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Biomolecular condensates form via coupled associative and segregative phase transitions of multivalent associative macromolecules. Phase separation coupled to percolation is one example of such transitions. Here, we characterize molecular and mesoscale structural descriptions of condensates formed by intrinsically disordered prion-like low complexity domains (PLCDs). These systems conform to sticker-and-spacers architectures. Stickers are cohesive motifs that drive associative interactions through reversible crosslinking and spacers affect the cooperativity of crosslinking and overall macromolecular solubility. Our computations reproduce experimentally measured sequence-specific phase behaviors of PLCDs. Within simulated condensates, networks of reversible inter-sticker crosslinks organize PLCDs into small-world topologies. The overall dimensions of PLCDs vary with spatial location, being most expanded at and preferring to be oriented perpendicular to the interface. Our results demonstrate that even simple condensates with one type of macromolecule feature inhomogeneous spatial organizations of molecules and interfacial features that likely prime them for biochemical activity. The authors provide a physical description for condensates as complex fluids defined by small-world network structures for their interiors, and of interfaces featuring a preference of locally and globally expanded conformations.

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via phase separation coupled to percolation. Intracellular condensates often encompass numerous distinct proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA-binding proteins, hnRNPA1 and FUS. Using simulations and experiments, we find that 1:1 mixtures of A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own due to complementary electrostatic interactions. Tie line analysis reveals that stoichiometric ratios of different components and their sequence-encoded interactions contribute jointly to the driving forces for condensate formation. Simulations also show that the spatial organization of PLCDs within condensates is governed by relative strengths of homotypic versus heterotypic interactions. We uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins. Mixtures of prion-like low complexity domains (PLCDs) are found in condensates such as stress granules. In this work, the authors report how the interplay between homotypic and heterotypic interactions contributes to condensate formation by mixtures of PLCDs.

Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif–linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions. Foutel et. al. identify conformational buffering as a mechanism for functional selection in intrinsically disordered protein regions that allows robust encoding of a tethering function by a hypervariable disordered linker through compensatory changes in sequence length and composition.

Prion-like low-complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behaviour of PLCDs. We interpret our results in the context of the stickers-and-spacers model for the phase separation of associative polymers. We find that tyrosine is a stronger sticker than phenylalanine, whereas arginine is a context-dependent auxiliary sticker. In contrast, lysine weakens sticker–sticker interactions. Increasing the net charge per residue destabilizes phase separation while also weakening the strong coupling between single-chain contraction in dilute phases and multichain interactions that give rise to phase separation. Finally, glycine and serine residues act as non-equivalent spacers, and thus make the glycine versus serine contents an important determinant of the driving forces for phase separation. The totality of our results leads to a set of rules that enable comparative estimates of composition-specific driving forces for PLCD phase separation. The complex link between protein sequence and phase behaviour for a family of prion-like low-complexity domains (PLCDs) has now been revealed. The results have uncovered a set of rules—which are interpreted using a stickers-and-spacers model—that govern the sequence-encoded phase behaviour of such PLCDs and enable physicochemical rationalizations that are connected to the underlying sequence composition.

Over the last decade, evidence has accumulated to suggest that numerous instances of cellular compartmentalization can be explained by the phenomenon of phase separation. This is a process by which a macromolecular solution separates spontaneously into dense and dilute coexisting phases. Semi-quantitative, in vitro approaches for measuring phase boundaries have proven very useful in determining some key features of biomolecular condensates, but these methods often lack the precision necessary for generating quantitative models. Therefore, there is a clear need for techniques that allow quantitation of coexisting dilute and dense phase concentrations of phase-separating biomolecules, especially in systems with more than one type of macromolecule. Here, we report the design and deployment of analytical High-Performance Liquid Chromatography (HPLC) for in vitro separation and quantification of distinct biomolecules that allows us to measure dilute and dense phase concentrations needed to reconstruct coexistence curves in multicomponent mixtures. This approach is label-free, detects lower amounts of material than is accessible with classic UV-spectrophotometers, is applicable to a broad range of macromolecules of interest, is a semi-high-throughput technique, and if needed, the macromolecules can be recovered for further use. The approach promises to provide quantitative insights into the balance of homotypic and heterotypic interactions in multicomponent phase-separating systems.

Biomolecular condensates are viscoelastic materials. Here we investigate the determinants of the sequence-encoded and age-dependent viscoelasticity of condensates formed by the prion-like low-complexity domain of the protein hnRNP A1 and its designed variants. We find that the dominantly viscous forms of the condensates are metastable Maxwell fluids. A Rouse–Zimm model that accounts for the network-like organization of proteins within condensates reproduces the measured viscoelastic moduli. We show that the strengths of aromatic inter-sticker interactions determine sequence-specific amplitudes of elastic and viscous moduli and the timescales over which elastic properties dominate. These condensates undergo physical ageing on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of dominantly viscous phases. The ageing of condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, β-sheet-containing, semi-crystalline, elastic, Kelvin–Voigt solids. Our results suggest that sequence grammars, which refer to amino acid identities of stickers versus spacers in prion-like low-complexity domains, have evolved to afford control over metastabilities of dominantly viscous fluid phases of condensates. This selection is likely to render barriers for conversion from metastable fluids to globally stable solids insurmountable on functionally relevant timescales. The time-dependent viscoelastic moduli of biomolecular condensates are connected to the functions that the condensates influence in cells. Now sticker and spacer residues in proteins are shown to regulate condensate viscoelasticity and ageing dynamics.

Several algorithms have been developed that use amino acid sequences to predict whether or not a protein or a region of a protein is disordered. These algorithms make accurate predictions for disordered regions that are 30 amino acids or longer, but it is unclear whether the predictions can be directly related to the backbone dynamics of individual amino acid residues. The nuclear Overhauser effect between the amide nitrogen and hydrogen (NHNOE) provides an unambiguous measure of backbone dynamics at single residue resolution and is an excellent tool for characterizing the dynamic behavior of disordered proteins. In this report, we show that the NHNOE values for several members of a family of disordered proteins are highly correlated with the output from three popular algorithms used to predict disordered regions from amino acid sequence. This is the first test between an experimental measure of residue specific backbone dynamics and disorder predictions. The results suggest that some disorder predictors can accurately estimate the backbone dynamics of individual amino acids in a long disordered region.

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via coupled associative and segregative phase transitions. We previously deciphered how evolutionarily conserved sequence features drive phase separation of PLCDs through homotypic interactions. However, condensates typically encompass a diverse mixture of proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA binding proteins namely, hnRNPA1 and FUS. We find that 1:1 mixtures of the A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own. The enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD arise partly from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic residues. Further, tie line analysis shows that stoichiometric ratios of different components and their sequence-encoded interactions jointly contribute to the driving forces for condensate formation. These results highlight how expression levels might be tuned to regulate the driving forces for condensate formation in vivo . Simulations also show that the organization of PLCDs within condensates deviates from expectations based on random mixture models. Instead, spatial organization within condensates will reflect the relative strengths of homotypic versus heterotypic interactions. We also uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins. Overall, our findings emphasize the network-like organization of molecules within multicomponent condensates, and the distinctive, composition-specific conformational features of condensate interfaces.Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via coupled associative and segregative phase transitions. We previously deciphered how evolutionarily conserved sequence features drive phase separation of PLCDs through homotypic interactions. However, condensates typically encompass a diverse mixture of proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA binding proteins namely, hnRNPA1 and FUS. We find that 1:1 mixtures of the A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own. The enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD arise partly from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic residues. Further, tie line analysis shows that stoichiometric ratios of different components and their sequence-encoded interactions jointly contribute to the driving forces for condensate formation. These results highlight how expression levels might be tuned to regulate the driving forces for condensate formation in vivo . Simulations also show that the organization of PLCDs within condensates deviates from expectations based on random mixture models. Instead, spatial organization within condensates will reflect the relative strengths of homotypic versus heterotypic interactions. We also uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins. Overall, our findings emphasize the network-like organization of molecules within multicomponent condensates, and the distinctive, composition-specific conformational features of condensate interfaces.Biomolecular condensates are mixtures of different protein and nucleic acid molecules that organize biochemical reactions in cells. Much of what we know about how condensates form comes from studies of phase transitions of individual components of condensates. Here, we report results from studies of phase transitions of mixtures of archetypal protein domains that feature in distinct condensates. Our investigations, aided by a blend of computations and experiments, show that the phase transitions of mixtures are governed by a complex interplay of homotypic and heterotypic interactions. The results point to how expression levels of different protein components can be tuned in cells to modulate internal structures, compositions, and interfaces of condensates, thus affording distinct ways to control the functions of condensates.Significance StatementBiomolecular condensates are mixtures of different protein and nucleic acid molecules that organize biochemical reactions in cells. Much of what we know about how condensates form comes from studies of phase transitions of individual components of condensates. Here, we report results from studies of phase transitions of mixtures of archetypal protein domains that feature in distinct condensates. Our investigations, aided by a blend of computations and experiments, show that the phase transitions of mixtures are governed by a complex interplay of homotypic and heterotypic interactions. The results point to how expression levels of different protein components can be tuned in cells to modulate internal structures, compositions, and interfaces of condensates, thus affording distinct ways to control the functions of condensates.

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques - Brillouin microscopy and quantitative phase imaging (QPI) - to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

Biomolecular condensates are viscoelastic materials. Here, we report results from investigations into molecular-scale determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by prion-like low-complexity domains (PLCDs). The terminally viscous forms of PLCD condensates are Maxwell fluids. Measured viscoelastic moduli of these condensates are reproducible using a Rouse-Zimm model that accounts for the network-like organization engendered by reversible physical crosslinks among PLCDs in the dense phase. Measurements and computations show that the strengths of aromatic inter-sticker interactions determine the sequence-specific amplitudes of elastic and viscous moduli as well as the timescales over which elastic properties dominate. PLCD condensates also undergo physical aging on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of terminally viscous phases. The aging of PLCD condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, terminally elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to the identities of stickers versus spacers in PLCDs, have evolved to afford control over the metastabilities of terminally viscous fluid phases of condensates. This selection can, in some cases, render barriers for conversion from metastable fluids to globally stable solids to be insurmountable on functionally relevant timescales.