Biology subjects

Mittal, J.

Publications and source records attributed to Mittal, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Scaffold Affinity Tunes Biomolecular Condensate Function

Biomolecular condensates (BMCs) organize cellular biochemistry by concentrating selected molecules into dynamic membrane-free compartments. Yet the molecular parameters that determine not only whether condensates form, but also how they behave and what they do, remain poorly defined. Here we show that scaffold binding affinity (Kd) is a quantitative determinant of condensate phase behavior, internal dynamics and biochemical output. Using a modular SUMO-SIM system in which scaffold valency was held constant while binding affinity was systematically varied, we found that affinity governs the phase boundary, resistance to chemical perturbation, and molecular mobility of condensates in vitro and in human cells. In multicomponent mixtures, the highest-affinity scaffold dominated dense-phase composition and dynamics, revealing a hierarchical rule for condensate organization. Finally, affinity-dependent changes in condensate dynamics translated into tunable enzyme activity, establishing binding energetics as an engineerable parameter for programming condensate biochemistry.

biochemistry↗

bioRxiv2026

Condensate Material Properties Influence Cargo Selection for Neuronal Extrusion via Large Extracellular Exopher Vesicles

Neurons can maintain proteostasis by extruding protein aggregates and damaged organelles via large extracellular vesicles called exophers. However, the biophysical rules governing exopher cargo selection remain poorly understood. Here, we discovered that biomolecular condensates can localize to exophers and investigated whether condensate material properties-ranging from liquids to dynamically arrested gels-determine their recruitment to exophers. By reprogramming interactions in a multidomain protein, we tuned condensate properties and linked them to neuronal exopher recruitment using a multidisciplinary approach combining in vivo dynamics, in vitro material properties, and molecular dynamics simulations. Our findings reveal that gel-like condensates with slower dynamics and higher viscoelasticity localize to exophers more avidly than dynamic liquid-like condensates, demonstrating a strong correlation between condensate dynamics and exopher recruitment. This work lays the framework for investigating the biophysical determinants of exopher cargo selection, providing critical insights into the influence of proteome material state on neuronal protein quality control.

biophysics↗

bioRxiv2026

The role of electrostatic interactions in the phase separation of HP1α and its protein binding partners

Heterochromatin protein 1 (HP1) is an intrinsic component of heterochromatin domains where it is involved in a diverse set of functions including heterochromatin spreading and organization, chromatin compaction and transcriptional silencing. It has been suggested that HP1 functions through a phase separation mechanism, a process that has been observed in vitro in the presence of N-terminal phosphorylation, nucleic acids and nucleosome arrays. HP1 can also interact with numerous binding partners that contain a specific motif called an HP1 access code (HAC). HACs recognize and bind to an interface formed by the chromoshadow (CSD) domains in the HP1 homodimer, the functional form of the protein. It has been shown that some HP1 binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1 and three binding partners, namely the p150 subunit of the chromatin assembly factor 1 (CAF-1), the N-terminal domain of the lamin B receptor (LBR), and the mitotic protein Shugoshin 1 (Sgo1). Using phase separation assays, we show that CAF-1 prevents HP1 phase separation while LBR and Sgo1 enhance it. Binding assays, mutational studies, NMR spectroscopy and computational analysis allow us to dissect the contributions of the HAC motifs, the charge patterns of the binding partner sequences and the role of N-terminal phosphorylation on HP1 in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1, while electrostatic interactions dominate the regulation of phosphorylated HP1. These results suggest that HP1s binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts. Statement of significanceCellular nuclei are organized into euchromatin and heterochromatin domains, encompassing actively transcribed genes or transcriptionally silent regions, respectively. The regulation of these domains is an active area of study with vast consequences for human health and disease. Here, we focus on the HP1 protein, a key component of heterochromatin compartments that is believed to function through a phase separation mechanism. HP1 interacts with a multitude of binding partners that recognize a specific interaction surface on the protein. While these specific interactions are important, our results suggest that the overall electrostatic network that forms between the proteins has a profound effect on the properties of HP1. These results have important implications for understanding how HP1 function is regulated in heterochromatin environments.

biophysics↗

bioRxiv2026

Molecular Origins of pH Gradients in Charge-Regulated Biomolecular Condensates

Biomolecular condensates exhibit spontaneous electrochemical microenvironments characterized by asymmetric ion distributions and pH gradients that emerge from protein-sequence-dependent charge regulation. Despite their biological importance, mechanistic understanding of these microenvironments has been constrained by the absence of computationally tractable frameworks capable of treating proton exchange, counterion partitioning, and buffer equilibria on consistent thermodynamic footing. Here, we introduce the buffered Charge-Regulation Monte Carlo (b-CR-MC) framework, which couples grand-canonical exchange of ions and buffer species with explicit charge regulation of titratable residues. By extending the CR-MC ion-merging strategy to multicomponent reservoirs and employing the Restricted Primitive Model, b-CR-MC achieves computational efficiency while maintaining thermodynamic rigor, with quantitative agreement to the more expensive generalized G-RxMC approach. Applied to full-length FUS (net positive) and PGL-3 (net negative) under physiological conditions, the framework reveals sequence-dependent pH gradients: the dense phase of FUS exhibits an alkaline shift, while PGL-3 exhibits an acidic shift, in both cases driving the condensate interior toward the proteins isoelectric point. Slab-geometry simulations further resolve the Donnan potential and continuous ion profiles across the condensate interface, confirming the direction and magnitude of these electrochemical shifts. Additionally, we identify spatially resolved buffer depletion within dense phases, establishing that dynamic charge regulation is a primary determinant rather than a secondary correction to condensate electrochemistry. By establishing a sequence-resolved, thermodynamically consistent computational platform, b-CR-MC enables quantitative prediction of how mutations and post-translational modifications reprogram condensate microenvironments across biological and pathophysiological contexts.

biophysics↗

bioRxiv2026

Learning molecular determinants of selective small-molecule partitioning across biomolecular condensates

The functional role of biomolecular condensates is shaped by the composition of constituent proteins, nucleic acids, ions, and small molecules. Selective partitioning of small molecules into condensates has therefore emerged as a potential route to condensate-specific chemical probes and therapeutics. Although partitioning is influenced by differences in solvation environments between coexisting dense and dilute phases, a molecular framework connecting small-molecule structure to condensate-specific enrichment remains lacking. Here, we use existing experimental partitioning data for a library of FDA-approved drugs and metabolites across four biomolecular condensates to develop an interpretable graph-based model of small-molecule partitioning. By combining multitask pretraining, condensate-specific fine-tuning, evidential uncertainty quantification, and atom-level attribution analysis, our model predicts continuous partition coefficients with improved accuracy over descriptor-based approaches. Atom-level attributions reveal that condensate partitioning is not governed by a universal chemical rule: the same molecular scaffold can be read differently by distinct condensate environments, with local atomic context and connectivity determining whether specific atoms promote or suppress enrichment. We further apply the trained model to ~1.7 million drug-like molecules from ChEMBL, identifying a chemically diverse space of predicted condensate-selective partitioners and mapping regions where predictions are confident versus where new measurements would be most informative. Together, this work establishes condensate partitioning as a chemically learnable property shaped by the interplay between small-molecule structure and condensate-specific microenvironments, providing an interpretable and uncertainty-aware framework for defining molecular determinants of partitioning and guiding the discovery of condensate-selective small molecules.

biochemistry↗

bioRxiv2026

Organization of core mitochondrial replication components into multiphasic condensates

Genomes are organized across several hierarchical levels. Intracellular phase transitions compartmentalize many genomic processes, including transcription and DNA repair. However, little is known regarding how phase transitions contribute to replication, in which long strands of double- and single-stranded DNA need to be coordinated. Here, we investigated the molecular interactions driving the condensation of core mitochondrial replication components into mt-nucleoids. Complex phase behavior emerged among purified mt-replication components: each nucleic acid colocalized with its cognate architectural protein within multiphasic condensates. Using single-molecule experiments, we found that formation of ssDNA increased the partitioning of its cognate protein mtSSB within the condensate, consistent with the preferential localization of mtSSB to replicating mt-nucleoids. To develop mechanistic insights, we built a minimalistic coarse-grained model of mt-replication components that showed how interactions between binary pairs dictate their assembly within condensates. The multiphasic organization of mt-nucleoids has implications for how replication can be spontaneously organized in cells. HighlightsO_LIComplex co-phase behavior of TFAM-mtDNA depends on their affinity and DNA length C_LIO_LIMt-replication architectural proteins segregate ssDNA and dsDNA within condensates C_LIO_LImtSSB selectively partitions into actively replicating nucleoids in vivo C_LIO_LIExposure of ssDNA promotes mtSSB partitioning into TFAM-mtDNA condensates C_LI

biophysics↗

bioRxiv2026

Breaking β-sheets in FUS prion-like domain preserves phase separation and function but prevents aggregation and toxicity

The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation associated with RNA processing. However, the prion-like low complexity (LC) domain of FUS forms solid-like aggregates in neurodegenerative diseases. Whether the formation of {beta}-sheet structure associated with pathology is also physiologically/functionally relevant is debated. Similarly, if mislocalization alone or concomitant aggregation is responsible for FUS gain-of-function toxicity remains to be probed. Here, we introduce {beta}-sheet breaking proline residues into FUS LC with the goal of preventing cross-{beta}-driven aggregation without disrupting essential functions and phase separation. {beta}-sheet-deficient FUS variants maintain native-like global motions, disorder, and phase separation, but no longer show a liquid-to-solid transition (LST). Biochemical partitioning, cellular localization, and auto- and cross-regulatory functions of FUS all remain essentially unchanged. Conversely, FUS-induced neurodegeneration in several Drosophila models is drastically reduced. These findings suggest a strategy for mitigating disease-related toxicity through backbone structure modulation to prevent prion-like domain protein aggregation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/706410v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@d15f63org.highwire.dtl.DTLVardef@1cd6221org.highwire.dtl.DTLVardef@e58126org.highwire.dtl.DTLVardef@181ec67_HPS_FORMAT_FIGEXP M_FIG C_FIG SUMMARYThe RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation as part of its physiological function but can aberrantly aggregate into solid-like assemblies in amyotrophic lateral sclerosis and frontotemporal dementia. To dissect the role of {beta}-sheets in both function and pathological transition, we engineered {beta}-sheet-preventing FUS variants via targeted proline residue insertions in the prion-like disordered region. These variants retained native structure, motions, and phase behavior yet showed dramatically reduced aggregation, both as an isolated prion-like domain and in full-length FUS. Crucially, these variants maintained a panel of FUS cellular functions that depend on FUS condensation but prevented FUS toxicity in fly models of neurodegeneration. Our findings implicate {beta}-sheets as key drivers of FUS condensate maturation and neuronal toxicity, highlighting {beta}-sheet modulation as a therapeutic strategy against FUS-related neurodegeneration. HIGHLIGHTSO_LITargeted proline additions disrupt {beta}-sheet formation in FUS without altering native conformations, dynamics, or phase separation behavior C_LIO_LI{beta}-sheet-deficient FUS variants prevent aggregation and liquid-to-solid transitions while retaining key biological functions C_LIO_LIIn vivo models reveal attenuated toxicity of {beta}-sheet-deficient FUS in Drosophila C_LIO_LI{beta}-sheets are identified as central drivers of condensate maturation and neuronal death, offering a therapeutic entry point for modulating prion-like domain pathology C_LI

biochemistry↗

bioRxiv2026

Biomolecular Condensates Act as Distinct Solvation Environments that Reshape Amino Acid pK Values

Biomolecular condensates create distinct solvation environments in which the ionization equilibria of amino acid side chains may differ markedly from those in bulk aqueous solution. Here, we use all-atom continuous constant pH molecular dynamics simulations to investigate the changes to the pKa values of titratable residues between the coexisting phases of biomolecular condensates. We find that protonated states are favored in the condensate, resulting in the stabilization of charged forms of cationic residues and neutral forms of anionic residues. The effect is consistent across condensates formed by five peptide sequences suggesting that the preference for protonated states is a universal feature of the condensate microenvironment. This highlights that differences in the solvation environments between the coexisting phases play a central role in charge regulation in phase-separating proteins.

biophysics↗

bioRxiv2025

The Structural Basis for RNA Binding and Recognition of the Disordered Prion-Like Domain of TDP-43

Though the structural details of how RNA interacts with folded RNA-binding domains are well established, how intrinsically disordered regions (IDRs) found in a large fraction of RNA-binding proteins mediate contacts with RNA and if they contribute to binding specificity has not been extensively characterized. The human RNA-binding protein TDP-43 is associated with many RNA processing functions that require its predominantly disordered C-terminal domain (CTD) that forms disease-associated inclusions in ALS, and other neurodegenerative conditions. Here, we demonstrate that TDP-43 CTD directly interacts with RNA primarily via a region of the IDR composed of clustered positively charged residues. Large RNAs act as a multivalent scaffold for CTD monomers, inducing the -helical segment of TDP-43 CTD to form multimeric protein-protein structures. Additionally, we probe the nucleotide base and amino acid specificity of CTD-RNA interactions, showing that arginine, aromatic and polar residues display a preference for U and G nucleic acid bases over C and A. Finally, we probe the molecular basis for the strong binding interaction between TDP-43 and G4 quadruplex structures and discover similarly avid interactions with cytosine-rich DNA I-motifs. This work deepens our understanding of how disordered regions of proteins contribute to RNA recognition, drive function, and contribute to disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/695782v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f1d256org.highwire.dtl.DTLVardef@284c2forg.highwire.dtl.DTLVardef@1a1d715org.highwire.dtl.DTLVardef@2fbb1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Site-specific methionine oxidation alters structure and phase separation of TDP-43 C-terminal domain

TAR DNA binding protein 43 (TDP-43), a key protein linked to ALS pathology, undergoes phase separation and forms functional assemblies via condensation within cells. The conserved region (CR) within its C-terminal domain (CTD) mediates self-assembly through helix-helix interactions, while the flanking intrinsically disordered regions (IDRs) contribute to phase separation through transient interactions involving aromatic and hydrophobic residues. The CTD contains ten methionine residues distributed equally between these regions, making it particularly susceptible to oxidative modifications. While methionine oxidation is known to impair phase separation, neither the precise mechanism nor the specific contribution of methionines in the CR compared to the IDRs has been determined. Here, we combine NMR spectroscopy and all-atom molecular dynamics (MD) simulations to reveal if and how methionine oxidation in each region differentially affects CTD structure and phase separation. We demonstrate that all methionine residues are vulnerable to oxidation, leading to distinct regional effects: oxidation of CR methionines disrupts helical structure and directly impairs intermolecular helical association, while oxidation of IDR methionines disrupts long-range contacts. Hence, oxidation of methionines in both regions contributes to impaired phase separation, albeit through different mechanisms. These findings establish methionines as critical redox-sensitive modulators of TDP-43 phase behavior and provide molecular insights into how oxidative stress may contribute to TDP-43 dysregulation in neurodegenerative diseases.

biochemistry↗

bioRxiv2025

RNA modulates FUS condensate assembly, dynamics, and aggregation through diverse molecular contacts

Fused in sarcoma (FUS) is an RNA-binding protein that undergoes phase separation with RNA and other cellular components, forming ribonucleoprotein (RNP) granules. While recent advances delineating the molecular forces that underlie phase separation have largely focused on protein-protein interactions (1-6), the molecular details of protein-RNA interactions within condensates remain limited. In this study, we demonstrate that RNA modulates the phase separation of the low-complexity (LC) and arginine-glycine-glycine motif (RGG1) domains of FUS: low RNA concentrations enhance protein phase separation and excess RNA disrupts it. By integrating biochemical assays, NMR spectroscopy, and molecular dynamics simulations, we show that RNA incorporates into FUS condensates, reducing condensate density while enhancing local relaxation and diffusional motion of FUS. Surprisingly, whereas RNA binding in the dispersed phase primarily involves the RGG1 domain, within the condensed phase, both LC and RGG1 domains contribute to interactions with RNA. NMR and simulation data show diverse interactions between amino acids and RNA moieties, including prominent glutamine-RNA contacts, that stabilize FUS-RNA co-condensates. Furthermore, we found that RNA accelerates the liquid-to-solid transition of FUS LC-RGG1 condensates, promoting fibrillar aggregate formation. Together, these results provide mechanistic insight into how RNA regulates the assembly, dynamics, and maturation of protein condensates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/694118v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@19168a9org.highwire.dtl.DTLVardef@16e6878org.highwire.dtl.DTLVardef@18cb8a9org.highwire.dtl.DTLVardef@1af6a4c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Using NMR and molecular simulations, we map how RNA engages FUS LC-RGG1 within condensates through electrostatic, {pi}-stacking, and hydrogen-bond contacts. We find that RNA incorporation dilutes condensate density, tunes protein mobility, remodels interaction networks, and accelerates the formation of fibrillar aggregates. C_FIG

biochemistry↗

bioRxiv2025

Sticky Interactions Govern Sequence-Dependent Dynamics in Biomolecular Condensates

Intrinsically disordered proteins (IDPs) play a central role in shaping the dynamics and material properties of biomolecular condensates. Understanding how sequence features determine these properties is critical for elucidating physiological function and guiding the rational design of synthetic condensates. Here, we use molecular dynamics simulations to investigate condensates formed by model IDPs with systematically varied chain length and charge patterning, two features characteristic of natural IDPs. Our results show that chain relaxation times, governed by sequence-dependent electrostatic interactions, quantitatively predict condensate viscosity and diffusivity. These condensates exhibit dynamics consistent with a crossover regime between Rouse and reptation behavior. While the Rouse model with idealized friction fails to capture sequence effects, the sticky Rouse model, which incorporates transient interchain contact lifetimes, accurately predicts chain reconfiguration times and, consequently, macroscopic material properties. This work establishes a predictive, sequence-resolved framework that links molecular interactions to condensate dynamics across length and time scales.

biophysics↗

bioRxiv2025

Heterotypic Protein Interactions Modulate the Condensate Dynamics and Aggregation of α-Synuclein

Multicomponent biomolecular condensates formed by diverse multivalent proteins underlie numerous cellular processes, from ribosome biogenesis to stress response regulation, and have recently been implicated in disease-related protein aggregation. Understanding how heterotypic protein interactions modulate condensate dynamics and transitions to amyloid states remains a major challenge. Here, we combine fluorescence -based ensemble and single-molecule approaches, molecular simulations, and systematic domain deletions to investigate how charge patterning and domain structure influence co-condensation between -synuclein, a disordered neuronal protein linked to Parkinsons disease, and the SARS-CoV-2 nucleocapsid protein (NP), a structured viral RNA-binding protein. We find that co-condensation is driven by multivalent electrostatic interactions and occurs when the heterotypic affinity exceeds a threshold, resulting in restricted protein dynamics and altered condensate material properties. These changes promote the formation of dense amyloid fibrils, as confirmed by atomic force microscopy and fluorescence assays. Our results elucidate how heterotypic interactions within multiphasic condensates can modulate phase behavior and aggregation, providing insight into broader mechanisms linking condensate dysregulation with neurodegenerative diseases.

biophysics↗

bioRxiv2025

Multiphasic Organization and Differential Dynamics of Proteins Within Protein-DNA Biomolecular Condensates

Biomolecular condensates formed through liquid-liquid phase separation are increasingly recognized as critical regulators of genome organization and gene expression. While the role of proteins in driving phase separation is well-established, how DNA modulates the structure and dynamics of protein-DNA condensates remains less well understood. Here, we employ a minimalist coarse-grained model to investigate the interplay between homotypic protein-protein and heterotypic protein-DNA interactions in governing condensate formation, composition, and internal dynamics. Our simulations reveal that DNA chain length and flexibility critically influence condensate morphology, leading to the emergence of multiphasic and core-shell organizations under strong heterotypic interactions. We find that DNA recruitment into the condensate significantly alters protein mobility, giving rise to differential dynamics of proteins within the condensate. By analyzing the distribution profiles of protein displacements, we identify up to five distinct diffusion modes, including proteins bound to DNA, confined within the dense phase, or freely diffusing. These results provide a mechanistic framework for interpreting spatially heterogeneous protein dynamics observed in chromatin condensates and emphasize the direct role of DNA in tuning condensate properties. Our findings provide new insights into how biophysical parameters may control the functional architecture of protein-DNA condensates in biological systems. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/658691v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@108a818org.highwire.dtl.DTLVardef@153f9a1org.highwire.dtl.DTLVardef@29f220org.highwire.dtl.DTLVardef@1d74d6e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Controlling Multicomponent Condensate Morphology via Additive-Modulated Interactions

The morphology of biomolecular condensates plays a critical role in regulating intracellular organization and function by enabling both spatial and temporal control over biochemical processes. Recent studies have shown that small-molecule cosolutes can not only modulate phase separation but also influence condensate morphology. However, the mechanistic understanding of how small molecules regulate condensate structure remains limited. In this study, we employ coarse-grained molecular dynamics simulations to investigate how the morphology of two-component condensates can be modulated through the introduction of additional small-molecule cosolutes. By systematically varying the interaction strengths between the small molecules and the macromolecular components, we observe morphological transitions between, e.g., core-shell and dewetted structures. To rationalize these transitions, we calculate second virial coefficients in the presence of the small molecules, providing a molecular-level framework to capture shifts in effective homotypic and heterotypic interactions. We further investigate the role of stoichiometry between the small molecules and macromolecules, demonstrating that stoichiometry and interaction strength jointly determine the condensate morphology by altering the relative interaction strengths among components. Additionally, we show that fully miscible two-component condensates can undergo transitions to microphase-separated morphologies, such as core-shell or dewetted, upon small molecule introduction. Together, these findings reveal that condensate morphology can be rationally tuned through interaction- and stoichiometry-dependent mechanisms, offering molecular-scale insights into how small-molecule cosolutes modulate condensate structure.

biophysics↗

bioRxiv2025

A High-Throughput, Flow Cytometry Approach to Measure Phase Behavior and Exchange in Biomolecular Condensates

Biomolecular condensates are essential for cellular organization, yet their formation dynamics and molecular content exchange properties remain poorly understood. In this study, we developed a high-throughput flow cytometry approach to quantify condensate formation, molecular colocalization, and dynamic exchange. Using self-interacting NPM1 condensates as a model system, we benchmarked the use of flow cytometry to broadly characterize their phase behavior across various protein concentrations and salt conditions. We further demonstrated that flow cytometry can assess the colocalization of macromolecules - including antibodies, lipids, small-molecule drugs, and RNAs, within NPM1 condensates. Importantly, we established the first assay to track real-time molecular exchange between preformed condensates and newly added, orthogonally tagged protein. This revealed that condensate aging significantly reduces molecular dynamisms, likely due to altered biophysical properties with time. Compared to conventional imaging techniques, which often require immobilized samples and complex experimental setups, our solution-based approach enables rapid, quantitative analysis of condensate behavior at the single-droplet level. This work provides a powerful framework for studying biomolecular condensates with enhanced precision and scalability, offering new insights into their dynamic properties and molecular interactions.

biophysics↗

bioRxiv2025

Multiscale simulations elucidate the mechanism of polyglutamine aggregation and the role of flanking domains in fibril polymorphism

Protein aggregation, which is implicated in aging and neurodegenerative diseases, typically involves a transition from soluble monomers and oligomers to insoluble fibrils. Polyglutamine (polyQ) tracts in proteins can form amyloid fibrils, which are linked to polyQ diseases, including Huntingtons disease (HD), where the length of the polyQ tract inversely correlates with the age of onset. Despite significant research on the mechanisms of Httex1 aggregation, atomistic information regarding the intermediate stages of its fibrillation and the morphological characteristics of the end-state amyloid fibrils remains limited. Recently, molecular dynamics (MD) simulations based on a hybrid multistate structure-based model, Multi-eGO, have shown promise in capturing the kinetics and mechanism of amyloid fibrillation with high computational efficiency while achieving qualitative agreement with experiments. Here, we utilize the multi-eGO simulation methodology to study the mechanism and kinetics of polyQ fibrillation and the effect of the N17 flanking domain of Huntingtin protein. Aggregation simulations of polyQ produced highly heterogeneous amyloid fibrils with variable-width branched morphologies by incorporating combinations of {beta}-turn, {beta}-arc, and {beta}-strand structures, while the presence of the N17 flanking domain reduces amyloid fibril heterogeneity by favoring {beta}-strand conformations. Our simulations reveal that the presence of N17 domain enhanced aggregation kinetics by promoting the formation of large, structurally stable oligomers. Furthermore, the early-stage aggregation process involves two distinct mechanisms: backbone interactions driving {beta}-sheet formation and side-chain interdigitation. Overall, our study provides detailed insights into fibrillation kinetics, mechanisms, and end-state polymorphism associated with Httex1 amyloid aggregation. SIGNIFICANCE STATEMENTPolyglutamine (polyQ) aggregation is central to Huntingtons disease and related neurodegenerative disorders. Despite extensive experimental efforts, a complete molecular understanding of this process--from early aggregation events to the origins of fibril polymorphism--has remained elusive, with varied interpretations of complex fibril architectures. Through multiscale simulations, we reveal how polyQ fibrils adopt diverse tertiary and quaternary structures and demonstrate how the N-terminal flanking domain (N17) modulates fibril architecture and accelerates aggregation. Our hybrid multi-eGO simulations capture early-stage fibrillation kinetics and identify distinct structural polymorphs that align with experimental observations. This work provides a molecular framework for understanding amyloid polymorphism and illuminates the role of flanking domains in shaping aggregation pathways-- offering valuable insights for therapeutic strategies targeting early toxic intermediates.

biophysics↗

bioRxiv2025

Elucidation of the molecular interaction network underlying full-length FUS conformational transitions and its phase separation using atomistic simulations

Fused in Sarcoma (FUS), a multi-domain RNA-binding protein, orchestrates cellular functions through liquid-liquid phase separation (LLPS), which promotes the formation of biomolecular condensates in vivo. While crucial to understanding cellular processes, an atomic-level view of the interdomain interactions associated with full-length (FL) FUS LLPS remains challenging due to its low solubility in vitro. Here, using all-atom (AA) molecular dynamics (MD) simulations, we examined the conformational dynamics and interdomain interactions of FL FUS in both dilute and condensed phases. Comparing two modern force fields (FFs) - Amber ff03ws and ff99SBws-STQ, we found that monomer simulation ensembles generated by both FFs exhibited qualitatively similar intramolecular interaction profiles dominated by intrinsically disordered regions (IDRs). While the two folded domains minimally participated in interdomain interactions, their stabilities significantly influenced the chain dimension and led to discrepancies compared to experimental data for both FFs. We observed that the Amber ff99SBws-STQ coupled with bond parameters adopted from the Zinc Amber force field (ZAFF) maintained stable folded domains and improved estimates of the chain dimensions. Finally, a microsecond-timescale simulation of FL FUS condensate revealed an extensive network of electrostatic interactions which are strongly correlated with those that modulate the dilute phase chain dimensions. Overall, insights from our all-atom simulations illuminate the interplay between folded domain stability and IDR interactions in modulating protein conformation and phase separation, advancing our understanding of FUS-related pathologies at the molecular level and aiding in the development of new therapeutics.

biophysics↗