Chain Collapse, Reduced Dielectric, and Water Release Drive Protein Phase Separation

Area of Science:

• Cellular Biology
• Biophysics
• Structural Biology

Background:

• Biomolecular condensates are essential microenvironments for cellular organization and biochemical reactions.
• Understanding the weak, transient interactions driving condensate phase separation is experimentally challenging.
• Current methods often rely on perturbative probes, limiting insights into native condensate properties.

Purpose of the Study:

• To develop and apply label-free techniques for investigating the structural biology and chemical properties of biomolecular condensate interiors.
• To elucidate the molecular mechanisms underlying phase separation in condensates, focusing on protein-protein and protein-solvent interactions.

Main Methods:

• Combined label-free optical scattering and vibrational spectroscopy across UV, visible, mid-IR, and THz wavelengths.
• Utilized deep-learning-based ensemble prediction for intrinsically disordered protein conformations.
• Investigated the N-terminal domain of DEAD-box helicase 4 (DDX4).

Main Results:

• Developed a suite of label-free approaches providing quantitative insights into condensate interiors.
• Observed protein chain collapse, reduced dielectric environment, and water release during DDX4 condensate formation.
• These events correlate with enhanced multivalent protein-protein interactions, promoting condensate growth.

Conclusions:

• The study presents a powerful, non-perturbative toolkit for analyzing biomolecular condensates.
• Phase separation involves specific molecular events that strengthen interactions, creating a positive feedback loop for condensate stability.
• Findings offer a deeper understanding of the physical principles governing intracellular organization.