Water Dynamics in the Formation of Single-Component-Based Multiphasic Droplets

Despite the significant presence of water in the liquid–liquid phase separation (LLPS) of biomolecular condensates, its critical role beyond the solvent effects has been largely overlooked. In this study, we investigate the synergistic effects of water on modulating the substructure and properties of biomolecular condensates. We designed and synthesized a series of peptide-based droplets with varying microstructures. Utilizing in situ Raman and Fourier transform infrared (FTIR) spectroscopy, we elucidate how an increased hydrogen bonding network between the peptide and water, coupled with a decreased hydrogen bonding network among peptides, drives the transition from multiphasic to homogeneous configurations. Furthermore, we demonstrate that this transformation can be dynamically regulated by adjusting peptide-water interactions through the application of Chaotropic and Kosmotropic agents. Our findings underscore the essential role of hydration water in the LLPS of single-component systems, paving the way for innovative strategies in the design and modulation of advanced hydrated materials.