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

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

Synthetic biomolecular condensates offer a route to engineer compartmentalized microenvironments with tunable physicochemical properties, yet design principles for controlling their internal organization remain limited. Here, we report a minimal two-component system based on thermoresponsive intrinsically disordered protein polymers with lower critical solution temperature behavior (LCST-IDPPs), in which electrostatic topology programs liquid-liquid phase separation (LLPS). Incorporating charged residues into LCST-IDPPs suppresses phase separation under physiological conditions, whereas mixing oppositely charged, individually non-coacervating IDPPs restores LLPS as an emergent, composition-dependent process driven by multivalent intermolecular charge compensation. We compare this two-component system with a covalently linked diblock containing the same charged chains. This change in chain connectivity alters the coupling between electrostatic pairing, counterion redistribution, and LCST-driven dehydration. As a result, the balance between inter- and intrachain ionic pairing encodes the residual charge and micropolarity of the dense phase. This topology-dependent microenvironment controls condensate miscibility and drives the formation of either homogeneous or internally demixed multiphase assemblies. The condensate interior also shifts the apparent pKa of ionizable residues, indicating that phase separation modifies local acid-base equilibria and alters the effective side-chain charge. Together, these findings show how electrostatic topology influences LLPS, the dense-phase microenvironment, and mesoscale organization in IDPP condensates.

Structural Fine-Tuning and Gel-Forming Properties of Peptides Containing Aromatic Amino Acid Residues

Programming Multidomain Peptides With Molecular Frustration Into Biomolecular Condensates

The discovery of biomolecular condensates, driven by liquid–liquid phase separation of intrinsically disordered proteins has significant impacts on both fundamental and applied science and engineering. Although most studies on biomolecular condensates focus on intrinsically disordered structures, research on the role of molecular ordering remains largely unexplored, however is beneficial for gaining new mechanistic understanding and further expand the design space of peptides for constructing functional condensates. Toward this goal, we conducted systematic studies on how molecular ordering impacts the phase behaviors of peptides using multidomain peptides (MDPs) as a model system. MDPs were designed using a molecular frustration principle in which parts of the peptides favored β-sheet assembly and parts favored disassembly. Through programming of each domain, it is evident that the phase behavior of MDPs is largely dictated by the secondary structure, and partially folded β-sheet plays a key role in driving MDPs to form condensates. We also discovered complex coacervates formed by MDPs and synthetic anionic polymers, which exhibited dramatically improved stability. Furthermore, we show enzyme-triggered condensation can be achieved using phosphorylated MDPs as the molecular precursor and alkaline phosphatase as a molecular switch, highlighting the potential of these materials for bacterial imaging and antimicrobial therapy development.

Intrinsically disordered regions as drivers of protein aggregation: mechanisms, phase separation, and emerging predictive frameworks

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) lack stable tertiary structures yet perform essential roles in cellular signaling, molecular recognition, transcriptional regulation, and biomolecular assembly. Their conformational flexibility enables functional adaptability but also increases susceptibility to aberrant intermolecular interactions and protein aggregation. Unlike folded proteins, aggregation in IDPs arises from transient conformational ensembles that expose cryptic aggregation-prone regions (APRs), facilitating oligomerization and fibril formation under specific cellular and environmental conditions. Several studies have further established a mechanistic relationship between intrinsic disorder, liquid–liquid phase separation (LLPS), and pathological aggregation, where dynamic condensates can undergo maturation into irreversible amyloid-like assemblies. These transitions are strongly influenced by sequence grammar, charge distribution, aromatic residue patterning, post-translational modifications, molecular crowding, and proteostasis regulation. This mini-review summarizes the molecular principles governing aggregation in disordered systems, with emphasis on conformational ensemble dynamics, disorder-to-order transitions, and the interplay between LLPS and fibrillization. The review further discusses computational approaches used to predict aggregation propensity in IDRs, including classical physicochemical predictors, ensemble-aware simulations, molecular dynamics frameworks, and emerging protein language model-based methods. Further, integration of artificial intelligence, structural biophysics, and multiscale modeling have substantially improved understanding of disorder-driven aggregation pathways. Collectively, these findings support a unified framework in which sequence composition, conformational heterogeneity, and cellular environment cooperatively regulate functional assembly and pathological aggregation in intrinsically disordered proteins.

Molecular mechanisms governing peptide nanodisc assembly and stability.

Apolipoprotein A-I mimetic 4F, an 18-residue amphipathic α-helix, can self-assemble with lipids to form peptide nanodiscs, yet the molecular determinants governing their assembly and stability remain poorly understood. Here, using coarse-grained molecular dynamics (CG-MD), we capture the de novo formation of 4F nanodiscs with DMPC and reveal a multistep assembly pathway involving nucleation, fusion, and ellipse-to-disc maturation. All-atom back-mapping shows that the nanodisc rim is structurally heterogeneous and stabilized by aromatic-acyl interactions, Lys headgroup anchoring, and inter-peptide electrostatic contacts. Lipid composition and temperature critically regulate nanodisc integrity: DMPC supports continuous peptide belts and long-term stability, whereas DPPC below its main phase transition temperature suppresses fusion and yields fragmented, non-uniform rims. These findings validate the ability of CG-MD to resolve nanodisc assembly mechanisms. Experimental measurements corroborate the simulations, demonstrating that 4F nanodiscs exhibit lower thermal resilience than MSP nanodiscs while retaining structural integrity at moderate temperatures. As a functional benchmark, MSP nanodiscs suppress the amyloid-binding thioflavin-T fluorescence signal associated with Aβ (1-40) fibrillar assembly, consistent with our previously reported findings for 4F nanodiscs and supporting the ability of amphipathic nanodisc rims to delay Aβ (1-40) aggregation. Together, these results establish a mechanistic framework and design principles for single-helix peptide nanodiscs and delineate the conditions under which they converge with or diverge from MSP-based scaffolds.

Tunable Thermotropic Liquid Crystals Using Amphiphilic Sequence-Defined Macromolecules

Liquid crystals (LCs) are a class of materials that combine molecular order and fluidity, making them crucial for technologies from displays to sensors. However, polymeric LC materials typically lack molecular precision, limiting systematic control over their phase behavior. Here, we report a modular synthetic strategy to prepare amphiphilic columnar liquid crystals based on hydrophobic di- and trialkylated galloyl cores attached to a sequence-defined polar oligomer grown by a thiolactone-based iterative protocol. This approach enables independent variation of (i) polar monomer type (e.g., hydroxyethyl acrylate and N,N-dimethylacrylamide), (ii) oligomer length (from 1-mer to tetramer), and (iii) core topology (two or three C18 chains). Differential scanning calorimetry, polarized optical microscopy, and X-ray scattering show that the number of hydrophobic C18 chains on the aromatic core is the dominant parameter governing mesophase stability. Derivatives with three C18 chains exhibit higher melting and isotropization temperatures and more pronounced transitions from lamellar crystals to hexagonally packed columnar mesophases than their two C18 analogues. The chemistry and length of the sequence-defined polar block further tune crystallization and mesophase behavior, i.e. hydrogen-bonding heads increase thermal stability, whereas bulkier PEG-like heads reduce crystallinity and can destabilize mesophases. Increasing oligomer length reduces crystallization enthalpies and can introduce competing liquid-crystalline states. These results demonstrate that the liquid crystalline behavior can be encoded at the molecular level, providing a basis for designing responsive and sequence-programmed LC materials.