Sequence-dependent material properties of biomolecular condensates and their relation to dilute phase conformations.

Material properties of phase-separated biomolecular condensates, enriched with disordered proteins, dictate many cellular functions. Contrary to the progress made in understanding the sequence-dependent phase separation of proteins, little is known about the sequence determinants of condensate material properties. Using the hydropathy scale and Martini models, we computationally decipher these relationships for charge-rich disordered protein condensates. Our computations yield dynamical, rheological, and interfacial properties of condensates that are quantitatively comparable with experimentally characterized condensates. Interestingly, we find that the material properties of model and natural proteins respond similarly to charge segregation, despite different sequence compositions. Molecular interactions within the condensates closely resemble those within the single-chain ensembles. Consequently, the material properties strongly correlate with molecular contact dynamics and single-chain structural properties. We demonstrate the potential to harness the sequence characteristics of disordered proteins for predicting and engineering the material properties of functional condensates, with insights from the dilute phase properties.

Conformational Properties of Polymers at Droplet Interfaces as Model Systems for Disordered Proteins.

Polymer models serve as useful tools for studying the formation and physical properties of biomolecular condensates. In recent years, the interface dividing the dense and dilute phases of condensates has been discovered to be closely related to their functionality, but the conformational preferences of the constituent proteins remain unclear. To elucidate this, we perform molecular simulations of a droplet formed by liquid‒liquid phase separation of homopolymers, as a surrogate model for the prion-like low-complexity domains. By systematically analyzing the polymer conformations at different locations in the droplet, we find that the chains become compact at the droplet interface compared to the droplet interior. Further, segmental analysis revealed that the end sections of the chains are enriched at the interface to maximize conformational entropy, and are more expanded than the middle sections of the chains. We find that the majority of chain segments lie tangential to the droplet surface and only the chain ends tend to align perpendicular to the interface. These trends also hold for the natural proteins FUC LC and LAF-1 RGG, which exhibit more compact chain conformations at the interface compared with the droplet interior. Our findings provide important insights into the interfacial properties of biomolecular condensates and highlight the value of using simple polymer physics models to understand the underlying mechanisms.

Role of Strong Localized vs Weak Distributed Interactions in Disordered Protein Phase Separation.

Interaction strength and localization are critical parameters controlling the single-chain and condensed-state properties of intrinsically disordered proteins (IDPs). Here, we decipher these relationships using coarse-grained heteropolymers comprised of hydrophobic (H) and polar (P) monomers as model IDPs. We systematically vary the fraction of P monomers XP and employ two distinct particle-based models that include either strong localized attractions between only H-H pairs (HP model) or weak distributed attractions between both H-H and H-P pairs (HP+ model). To compare different sequences and models, we first carefully tune the attraction strength for all sequences to match the single-chain radius of gyration. Interestingly, we find that this procedure produces similar conformational ensembles, nonbonded potential energies, and chain-level dynamics for single chains of almost all sequences in both models, with some deviations for the HP model at large XP. However, we observe a surprisingly rich phase behavior for the sequences in both models that deviates from the expectation that similarity at the single-chain level will translate to a similar phase-separation propensity. Coexistence between dilute and dense phases is only observed up to a model-dependent XP, despite the presence of favorable interchain interactions, which we quantify using the second virial coefficient. Instead, the limited number of attractive sites (H monomers) leads to the self-assembly of finite-sized clusters of different sizes depending on XP. Our findings strongly suggest that models with distributed interactions favor the formation of liquid-like condensates over a much larger range of sequence compositions compared to models with localized interactions.

Principles Governing the Phase Separation of Multidomain Proteins.

A variety of membraneless organelles, often termed "biological condensates", play an important role in the regulation of cellular processes such as gene transcription, translation, and protein quality control. On the basis of experimental and theoretical investigations, liquid-liquid phase separation (LLPS) has been proposed as a possible mechanism for the origin of biological condensates. LLPS requires multivalent macromolecules that template the formation of long-range, intermolecular interaction networks and results in the formation of condensates with defined composition and material properties. Multivalent interactions driving LLPS exhibit a wide range of modes from highly stereospecific to nonspecific and involve both folded and disordered regions. Multidomain proteins serve as suitable macromolecules for promoting phase separation and achieving disparate functions due to their potential for multivalent interactions and regulation. Here, we aim to highlight the influence of the domain architecture and interdomain interactions on the phase separation of multidomain protein condensates. First, the general principles underlying these interactions are illustrated on the basis of examples of multidomain proteins that are predominantly associated with nucleic acid binding and protein quality control and contain both folded and disordered regions. Next, the examples showcase how LLPS properties of folded and disordered regions can be leveraged to engineer multidomain constructs that form condensates with the desired assembly and functional properties. Finally, we highlight the need for improvements in coarse-grained computational models that can provide molecular-level insights into multidomain protein condensates in conjunction with experimental efforts.

Cosolvents as Liquid Surfactants for Boron Nitride Nanosheet (BNNS) Dispersions.

Despite a range of promising applications, liquid-phase exfoliation of boron nitride nanosheets (BNNSs) is limited, both by low yield in common solvents as well as the disadvantages of using dissolved surfactants. One recently reported approach is the use of cosolvent systems to increase the as-obtained concentration of BNNS; the role of these solvents in aiding exfoliation and/or aiding colloidal stability of BNNSs is difficult to distinguish. In this paper, we have investigated the use of a t-butanol/water cosolvent to disperse BNNSs. We utilize solvent-exchange experiments to demonstrate that the t-butanol is in fact essential to colloidal stability; we then utilized molecular dynamics simulations to explore the mechanism of t-butanol/BNNS interactions. Taken together, the experimental and simulation results show that the key to the success of t-butanol (as compared to the other alcohols of higher or lower molecular weight) lies in its ability to act as a "liquid dispersant" which allows it to favorably interact with both water and BNNSs. Additionally, we show that the stable dispersions of BNNS in water/t-butanol systems may be freeze-dried to yield nonaggregated, redispersible BNNS powders, which would be useful in an array of industrial processes.