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.

The transition of peptides and proteins from the solution phase into fibrillar structures is ageneral phenomenon encountered in functional and aberrant biology and is increasingly exploited in soft materials science.H owever,t he fundamental molecular events underpinning the early stages of their assembly and subsequent growth have remained challenging to elucidate.H ere,w es how that liquid-liquid phase separation into solute-rich and solute-poor phases is af undamental step leading to the nucleation of supramolecular nanofibrils from molecular building blocks,including peptides and even amphiphilic amino acids.T he solute-rich liquid droplets act as nucleation sites,a llowing the formation of thermodynamically favorable nanofibrils following Ostwalds step rule.T he transition from solution to liquid droplets is entropyd riven while the transition from liquid droplets to nanofibrils is mediated by enthalpic interactions and characterizedb ys tructural reorganization. These findings shed light on howt he nucleation barrier towardt he formation of solid phases can be lowered through ak inetic mechanism which proceeds through ametastable liquid phase.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell.Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein. Our findings illustrate the importance of the kinetics of binding of molecular chaperones, and also of potential therapeutic molecules, in the efficient suppression of specific pathogenic events linked to neurodegeneration.

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that has been shown to modify the properties of their monomeric states. Here, we identify a small molecule capable of binding and sequestering the amyloid-β peptide (Aβ) in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterise this interaction using biophysical experiments and integrative structural ensemble determination methods. We thus observe that this small molecule has the remarkable effect of increasing the conformational entropy of monomeric Aβ while decreasing its hydrophobic surface area. We then show that this small molecule rescues a Caenorhabditis elegans model of Aβ-associated toxicity in a manner consistent with the mechanism of action identified from the in silico and in vitro studies. These results provide an illustration of the strategy of targeting the monomeric states of disordered proteins with small molecules to alter their behaviour for therapeutic purposes.Previous studies have suggested that effective strategies for inhibiting Aβ aggregation could be based on targeting fibril surfaces to supress the generation of oligomers, or on the reduction of the toxicity of the oligomers 17-21 . It is unclear, however, whether sequestering Aβ in its soluble state could be an effective drug discovery strategy against Alzheimer's disease. Stabilisation of monomeric Aβ into a β-hairpin conformation with large biomolecules has been previously demonstrated to inhibit aggregation, for example using an affibody protein 22 . However, whether such stabilisation of Aβ in its monomeric form can be achieved via small molecule binding in a drug-like manner is still under debate. While there is research indicating a stabilising effect of small molecules on the soluble state of Aβ, there are contradictory reports of their effects on its aggregation [23][24][25] . It should also be considered that such molecules may not be specific, as for example some appear to bind monomeric Aβ in a manner similar to low concentrations of sodium dodecyl sulphate (SDS) 23-25 . Furthermore, it has been proposed that the binding of these small molecules to monomeric Aβ may be mediated by colloidal particles formed by the small molecules 26 , although this observation has also been disputed 23,24,27 . The uncertainty of whether monomeric Aβ is a viable drug target is caused, in part, by a lack of understanding of the molecular properties of monomeric Aβ and how to stabilise this peptide with specific small molecules that have the potential to be developed as drugs.The complexity of targeting monomeric Aβ is caused, in part, by the fact that Aβ is intrinsically disordered, as it lacks a well-defined structure and instead exists as a heterogeneous ensemble of conformationally distinct states 28 . The dynamic nature of disordered proteins, and the consequent absence of stable and persistent binding poc...

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