Structure-dependent recruitment and diffusion of guest proteins in liquid droplets of FUS.

Liquid droplets of a host protein, formed by liquid-liquid phase separation, recruit guest proteins and provide functional fields. Recruitment into p53 droplets is similar between disordered and folded guest proteins, whereas the diffusion of guest proteins inside droplets depends on their structural types. In this study, to elucidate how the recruitment and diffusion properties of guest proteins are affected by a host protein, we characterized the properties of guest proteins in fused in sarcoma (FUS) droplets using single-molecule fluorescence microscopy in comparison with p53 droplets. Unlike p53 droplets, disordered guest proteins were recruited into FUS droplets more efficiently than folded guest proteins, suggesting physical exclusion of the folded proteins from the small voids of the droplet. The recruitment did not appear to depend on the physical parameters (electrostatic or cation-π) of guests, implying that molecular size exclusion limits intermolecular interaction-assisted uptake. The diffusion of disordered guest proteins was comparable to that of the host FUS, whereas that of folded proteins varied widely, similar to the results for host p53. The scaling exponent of diffusion highlights the molecular sieving of large folded proteins in droplets. Finally, we proposed a molecular recruitment and diffusion model for guest proteins in FUS droplets.

Molecular principles of recruitment and dynamics of guest proteins in liquid droplets.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host-guest interactions hinder the understanding of the molecular grammar governing liquid-liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.