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.
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.
Rohit Kumar, Sukantha Dey, P. Rajput et al.· Advances in Materials· 1 citation
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.
Yulia Shmidov, Lixin Fan, Max Ney et al.· Biomacromolecules· 0 citations
This work investigates how protein folding landscapes are altered inside condensates, using the protein α-helix as a model folded domain and develops a chemically specific, residue-resolution model for quantification of α-helical folding and applies it to characterize diverse helices within condensates of varying physicochemical properties.
Nathaniel Hess, Jerelle A. Joseph· Journal of the American Chem...· 0 citations
The formation of fibrous architectures via peptide self-assembly underpins numerous biological functions and biomaterial applications; however, the thermodynamic origins of multistep assembly pathways remain elusive. Here, we map the complete free-energy landscape governing the liquid-liquid phase separation (LLPS)-mediated self-assembly of an amphiphilic peptide by exploiting temperature as a tunable parameter. We discover an unexpected thermodynamic mechanism: the initial LLPS-like clustering is enthalpy-driven but limited by a positive enthalpic barrier (+121 kJ mol-1), arising from the endothermic disruption of intramolecular hydrogen bonds before interpeptide contacts can form. Subsequent nucleation and fibril growth are governed by negative entropic barriers (-56 and -39 kJ mol-1, respectively), reflecting the reorganization cost of partially ordered oligomers. The energy landscape identifies LLPS as the rate-limiting step with the highest Gibbs free-energy barrier (+26 kJ mol-1). Our findings establish a generalizable framework for decoding multistep biomolecular self-organization, with implications for designing adaptive biomaterials and understanding aberrant phase transitions in diseases.
Yufan Yang, Haoning Gong, Peng Zhou et al.· Journal of Physical Chemistr...· 0 citations