Computational rheometry of viscoelastic networks: From random graphs to biomolecular condensates.

Multivalent biomacromolecules including multi-domain and intrinsically disordered proteins form biomolecular condensates via reversible phase transitions. Condensates are viscoelastic materials that display composition-specific rheological properties and responses to mechanical forces. Graph-based descriptions of microstructures can be combined with computational rheometry to model the outcomes of passive and active mechanical measurements.

Current practices in the study of biomolecular condensates: a community comment.

The realization that the cell is abundantly compartmentalized into biomolecular condensates has opened new opportunities for understanding the physics and chemistry underlying many cellular processes, fundamentally changing the study of biology. The term biomolecular condensate refers to non-stoichiometric assemblies that are composed of multiple types of macromolecules in cells, occur through phase transitions, and can be investigated by using concepts from soft matter physics. As such, they are intimately related to aqueous two-phase systems and water-in-water emulsions.

Quantifying collective interactions in biomolecular phase separation.

Biomolecular phase separation is an emerging theme for protein assembly and cellular organisation. The collective forces driving such condensation, however, remain challenging to characterise. Here we show that tracking the dilute phase concentration of only one component suffices to quantify composition and energetics of multicomponent condensates.

Tunable metastability of condensates reconciles their dual roles in amyloid fibril formation.

Stress granules form via co-condensation of RNA-binding proteins (RBPs) containing prion-like low-complexity domains (PLCDs) with RNA molecules. Homotypic interactions among PLCDs can drive amyloid fibril formation that is enhanced by amyotrophic lateral sclerosis (ALS)-associated mutations. We report that condensation- versus fibril-driving homotypic interactions are separable for A1-LCD, the PLCD of hnRNPA1.

Active transport enables protein condensation in cells.

Multiple factors drive biomolecular condensate formation. In plants, condensation of the transcription factors AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 attenuates response to the plant hormone auxin. Here, we report that actin-mediated movement of cytoplasmic ARF condensates enhances condensation.

Single-fluorogen imaging reveals distinct environmental and structural features of biomolecular condensates.

Biomolecular condensates are viscoelastic materials. Simulations predict that condensates formed by intrinsically disordered proteins are network fluids defined by spatially inhomogeneous organization of the underlying molecules. Here, we test these predictions and find that molecules within condensates are organized into slow-moving nanoscale clusters and fast-moving dispersed molecules.

Differential interactions determine anisotropies at interfaces of RNA-based biomolecular condensates.

Biomolecular condensates form via macromolecular phase separation. Here, we report results from our characterization of synthetic condensates formed by phase separation of mixtures comprising two types of RNA molecules and the biocompatible polymer polyethylene glycol. Purine-rich RNAs are scaffolds that drive phase separation via heterotypic interactions.

Backbone-mediated weakening of pairwise interactions enables percolation in peptide-based mimics of protein condensates.

Biomolecular condensates formed by intrinsically disordered proteins (IDPs) are semidilute solutions. These can be approximated as solutions of blob-sized segments, which are peptide-sized motifs. We leveraged the blob picture and molecular dynamics simulations to quantify differences between inter-residue interactions in model compound and peptide-based mimics of dense versus dilute phases.

Molecular grammars of intrinsically disordered regions that span the human proteome.

Intrinsically disordered regions (IDRs) of proteins are defined by functionally relevant molecular grammars. This refers to IDR-specific non-random amino acid compositions and non-random patterning of distinct pairs of amino acid types. Here, we introduce GIN (Grammars Inferred using NARDINI+) as a resource, which we have used to extract the molecular grammars of all human IDRs and classified them into thirty distinct clusters.

Nuclear speckle proteins form intrinsic and -dependent microphases.

Nuclear speckles are enriched in serine / arginine rich splicing factors (SRSFs), such as SRSF1. Splicing factors and proteins such as TDP-43 concentrate into distinct speckle territories to enable pre-mRNA processing. We have discovered that SRSFs and TDP-43 are block copolymers and the protein-specific interplay of inter-block repulsions and attractions drives spontaneous microphase separation.

Synapsin Condensation is Governed by Sequence-Encoded Molecular Grammars.

Multiple biomolecular condensates coexist at the pre- and post- synapse to enable vesicle dynamics and controlled neurotransmitter release in the brain. In pre-synapses, intrinsically disordered regions (IDRs) of synaptic proteins are drivers of condensation that enable clustering of synaptic vesicles (SVs). Using computational analysis, we show that the IDRs of SV proteins feature evolutionarily conserved non-random compositional biases and sequence patterns.

Identifying Sequence Effects on Chain Dimensions of Disordered Proteins by Integrating Experiments and Simulations.

It has become increasingly evident that the conformational distributions of intrinsically disordered proteins or regions are strongly dependent on their amino acid compositions and sequence. To facilitate a systematic investigation of these sequence-ensemble relationships, we selected a set of 16 naturally occurring intrinsically disordered regions of identical length but with large differences in amino acid composition, hydrophobicity, and charge patterning. We probed their conformational ensembles with single-molecule Förster resonance energy transfer (FRET), complemented by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as small-angle X-ray scattering (SAXS).

Chromatin-associated lncRNA-splicing factor condensates regulate hypoxia responsive RNA processing of genes pre-positioned near nuclear speckles.

Hypoxia-induced alternative splicing (AS) regulates tumor progression and metastasis. Little is known about how such AS is controlled and whether higher-order genome and nuclear domain (ND) organizations dictate these processes. We observe that hypoxia-responsive alternatively spliced genes position near nuclear speckle (NS), the ND that enhances splicing efficiency.

Sequence-specific interactions determine viscoelasticity and aging dynamics of protein condensates.

Biomolecular condensates are viscoelastic materials. Here, we investigate the determinants of 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.

Biomolecular Condensates are Characterized by Interphase Electric Potentials.

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates can be two- or multiphase systems defined by coexisting dense and dilute phases. Here, we show that solution ions partition asymmetrically across coexisting phases defined by condensates formed by intrinsically disordered proteins or homopolymeric RNA molecules.

Direct computations of viscoelastic moduli of biomolecular condensates.

Biomolecular condensates are viscoelastic materials defined by time-dependent, sequence-specific complex shear moduli. Here, we show that viscoelastic moduli can be computed directly using a generalization of the Rouse model that leverages information regarding intra- and inter-chain contacts, which we extract from equilibrium configurations of lattice-based Metropolis Monte Carlo (MMC) simulations of phase separation. The key ingredient of the generalized Rouse model is a graph Laplacian that we compute from equilibrium MMC simulations.

Synapsin condensation is governed by sequence-encoded molecular grammars.

Multiple biomolecular condensates coexist at the pre- and post- synapse to enable vesicle dynamics and controlled neurotransmitter release in the brain. In pre-synapses, intrinsically disordered regions (IDRs) of synaptic proteins are drivers of condensation that enable clustering of synaptic vesicles (SVs). Using computational analysis, we show that the IDRs of SV proteins feature evolutionarily conserved non-random compositional biases and sequence patterns.

Dominance analysis to assess solute contributions to multicomponent phase equilibria.

Phase separation in aqueous solutions of macromolecules underlies the generation of biomolecular condensates in cells. Condensates are membraneless bodies, representing dense, macromolecule-rich phases that coexist with the dilute, macromolecule-deficient phases. In cells, condensates comprise hundreds of different macromolecular and small molecule solutes.

Condenzymes: Biomolecular condensates with inherent catalytic activities.

We report the discovery that chemical reactions can be catalyzed by condensates formed by intrinsically disordered proteins (IDPs). The proteins themselves lack any catalytic activities. Catalytic functions of condensates emerge as a consequence of sequence-dependent mesoscale electrochemical microenvironments created by phase separation.

Biomolecular condensates are characterized by interphase electric potentials.

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates can be two- or multi-phase systems defined by coexisting dense and dilute phases. Here, we show that solution ions can partition asymmetrically across coexisting phases defined by condensates formed by intrinsically disordered proteins or homopolymeric RNA molecules.

Direct computations of viscoelastic moduli of biomolecular condensates.

facsimiles of biomolecular condensates are formed by different types of intrinsically disordered proteins including prion-like low complexity domains (PLCDs). PLCD condensates are viscoelastic materials defined by time-dependent, sequence-specific complex shear moduli. Here, we show that viscoelastic moduli can be computed directly using a generalization of the Rouse model and information regarding intra- and inter-chain contacts that is extracted from equilibrium configurations of lattice-based Metropolis Monte Carlo (MMC) simulations.

Solutes unmask differences in clustering versus phase separation of FET proteins.

Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride.

A High-Avidity, Thermostable, and Low-Cost Synthetic Capture for Ultrasensitive Detection and Quantification of Viral Antigens and Aerosols.

Lateral flow assays (LFAs) are currently the most popular point-of-care diagnostics, rapidly transforming disease diagnosis from expensive doctor checkups and laboratory-based tests to potential on-the-shelf commodities. Yet, their sensitive element, a monoclonal antibody, is expensive to formulate, and their long-term storage depends on refrigeration technology that cannot be met in resource-limited areas. In this work, LCB1 affibodies (antibody mimetic miniproteins) were conjugated to bovine serum albumin (BSA) to afford a high-avidity synthetic capture (LCB1-BSA) capable of detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and virus like particles (VLPs).

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs).