Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

We present improvements to the hydropathy scale (HPS) coarse‐grained (CG) model for simulating sequence‐specific behavior of intrinsically disordered proteins (IDPs), including their liquid–liquid phase separation (LLPS). The previous model based on an atomistic hydropathy scale by Kapcha and Rossky (KR scale) is not able to capture some well‐known LLPS trends such as reduced phase separation propensity upon mutations (R‐to‐K and Y‐to‐F). Here, we propose to use the Urry hydropathy scale instead, which was derived from the inverse temperature transitions in a model polypeptide with guest residues X. We introduce two free parameters to shift (Δ) and scale (µ) the overall interaction strengths for the new model (HPS‐Urry) and use the experimental radius of gyration for a diverse group of IDPs to find their optimal values. Interestingly, many possible (Δ, µ) combinations can be used for typical IDPs, but the phase behavior of a low‐complexity (LC) sequence FUS is only well described by one of these models, which highlights the need for a careful validation strategy based on multiple proteins. The CG HPS‐Urry model should enable accurate simulations of protein LLPS and provide a microscopically detailed view of molecular interactions.

Ribonucleoprotein (RNP) granules are membraneless organelles (MLOs), which majorly consist of RNA and RNA-binding proteins and are formed via liquid–liquid phase separation (LLPS). Experimental studies investigating the drivers of LLPS have shown that intrinsically disordered proteins (IDPs) and nucleic acids like RNA and other polynucleotides play a key role in modulating protein phase separation. There is currently a dearth of modelling techniques which allow one to delve deeper into how polynucleotides play the role of a modulator/promoter of LLPS in cells using computational methods. Here, we present a coarse-grained polynucleotide model developed to fill this gap, which together with our recently developed HPS model for protein LLPS, allows us to capture the factors driving protein-polynucleotide phase separation. We explore the capabilities of the modelling framework with the LAF-1 RGG system which has been well studied in experiments and also with the HPS model previously. Further taking advantage of the fact that the HPS model maintains sequence specificity we explore the role of charge patterning on controlling polynucleotide incorporation into condensates. With increased charge patterning we observe formation of structured or patterned condensates which suggests the possible roles of polynucleotides in not only shifting the phase boundaries but also introducing microscopic organization in MLOs.

The formation of membraneless organelles in cells commonly occurs via liquid−liquid phase separation (LLPS) and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Investigating the nature of these interactions, and their effect on dynamics within the condensed phase, is therefore of critical importance but very challenging for either simulation or experiment. Here, we study these interactions and their dynamics by pairing a novel multiscale simulation strategy with microsecond all-atom MD simulations of a condensed, IDP-rich phase. We simulate two IDPs this way, the low complexity domain of FUS and the N-terminal disordered domain of LAF-1, and find good agreement with experimental information about average density, water content, and residue−residue contacts. We go significantly beyond what is known from experiments by showing that ion partitioning within the condensed phase is largely driven by the charge distribution of the proteins andin the cases consideredshows little evidence of preferential interactions of the ions with the proteins. Furthermore, we can probe the microscopic diffusive dynamics within the condensed phase, showing that water and ions are in dynamic equilibrium between dense and dilute phases, and their diffusion is reduced in the dense phase. Despite their high concentration in the condensate, the protein molecules also remain mobile, explaining the observed liquid-like properties of this phase. We finally show that IDP self-association is driven by a combination of nonspecific hydrophobic interactions as well as hydrogen bonds, salt bridges, and π−π and cation−π interactions. The simulation approach presented here allows the structural and dynamical properties of biomolecular condensates to be studied in microscopic detail and is generally applicable to single-and multicomponent systems of proteins and nucleic acids involved in LLPS.

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.