Topology-Encoded Charge Polarity Governs Multiphase Organization in Intrinsically Disordered Protein Polymer Condensates.

Temperature-Triggered Nanoscale Morphological Transitions in a Synthetic Intrinsically Disordered Protein

Peptide self-assembly and liquid–liquid phase separation (LLPS), often mediated by intrinsically disordered regions (IDRs), are natural mechanisms that translate protein molecular features into complex nano- and mesoscale architectures. Although the thermodynamics and kinetics of these processes are well understood, synthetic materials integrating both functionalities remain rare. Inspired by the conserved IDR–assembly domain (AD) architecture of amyloidogenic proteins, we hypothesized that modular recombinant constructs combining LLPS-capable IDRs with β-sheet-forming ADs could generate materials with tunable structural properties. To test this, we engineered a library of elastin-like polypeptides (ELPs) fused to amphiphilic anionic or cationic amyloidogenic peptides, enabling systematic investigation of how sequence parameters─including ELP length, AD charge, and hydrophilicity─and environmental conditions, including temperature, pH, and salt concentration, influence material behavior. Our results reveal links between molecular design and emergent multiscale structures, including micelles and vesicles embedded within coacervates. This work provides a framework for designing hybrid proteins coupling LLPS and self-assembly.

Engineering Heterotypic Biomolecular Condensates with Synthetic Peptides for Controlled Spatial Organization and Liquid-like Nature

Sequence-dependent molecular asymmetry and architecture define electric potential profiles of biomolecular condensates

Biomolecular condensates, which regulate diverse cellular processes, exhibit distinct electric potential profiles. This potential gradient between the dilute and the dense phases serves as the underlying driving force mediating the unique microenvironment and electrochemical activity of condensates. However, the molecular principles encoding the electric potential profiles of condensates remain unclear. In this study, we show that molecular asymmetry is a unifying origin of electric polarization in condensates. Asymmetric protein–cation and protein–anion affinities alone generate an interfacial electric double layer and a finite potential even in condensates formed by charge-free proteins. The sign of potential gradient follows the direction of the affinity bias, and the magnitude collapses onto a single linear function of dense-phase protein volume fraction across changes in chain length, interaction strength and salt concentration. Further, chain termini preferentially occupy the condensate interface, so charges positioned asymmetrically with respect to the termini create spatial charge separation even in neutral polyampholytes. These interaction-encoded and sequence architecture-encoded asymmetries can reinforce, screen or reverse one another, allowing the magnitude and polarity of the interphase potential to be tuned through sequence design or solvent environments.

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 protein’s 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.

Programmable Multiphasic Condensates Formed via Evaporation–Induced Phase Separation of Minimal Peptide Model

Biomolecular self‐assembly is ubiquitous in nature, encompassing both ordered and disordered structures to create sophisticated superstructures essential for complex biological functions. Protein and peptide condensates formed via liquid–liquid phase separation (LLPS) are characterize by disordered assembly, gaining significant interest due to their crucial role in physiological events and potential applications from drug delivery to biosensing. Short peptides with ordered structures have been widely explored as building blocks for nanoarchitectured materials, but they lack the disordered features that endow biological systems with flexibility and adaptability. Here we introduce a minimalistic peptide sticker‐and‐spacer model that forms biomolecular condensates with core–shell structure through phase separation and spontaneous evaporation. The design allows to derive the guidelines for programming condensate's architecture from homogeneous to multiphasic state via the selection of sticker and spacer. Furthermore, we demonstrate control over compartmentalization driven by intrinsic redox chemistry and post‐assembly modification. The condensates efficiently encapsulate and protect small‐molecule payloads and function as microreactors. The evaporation‐induced spontaneous phase separation results in solidified condensates enriched with redox‐active tyrosine, which serve as novel nano‐bioreactors, promoting selective biomineralization and formation of uniform metal–peptide nanohybrids. Therefore, our study provides a framework for the artificial design of protocells mimetic multicompartmental condensates endowed with on‐demand functionality.

Homeostatic Noise Buffering in Biomolecular Condensates Hinges on Phase Multiplicity Modulated by Interfacial and Droplet Size Effects

Specific mixing or demixing of molecular species is a characteristic feature of condensed intracellular membraneless compartments. How sequence patterns of intrinsically disordered proteins (IDPs) fundamentally impact subcompartmentalization of biomolecular condensates and their role in buffering against concentration fluctuations are hereby addressed by modeling liquid-liquid phase separation (LLPS) of polyampholytic sequence pairs using random phase approximation (RPA) polymer theory and molecular dynamics (MD). RPA theory predicts both binary and ternary LLPS in a temperature-sensitive manner. We observe demixing underpinned by ternary LLPS for pairs with dissimilar sequence charge patterns but not for pairs with similar sequence charge patterns. Notably, the predicted behaviors are corroborated by MD when RPA is augmented with interfacial tension and/or a finite-size formalism commensurating with the typical small sizes of MD model systems, supporting our stipulation that RPA theory is a useful sequence-specific modeling tool for biomolecular condensates with larger, more realistic sizes when finite-size effects are much less significant. In principle, when the condensate size is sufficiently large, ternary LLPS is superior to binary LLPS in noise buffering because the IDP compositions of the three coexisting phases in ternary LLPS remain unchanged over an extended two-dimensional concentration regime, whereas the two coexisting phases in binary LLPS are fixed only along a tieline. However, when condensate sizes are sufficiently small, the buffering capacities of ternary versus binary LLPSs are more complex as they are modulated differently by finite-size effects. Biophysical ramifications of this interplay are discussed in view of the size diversity of natural biomolecular condensates.