Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy.

The phenomenon of reversible liquid-liquid phase separation of proteins underlies the formation of membraneless organelles, which are crucial for cellular processes such as signalling and transport. In addition, it is also of great interest to uncover the mechanisms of further irreversible maturation of the functional dense liquid phase into aberrant insoluble assemblies due to its implication in human disease. Recent advances in methods based on atomic force microscopy (AFM) have made it possible to study protein condensates at the nanometer level, providing unprecedented information on the nature of the intermolecular interactions governing phase separation. Here, we provide an in-depth description of a protocol for the characterisation of the morphology, stiffness, and chemical properties of protein condensates using infrared nanospectroscopy (AFM-IR).