Biophysical exploration into the water phase within biomolecular condensates

Abstract :

Intrinsically disordered proteins (IDPs), rich in positively charged residues such as Lysine and Arginine, are key drivers of liquid-liquid phase separation (LLPS), forming biomolecular condensates through electrostatic interactions with the negatively charged phosphate backbone of nucleic acids (UTP/ATP) and additional non-covalent interactions with nucleobases. Biomolecular condensates are known to contain 50-70% water along with biomacromolecules. Spectroscopic studies reveal that this internal water is restricted and less mobile, suggesting it is not merely a passive solvent but rather an active structural component that modulates intermolecular interactions in condensates. Here, we introduce new fluorescent probes to investigate the aqueous phase within biomolecular condensates. These probes act as photoacids and photobases, with pyranine (HPTS) serving as the photoacid and 6-aminoquinoline (6-AQ) as the photobase. Upon excitation, the photoacid (HPTS) undergoes a pKa drop, enabling excited-state proton transfer (ESPT) to nearby proton acceptors, a process highly sensitive to the local environment and water dynamics. In contrast, the photobase (6-AQ) becomes protonated by nearby proton donors in its excited state. By incorporating these complementary probes into the condensates, we directly monitor water dynamics and gain insight into its functional role in condensate organization, hydration, and stability. By combining time-correlated single photon counting (TCSPC) and steady-state fluorescence spectroscopies, we quantitatively assess key parameters such as the effective radius of aqueous microcavities and the kinetics of proton transfer within the condensates. We find that the biomolecular condensates contain nanocavities on the nanometer scale with water organization that slightly inhibits free proton diffusion. These measurements provide a unique window into the nanoscale organization of water and its influence on proton mobility, which is fundamental to many biochemical processes occurring within these membraneless compartments.