Liquid-Liquid Phase Separation is Driven by Large-Scale Conformational Unwinding and Fluctuations of Intrinsically Disordered Protein Molecules

Liquid-liquid phase separation occurs via a multitude of transient, non-covalent, intermolecular interactions resulting in phase transition of intrinsically disordered proteins/regions (IDPs/IDRs) and other biopolymers into mesoscopic, dynamic, non-stoichiometric, supramolecular condensates. IDPs resemble associative polymers possessing stereospecific \"stickers\" and flexible \"spacers\" that govern the transient chain-chain interactions and fluidity in phase-separated liquid droplets. However, the fundamental molecular origin of phase separation remains elusive. Here we present a unique case to demonstrate that unusual conformational expansion events coupled with solvation and fluctuations drive phase separation of tau, an IDP associated with Alzheimers disease. Using intramolecular excimer emission as a powerful proximity readout, we show the unraveling of polypeptide chains within the protein-rich interior environment that can promote critical interchain contacts. Using highly-sensitive picosecond time-resolved fluorescence depolarization measurements, we directly capture rapid large-amplitude torsional fluctuations in the extended chains that can control the relay of making-and-breaking of noncovalent intermolecular contacts maintaining the internal fluidity. Our observations, together with the existing polymer theories, suggest that such an orchestra of concerted molecular shapeshifting events involving chain expansion, solvation, and fluctuations can provide additional favorable free energies to overcome the entropy of mixing term during phase separation. The interplay of these key molecular parameters can also be of prime importance in modulating the mesoscale material property of liquid-like condensates and their maturation of into pathological gel-like and solid-like aggregates.