate maturation and for exploiting LLPS in the design of functional biomaterials. Here, we investigate the phase behaviour of β-lactoglobulin (BLG) under polyethylene glycol (PEG)-induced crowding conditions, following the evolution of protein-rich condensates from their formation and growth to the emergence of intermolecular β-sheet-rich solid assemblies. By combining environment-sensitive fluorescent probes with fluorescence lifetime, spectral-phasor, and fluorescence recovery after photobleaching (FRAP) microscopy, we monitored changes in the local molecular environment, protein mobility, and internal organization of individual condensates. These complementary approaches reveal the progressive emergence of structural heterogeneity and localized β-sheet-rich domains within individual condensates. Notably, these transformations occur within hours under mild, non-denaturing conditions and at relatively low protein concentrations, where BLG is generally regarded as a stable globular protein. Presented results provide a comprehensive picture of condensate maturation in this model system and demonstrate how multimodal fluorescence imaging enables the spatially resolved characterization of dynamic structural transitions in protein condensates, offering a valuable framework for investigating and engineering protein-based soft materials.