Biomolecular condensates formed through liquid-liquid phase separation play crucial roles in cellular organization and are implicated in neurodegenerative diseases when aberrant phase transitions occur. Understanding molecular mobility and potential heterogeneous behavior within these condensates is essential for elucidating how macromolecular crowding influences protein dynamics, interactions, and function. Here, we employ a model system using bovine serum albumin (BSA) proteins, which form condensate droplets in the presence of polyethylene glycol (PEG) as a crowding agent, mimicking the dense cytoplasmic environment. Recent studies have shown that such protein-rich droplets can sustain enzymatic activity and exhibit dynamic behavior similar to intracellular condensates with viscoelastic properties resembling the soft glassy rheology observed in supercooled molecular liquids. We aimed to characterize protein dynamics and investigate spatial heterogeneity within these droplets using complementary fluorescence techniques: Fluorescence correlation spectroscopy (FCS), raster image correlation spectroscopy (RICS), and fluorescence recovery after photobleaching (FRAP). Our findings demonstrate that BSA is highly segregated into droplets, creating a 100× more viscous environment compared to the supernatant phase. FRAP is powerful at detecting the slow mobility components, but a quantitative analysis is tricky. FCS and RICS provide robust protein mobility measurements in biomolecular condensates, revealing substantially reduced molecular diffusion within protein-rich condensates. RICS offers distinct advantages through distributed excitation that reduces photobleaching and captures spatial heterogeneity.
Priyanshi Kalra (Sun,) studied this question.