Elucidating the dynamics of individual IDPs in a biomolecular condensate using single-molecule FRET and molecular simulation.

Abstract

A wide range of biomolecules in solution can phase-separate and form membraneless organelles in the cell. These assemblies often have liquid-like properties, and the corresponding dynamics and exchange of molecules with the environment are important for biological function. The dynamics and materials properties of these systems are commonly assessed based on translational diffusion or rheological properties, typically covering timescales of milliseconds and longer. However, information on the structure and dynamics at the molecular level is lacking. We have studied coacervates of two intrinsically disordered highly oppositely charged nuclear proteins as a prototypical example of heterotypic liquid-liquid phase separation of components abundant in the cell nucleus. Using single-molecule fluorescence spectroscopy, we show that the proteins in the condensates remain disordered, and that their chain dynamics occur on timescale surprisingly close to the ∼100-nanosecond dynamics of the 1:1 complex in dilute solution, even though the condensate is almost three orders of magnitude more viscous than the dilute solution and translational diffusion of the same proteins is ∼50 times slower in the dense than in the dilute phase. The experimental results are in good agreement with large-scale all-atom explicit-solvent molecular dynamics simulations consisting of ∼200 proteins and ∼4 million atoms in total, which can thus provide detailed insights into the protein interactions and dynamics within the condensate. The simulations indicate that the rapid protein chain dynamics in the dense phase are linked to the similarity of the local environment experienced by the proteins in the dilute and dense phases and the short lifetime of intermolecular contacts in the dense phase.