Raman Dissection of Water-Interaction Coupling in Condensate-Relevant Peptides

Abstract

Biomolecular condensates formed through liquid–liquid phase separation are primarily driven by noncovalent interactions among intrinsically disordered proteins and nucleic acids. Despite water constituting the majority of the condensate volume, its role in modulating these molecular interactions remains poorly understood. Here, we employ time-resolved Raman spectroscopy to investigate amino acids and IDP-derived peptides, each designed to be dominated by a single type of noncovalent interaction. By simulating molecular crowding through controlled solution evaporation, we track changes in molecular vibrational modes and the water structure. Distinct responses were observed depending on the interaction type, with differences in backbone and side-chain behaviors as well as in the retention and loss of tetrahedral versus distorted water populations. Peptides driven by hydrogen bonding retained more tetrahedral water, while hydrophobic peptides showed stepwise water depletion. Our findings offer direct molecular-level insight into how hydration influences the behavior of condensate-relevant molecules and provide a framework for understanding the physicochemical basis of biomolecular condensate formation.