Seeing the chemistry of biomolecular condensates: in situ mapping of composition and water content

Biomolecular condensates are cellular organelles that form via liquid-liquid phase separation of proteins and nucleic acids. The functional role of condensates is tightly coupled to their material properties such as viscosity and hydrophobicity, which serve as phenomenological markers of cellular state in health and disease. These properties depend critically on condensate composition and water content. However, available approaches to determine condensate composition typically rely on invasive procedures that can disrupt or destroy the tested sample. Here, we introduce Raman spectroscopy coupled with spectral phasor analysis as an in situ, label-free approach to resolve the chemical profile and molecular concentrations within aqueous polymer solutions and within the dense and dilute phases of biomolecular condensate systems. In addition to quantifying the protein and water volume fractions, our method yields a precise readout of client molecule partitioning inside condensates. Across a wide range of condensate systems, we find that the dense phase remains overwhelmingly water-rich, even in condensates that exhibit low apparent dielectric constants. By explicitly accounting for protein backbone contributions to the Raman spectrum, we assess the contribution of "solid-like" hydrogen-bonded water resulting from protein hydration and find that the overwhelming majority of water molecules within condensates largely retain bulk "liquid-like" vibrational properties. Finally, by combining Raman-based compositional analysis with environment-sensitive fluorescent probes, we investigate the microscopic determinants of condensate hydrophobicity. We show that condensate hydrophobicity emerges from a combined contribution of the macromolecular structural features and water partitioning inside condensates, rather than from water content or hydrogen bonding alone.