Reversible Kinetic Trapping of FUS Biomolecular Condensates

Abstract

Formation of membrane-less organelles by self-assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain. Forming FUS LC condensates directly at low temperature leads to formation of metastable, kinetically trapped condensates that show arrested coalescence, escape from which to untrapped condensates can be achieved via thermal annealing. Using experimental and computational approaches, the authors find that molecular structure of interfacial FUS LC in kinetically trapped condensates is distinct (more 𝜷-sheet like) compared to untrapped FUS LC condensates. Moreover, molecular motion within kinetically trapped condensates is substantially slower compared to that in untrapped condensates thereby demonstrating two unique liquid FUS condensates. Controlling condensate thermodynamic state, stability, and structure with a simple thermal switch may contribute to pathological protein aggregate stability and provides a facile method to trigger condensate mixing for biotechnology applications.

Protein structural transitions critically transform the network connectivity and viscoelasticity of RNA-binding protein condensates but RNA can prevent it

“…1f and 2a). These results are consistent with recent experimental observations of FUS condensates increasing their density upon ageing [117].…”

“…Only a minor densification of the condensates over time is found when a very high percentage of β-sheet transitions has occurred (i.e., 75% of the available disorder LARKS have transitioned into inter-protein β-sheets). However, such modest increment of density [117], compared to the more prominent variation found in pure A-LCD-hnRNPA1 and FUS-PLD aged droplets (Figs. 1d and 2a respectively) is due to the small region in which the three LARKS are located within the full-FUS sequence (50-residue region within the 526-residue whole FUS sequence).…”

Single-Droplet Surface-Enhanced Raman Scattering Decodes the Molecular Language of Liquid-Liquid Phase Separation

“…Vibrational Raman spectroscopy performed in a microscopy format allows us to uniquely and elegantly combine the aforesaid capabilities to obtain the protein structural information from a well-defined spatial location by focusing the laser beam into a sub-micron spot. Such non-invasive and label-free laser micro-Raman measurements permit us to access the wealth of structural information by monitoring a range of bond vibrational frequencies while retaining the spatial resolution 24,25,26 . However, owing to a low Raman scattering cross-section, Raman spectroscopy is a highly insensitive technique, especially for biomolecules under physiological conditions in aqueous solutions 27 .…”

The stoichiometric interaction model for mesoscopic molecular dynamics simulations of liquid-liquid phase separation

“…First, given the number of proteins and timescales involved, conventional all-atom MD simulations are currently beyond the reach. Coarse-grained protein models in which each amino acid is represented by one particle have been successfully applied to simulate LLPS and condensates made by the promiscuous interaction of intrinsically disordered proteins (Dignon et al(13), Das et al(14), Lafage et al(15), Chatterjee et al(16), Tesei et al(17))(18).…”