Unravelling the molecular mechanisms of LLPS in neurobiology with Raman Spectroscopy

Several proteins linked to neurodegenerative and neurodevelopmental disorders have been shown to undergo liquid-liquid phase separation (LLPS), a physicochemical process which drives the formation of membraneless condensates. However, identifying specific secondary protein structures (e.g., helices, beta-sheets), small conformational changes, or individual amino acid residues that govern LLPS remains a challenge. To elucidate how protein structures conform within condensates and how they evolve during condensate formation, there is a need for methods that can report directly and non-invasively on biomolecular states. Here, we review studies that have demonstrated Raman spectroscopy as a powerful tool for the investigation of LLPS both in vitro and in cellulo . The concentration of proteins and nucleic acids along with monitoring condensate maturation and progression toward aggregates is discussed. We highlight the importance of rigorous spectral processing to accurately extract reliable structural and molecular information. This review aims to present the latest advances in applying Raman Spectroscopy to the investigation of LLPS in neurobiology. Such spectroscopic information contributes to the thorough understanding of the mechanisms underlying LLPS, a mechanism which may prove key to the development of effective therapeutic strategies for neurodegenerative disorders. • Biomolecules form membraneless condensates via liquid-liquid phase separation. • Raman Spectroscopy is used to study proteins undergoing LLPS in neurobiology. • Raman signatures can be obtained for in vitro and in cellulo LLPS. • Proper spectral processing is key to disentangle complex Raman signature maps. • RS allows monitoring functional vs pathological assemblies.