The liquid-to-solid transition of FUS is promoted by the condensate surface.

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Theoretical and Data-Driven Approaches for Biomolecular Condensates.

Biomolecular condensation processes are increasingly recognized as a fundamental mechanism that living cells use to organize biomolecules in time and space. These processes can lead to the formation of membraneless organelles that enable cells to perform distinct biochemical processes in controlled local environments, thereby supplying them with an additional degree of spatial control relative to that achieved by membrane-bound organelles. This fundamental importance of biomolecular condensation has motivated a quest to discover and understand the molecular mechanisms and determinants that drive and control this process. Within this molecular viewpoint, computational methods can provide a unique angle to studying biomolecular condensation processes by contributing the resolution and scale that are challenging to reach with experimental techniques alone. In this Review, we focus on three types of dry-lab approaches: theoretical methods, physics-driven simulations and data-driven machine learning methods. We review recent progress in using these tools for probing biomolecular condensation across all three fields and outline the key advantages and limitations of each of the approaches. We further discuss some of the key outstanding challenges that we foresee the community addressing next in order to develop a more complete picture of the molecular driving forces behind biomolecular condensation processes and their biological roles in health and disease.

Aging can transform single-component protein condensates into multiphase architectures.

Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multicomponent mixtures of biomolecules (e.g., proteins and nucleic acids) when the different components present sufficient physicochemical diversity (e.g., in intermolecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g., temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli or emerge progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the progressive intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of aging. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically aging condensates with nonconservative intermolecular forces. Our nonequilibrium simulations of condensate aging predict that single-component condensates that are initially homogeneous and liquid like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age due to gradual and irreversible enhancement of interprotein interactions. The type of multiphase architecture is determined by the aging mechanism, the molecular organization of the gel and liquid phases, and the chemical makeup of the protein. Notably, we predict that interprotein disorder to order transitions within the prion-like domains of intracellular proteins can lead to the required nonconservative enhancement of intermolecular interactions. Our study, therefore, predicts a potential mechanism by which the nonequilibrium process of aging results in single-component multiphase condensates.

Aromatic and arginine content drives multiphasic condensation of protein-RNA mixtures.

Multiphasic architectures are found ubiquitously in biomolecular condensates and are thought to have important implications for the organization of multiple chemical reactions within the same compartment. Many of these multiphasic condensates contain RNA in addition to proteins. Here, we investigate the importance of different interactions in multiphasic condensates comprising two different proteins and RNA using computer simulations with a residue-resolution coarse-grained model of proteins and RNA. We find that in multilayered condensates containing RNA in both phases, protein-RNA interactions dominate, with aromatic residues and arginine forming the key stabilizing interactions. The total aromatic and arginine content of the two proteins must be appreciably different for distinct phases to form, and we show that this difference increases as the system is driven toward greater multiphasicity. Using the trends observed in the different interaction energies of this system, we demonstrate that we can also construct multilayered condensates with RNA preferentially concentrated in one phase. The "rules" identified can thus enable the design of synthetic multiphasic condensates to facilitate further study of their organization and function.

Surfactants or scaffolds? RNAs of varying lengths control the thermodynamic stability of condensates differently.

Biomolecular condensates, thought to form via liquid-liquid phase separation of intracellular mixtures, are multicomponent systems that can include diverse types of proteins and RNAs. RNA is a critical modulator of RNA-protein condensate stability, as it induces an RNA concentration-dependent reentrant phase transition-increasing stability at low RNA concentrations and decreasing it at high concentrations. Beyond concentration, RNAs inside condensates can be heterogeneous in length, sequence, and structure. Here, we use multiscale simulations to understand how different RNA parameters interact with one another to modulate the properties of RNA-protein condensates. To do so, we perform residue/nucleotide resolution coarse-grained molecular dynamics simulations of multicomponent RNA-protein condensates containing RNAs of different lengths and concentrations, and either FUS or PR25 proteins. Our simulations reveal that RNA length regulates the reentrant phase behavior of RNA-protein condensates: increasing RNA length sensitively rises the maximum value that the critical temperature of the mixture reaches, and the maximum concentration of RNA that the condensate can incorporate before beginning to become unstable. Strikingly, RNAs of different lengths are organized heterogeneously inside condensates, which allows them to enhance condensate stability via two distinct mechanisms: shorter RNA chains accumulate at the condensate's surface acting as natural biomolecular surfactants, while longer RNA chains concentrate inside the core to saturate their bonds and enhance the density of molecular connections in the condensate. Using a patchy particle model, we additionally demonstrate that the combined impact of RNA length and concentration on condensate properties is dictated by the valency, binding affinity, and polymer length of the various biomolecules involved. Our results postulate that diversity on RNA parameters within condensates allows RNAs to increase condensate stability by fulfilling two different criteria: maximizing enthalpic gain and minimizing interfacial free energy; hence, RNA diversity should be considered when assessing the impact of RNA on biomolecular condensates regulation.

Photogeneration of Spin Quintet Triplet-Triplet Excitations in DNA-Assembled Pentacene Stacks.

Singlet fission (SF), an exciton-doubling process observed in certain molecular semiconductors where two triplet excitons are generated from one singlet exciton, requires correctly tuned intermolecular coupling to allow separation of the two triplets to different molecular units. We explore this using DNA-encoded assembly of SF-capable pentacenes into discrete π-stacked constructs of defined size and geometry. Precise structural control is achieved via a combination of the DNA duplex formation between complementary single-stranded DNA and the local molecular geometry that directs the SF chromophores into a stable and predictable slip-stacked configuration, as confirmed by molecular dynamics (MD) modeling. Transient electron spin resonance spectroscopy revealed that within these DNA-assembled pentacene stacks, SF evolves via a bound triplet pair quintet state, which subsequently converts into free triplets. SF evolution via a long-lived quintet state sets specific requirements on intermolecular coupling, rendering the quintet spectrum and its zero-field-splitting parameters highly sensitive to intermolecular geometry. We have found that the experimental spectra and zero-field-splitting parameters are consistent with a slight systematic strain relative to the MD-optimized geometry. Thus, the transient electron spin resonance analysis is a powerful tool to test and refine the MD-derived structure models. DNA-encoded assembly of coupled semiconductor molecules allows controlled construction of electronically functional structures, but brings with it significant dynamic and polar disorders. Our findings here of efficient SF through quintet states demonstrate that these conditions still allow efficient and controlled semiconductor operation and point toward future opportunities for constructing functional optoelectronic systems.

The Chromatin Regulator HMGA1a Undergoes Phase Separation in the Nucleus.

The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through electrostatic interactions via AT hooks 2 and 3. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.

RNA length has a non-trivial effect in the stability of biomolecular condensates formed by RNA-binding proteins.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) play a crucial role in the spatiotemporal organization of the cell material. Nucleic acids can act as critical modulators in the stability of these protein condensates. To unveil the role of RNA length in regulating the stability of RNA binding protein (RBP) condensates, we present a multiscale computational strategy that exploits the advantages of a sequence-dependent coarse-grained representation of proteins and a minimal coarse-grained model wherein proteins are described as patchy colloids. We find that for a constant nucleotide/protein ratio, the protein fused in sarcoma (FUS), which can phase separate on its own-i.e., via homotypic interactions-only exhibits a mild dependency on the RNA strand length. In contrast, the 25-repeat proline-arginine peptide (PR25), which does not undergo LLPS on its own at physiological conditions but instead exhibits complex coacervation with RNA-i.e., via heterotypic interactions-shows a strong dependence on the length of the RNA strands. Our minimal patchy particle simulations suggest that the strikingly different effect of RNA length on homotypic LLPS versus RBP-RNA complex coacervation is general. Phase separation is RNA-length dependent whenever the relative contribution of heterotypic interactions sustaining LLPS is comparable or higher than those stemming from protein homotypic interactions. Taken together, our results contribute to illuminate the intricate physicochemical mechanisms that influence the stability of RBP condensates through RNA inclusion.

Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components.

One of the key mechanisms used by cells to control the spatiotemporal organization of their many components is the formation and dissolution of biomolecular condensates through liquid-liquid phase separation (LLPS). Using a minimal coarse-grained model that allows us to simulate thousands of interacting multivalent proteins, we investigate the physical parameters dictating the stability and composition of multicomponent biomolecular condensates. We demonstrate that the molecular connectivity of the condensed-liquid network-i.e., the number of weak attractive protein-protein interactions per unit of volume-determines the stability (e.g., in temperature, pH, salt concentration) of multicomponent condensates, where stability is positively correlated with connectivity. While the connectivity of scaffolds (biomolecules essential for LLPS) dominates the phase landscape, introduction of clients (species recruited via scaffold-client interactions) fine-tunes it by transforming the scaffold-scaffold bond network. Whereas low-valency clients that compete for scaffold-scaffold binding sites decrease connectivity and stability, those that bind to alternate scaffold sites not required for LLPS or that have higher-than-scaffold valencies form additional scaffold-client-scaffold bridges increasing stability. Proteins that establish more connections (via increased valencies, promiscuous binding, and topologies that enable multivalent interactions) support the stability of and are enriched within multicomponent condensates. Importantly, proteins that increase the connectivity of multicomponent condensates have higher critical points as pure systems or, if pure LLPS is unfeasible, as binary scaffold-client mixtures. Hence, critical points of accessible systems (i.e., with just a few components) might serve as a unified thermodynamic parameter to predict the composition of multicomponent condensates.

Emergence of chromatin hierarchical loops from protein disorder and nucleosome asymmetry.

Protein flexibility and disorder is emerging as a crucial modulator of chromatin structure. Histone tail disorder enables transient binding of different molecules to the nucleosomes, thereby promoting heterogeneous and dynamic internucleosome interactions and making possible recruitment of a wide-range of regulatory and remodeling proteins. On the basis of extensive multiscale modeling we reveal the importance of linker histone H1 protein disorder for chromatin hierarchical looping. Our multiscale approach bridges microsecond-long bias-exchange metadynamics molecular dynamics simulations of atomistic 211-bp nucleosomes with coarse-grained Monte Carlo simulations of 100-nucleosome systems. We show that the long C-terminal domain (CTD) of H1-a ubiquitous nucleosome-binding protein-remains disordered when bound to the nucleosome. Notably, such CTD disorder leads to an asymmetric and dynamical nucleosome conformation that promotes chromatin structural flexibility and establishes long-range hierarchical loops. Furthermore, the degree of condensation and flexibility of H1 can be fine-tuned, explaining chromosomal differences of interphase versus metaphase states that correspond to partial and hyperphosphorylated H1, respectively. This important role of H1 protein disorder in large-scale chromatin organization has a wide range of biological implications.

Surface Electrostatics Govern the Emulsion Stability of Biomolecular Condensates.

Liquid-liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions that in general have a propensity to fuse and coalesce; however, many condensates persist as independent droplets in the test tube and inside cells. This stability is crucial for their function, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modeling, we investigate how the nanoscale forces that sustain condensates impact their stability against fusion. By comparing peptide-RNA (PR25:PolyU) and proteinaceous (FUS) condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity. Moreover, measurements of single condensate zeta potentials reveal that such systems can constitute classically stable emulsions. Taken together, these results highlight the role of passive stabilization mechanisms in protecting biomolecular condensates against coalescence.

Deoxyribonucleic Acid Encoded and Size-Defined π-Stacking of Perylene Diimides.

Natural photosystems use protein scaffolds to control intermolecular interactions that enable exciton flow, charge generation, and long-range charge separation. In contrast, there is limited structural control in current organic electronic devices such as OLEDs and solar cells. We report here the DNA-encoded assembly of π-conjugated perylene diimides (PDIs) with deterministic control over the number of electronically coupled molecules. The PDIs are integrated within DNA chains using phosphoramidite coupling chemistry, allowing selection of the DNA sequence to either side, and specification of intermolecular DNA hybridization. In this way, we have developed a "toolbox" for construction of any stacking sequence of these semiconducting molecules. We have discovered that we need to use a full hierarchy of interactions: DNA guides the semiconductors into specified close proximity, hydrophobic-hydrophilic differentiation drives aggregation of the semiconductor moieties, and local geometry and electrostatic interactions define intermolecular positioning. As a result, the PDIs pack to give substantial intermolecular π wave function overlap, leading to an evolution of singlet excited states from localized excitons in the PDI monomer to excimers with wave functions delocalized over all five PDIs in the pentamer. This is accompanied by a change in the dominant triplet forming mechanism from localized spin-orbit charge transfer mediated intersystem crossing for the monomer toward a delocalized excimer process for the pentamer. Our modular DNA-based assembly reveals real opportunities for the rapid development of bespoke semiconductor architectures with molecule-by-molecule precision.

Targeted modulation of protein liquid-liquid phase separation by evolution of amino-acid sequence.

Rationally and efficiently modifying the amino-acid sequence of proteins to control their ability to undergo liquid-liquid phase separation (LLPS) on demand is not only highly desirable, but can also help to elucidate which protein features are important for LLPS. Here, we propose a computational method that couples a genetic algorithm to a sequence-dependent coarse-grained protein model to evolve the amino-acid sequences of phase-separating intrinsically disordered protein regions (IDRs), and purposely enhance or inhibit their capacity to phase-separate. We validate the predicted critical solution temperatures of the mutated sequences with ABSINTH, a more accurate all-atom model. We apply the algorithm to the phase-separating IDRs of three naturally occurring proteins, namely FUS, hnRNPA1 and LAF1, as prototypes of regions that exist in cells and undergo homotypic LLPS driven by different types of intermolecular interaction, and we find that the evolution of amino-acid sequences towards enhanced LLPS is driven in these three cases, among other factors, by an increase in the average size of the amino acids. However, the direction of change in the molecular driving forces that enhance LLPS (such as hydrophobicity, aromaticity and charge) depends on the initial amino-acid sequence. Finally, we show that the evolution of amino-acid sequences to modulate LLPS is strongly coupled to the make-up of the medium (e.g. the presence or absence of RNA), which may have significant implications for our understanding of phase separation within the many-component mixtures of biological systems.

Protein disorder-to-order transition enhances the nucleosome-binding affinity of H1.

Intrinsically disordered proteins are crucial elements of chromatin heterogenous organization. While disorder in the histone tails enables a large variation of inter-nucleosome arrangements, disorder within the chromatin-binding proteins facilitates promiscuous binding to a wide range of different molecular targets, consistent with structural heterogeneity. Among the partially disordered chromatin-binding proteins, the H1 linker histone influences a myriad of chromatin characteristics including compaction, nucleosome spacing, transcription regulation, and the recruitment of other chromatin regulating proteins. Although it is now established that the long C-terminal domain (CTD) of H1 remains disordered upon nucleosome binding and that such disorder favours chromatin fluidity, the structural behaviour and thereby the role/function of the N-terminal domain (NTD) within chromatin is yet unresolved. On the basis of microsecond-long parallel-tempering metadynamics and temperature-replica exchange atomistic molecular dynamics simulations of different H1 NTD subtypes, we demonstrate that the NTD is completely unstructured in solution but undergoes an important disorder-to-order transition upon nucleosome binding: it forms a helix that enhances its DNA binding ability. Further, we show that the helical propensity of the H1 NTD is subtype-dependent and correlates with the experimentally observed binding affinity of H1 subtypes, suggesting an important functional implication of this disorder-to-order transition.

DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses.

Thermodynamics and kinetics of phase separation of protein-RNA mixtures by a minimal model.

Intracellular liquid-liquid phase separation enables the formation of biomolecular condensates, such as ribonucleoprotein granules, which play a crucial role in the spatiotemporal organization of biomolecules (e.g., proteins and RNAs). Here, we introduce a patchy-particle polymer model to investigate liquid-liquid phase separation of protein-RNA mixtures. We demonstrate that at low to moderate concentrations, RNA enhances the stability of RNA-binding protein condensates because it increases the molecular connectivity of the condensed-liquid phase. Importantly, we find that RNA can also accelerate the nucleation stage of phase separation. Additionally, we assess how the capacity of RNA to increase the stability of condensates is modulated by the relative protein-protein/protein-RNA binding strengths. We find that phase separation and multiphase organization of multicomponent condensates is favored when the RNA binds with higher affinity to the lower-valency proteins in the mixture than to the cognate higher-valency proteins. Collectively, our results shed light on the roles of RNA in ribonucleoprotein granule formation and the internal structuring of stress granules.

Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid-Liquid Phase Separation.

Proteins containing intrinsically disordered regions (IDRs) are ubiquitous within biomolecular condensates, which are liquid-like compartments within cells formed through liquid-liquid phase separation (LLPS). The sequence of amino acids of a protein encodes its phase behaviour, not only by establishing the patterning and chemical nature (e.g., hydrophobic, polar, charged) of the various binding sites that facilitate multivalent interactions, but also by dictating the protein conformational dynamics. Besides behaving as random coils, IDRs can exhibit a wide-range of structural behaviours, including conformational switching, where they transition between alternate conformational ensembles. Using Molecular Dynamics simulations of a minimal coarse-grained model for IDRs, we show that the role of protein conformation has a non-trivial effect in the liquid-liquid phase behaviour of IDRs. When an IDR transitions to a conformational ensemble enriched in disordered extended states, LLPS is enhanced. In contrast, IDRs that switch to ensembles that preferentially sample more compact and structured states show inhibited LLPS. This occurs because extended and disordered protein conformations facilitate LLPS-stabilising multivalent protein-protein interactions by reducing steric hindrance; thereby, such conformations maximize the molecular connectivity of the condensed liquid network. Extended protein configurations promote phase separation regardless of whether LLPS is driven by homotypic and/or heterotypic protein-protein interactions. This study sheds light on the link between the dynamic conformational plasticity of IDRs and their liquid-liquid phase behaviour.

Breakdown of the law of rectilinear diameter and related surprises in the liquid-vapor coexistence in systems of patchy particles.

The phase diagram of molecular or colloidal systems depends strongly on the range and angular dependence of the interactions between the constituent particles. For instance, it is well known that the critical density of particles with "patchy" interactions shifts to lower values as the number of patches is decreased [see Bianchi et al. Phys. Rev. Lett. 97, 168301 (2006)]. Here, we present simulations that show that the phase behavior of patchy particles is even more interesting than had been appreciated. In particular, we find that, upon cooling below the critical point, the width of the liquid-vapor coexistence region of a system of particles with tetrahedrally arranged patches first increases, then decreases, and finally increases again. In other words, this system exhibits a doubly re-entrant liquid-vapor transition. As a consequence, the system exhibits a very large deviation from the law of rectilinear diameter, which assumes that the critical density can be obtained by linear extrapolation of the averages of the densities of the coexisting liquid and vapor phases. We argue that the unusual behavior of this system has the same origin as the density maximum in liquid water and is not captured by the Wertheim theory. The Wertheim theory also cannot account for our observation that the phase diagram of particles with three patches depends strongly on the geometrical distribution of the patches and on the degree to which their position on the particle surface is rigidly constrained. However, the phase diagram is less sensitive to small angular spreads in the patch locations. We argue that the phase behavior reported in this paper should be observable in experiments on patchy colloids and may be relevant for the liquid-liquid equilibrium in solutions of properly functionalized dendrimers.