Intracellular membraneless organelles and their myriad cellular functions have garnered tremendous recent interest. It is becoming well accepted that they form via liquid-liquid phase separation (LLPS) of protein mixtures (often including RNA), where the organelles correspond to a protein-rich droplet phase coexisting with a protein-poor bulk phase. The major protein components contain disordered regions and often also RNA-binding domains, and the disordered fragments on their own easily undergo LLPS. By contrast, LLPS for structured proteins has been observed infrequently. The contrasting phase behaviors can be explained by modeling disordered and structured proteins, respectively, as polymers and colloids. These physical models also provide a better understanding of the regulation of droplet formation by cellular signals and its dysregulation leading to diseases.

Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA and other macromolecular regulators, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with regulatory components. Protein-like particles have four patches, with attraction strength εPP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength εPR tunable. At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.

Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with RNA or other regulatory components. Protein-like particles have four patches, with attraction strength ε PP ; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength ε PR tunable. At low ε PR , the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate ε PR , the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At ε PR > ε PP , the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.

The malleability of intrinsically disordered proteins (IDPs) has generated great interest in understanding how their conformations respond to crowded cellular environments. Experiments can report gross properties such as fluorescence resonance energy transfer (FRET) efficiency but cannot resolve the conformational ensembles of IDPs and their interactions with macromolecular crowders. Computation can in principle provide the latter information but in practice has been hampered by the enormous expense for realistic modeling of IDPs and crowders and for sufficient conformational sampling. Here, taking advantage of a powerful method called FMAP (fast Fourier transform-based modeling of atomistic protein-crowder interactions), we computed how the conformational ensembles of three IDPs are modified in concentrated polyethylene glycol (PEG) 6000 solutions. We represented the IDPs at the all-atom level and the PEG molecules at a coarse-grained level and calculated the experimental observable, i.e., FRET efficiency. Whereas accounting for only steric repulsion of PEG led to overestimation of crowding effects, quantitative agreement with experimental data was obtained upon including mild IDP-PEG attraction. The present work demonstrates that realistic modeling of IDPs under crowded conditions for direct comparison with experiments is now achievable.

We demonstrate, to the best of our knowledge, a first accurate empirical model for reflectance measurements from highly turbid media over the full range of incident angles, i.e., for reflectivity values going from unity in the total internal reflection regime to nearly zero when almost all the light is transmitted. Evidence that our model is accurate is provided by extraction of the particle size, followed by independent verification with dynamic light scattering. Our methodology is in direct contrast with the prevalent approach in turbid media of focusing on only the critical angle region, which is just a small subset of the entire reflectance data.