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

Abstract

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, nonstoichiometric, 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 Alzheimer's 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.

Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid–Liquid Phase Separation

“…As we approach the critical temperature, the radii of gyration of the proteins among the two phases become increasingly similar (see Boxplots in Figure 5 ). Phase-separation driven expansion for proteins undergoing homotypic LLPS was observed for -IDP [ 114 ] using steady-state fluorescence measurements of pyrene and fluorescein-labeled tau-K18 proteins, a protein associated with Alzheimer’s disease. Even if modest, phase-separation induced expansion enables IDRs to establish a surplus of enthalpy maximizing inter-protein contacts in the condensed phase than those they would establish if they remained unchanged or underwent collapse.…”

IM30 IDPs form a membrane protective carpet upon super-complex disassembly

“…These assemblies, which likely correspond to the IM30 carpet structures observed in the present study, have been identified in vivo to dynamically localize, preferably at stressed TM regions 3 . In fact, dynamic self-assembly is typically observed with IDPs, often involving liquidliquid phase separation 33,41,42 . In contrast to the formation of membrane-less organelles in cells, induced by liquid-liquid phase separation of IDPs, demixing into a condensed and a protein-light fraction (i.e.…”

IM30 IDPs form a membrane-protective carpet upon super-complex disassembly

“…These assemblies, which likely correspond to the IM30 carpet structures observed in the present study, have been identified in vivo to dynamically localize, preferably at stressed TM regions 3 . In fact, dynamic self-assembly is typically observed with IDPs, often involving liquid-liquid phase separation 33,41,42 . In contrast to the formation of membrane-less organelles in cells, induced by liquid-liquid phase separation of IDPs, demixing into a condensed and a protein-light fraction (i.e., carpets and unassociated but membrane-attached protomers) appears to take place on the membrane surface in case of IM30.…”