Jul 2026· Journal of the American Chemical Society· Vol 148, pp. 30920-30929· 0 citations· 50 references
Medicine
TL;DR
Investigating the first microsecond of peptide condensation through temperature jump infrared spectroscopy and molecular dynamics simulations reveals that hydrogen bonding could enable hydrophobic disordered proteins to adopt preorganized conformations in response to environmental stimuli and serve as a key factor in mediating the assembly kinetics in complex cellular environments.
Abstract
Biomolecular condensation is a key process for cells to maintain their normal physiological activities. However, the process of phase transition remains mysterious, especially for the initial moments of condensation. Herein, we investigated the first microsecond of peptide condensation through temperature jump infrared spectroscopy and molecular dynamics simulations. These techniques overcome the limited spatiotemporal resolution of traditional approaches, allowing us to capture the molecular events and kinetic information on the initial moment for phase transition. The results reveal that structural transitions and early assembly of intrinsically disordered proteins occur on ultrafast time scales. Unexpectedly, backbone hydrogen bonding emerges as the overlooked key mediator to stabilize the local structure for the ultrafast condensation of hydrophobic polypeptides compared to hydrophobicity. By locking local structures, hydrogen bonds help to form more stable interaction interfaces. These findings indicate that hydrogen bonding could enable hydrophobic disordered proteins to adopt preorganized conformations in response to environmental stimuli and serve as a key factor in mediating the assembly kinetics in complex cellular environments.
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.
Debdatta Das, Jenny N Nguyen, Navneet Sahoo et al.· Small· 0 citations
Self-assembly, the spontaneous organization of molecular components into ordered structures without external intervention, offers a powerful route to complex nanomaterials. Yet the molecular pathways that govern hierarchical assembly, particularly under non-equilibrium conditions, often remain poorly understood. Here we establish an integrated experimental platform that couples microfluidic control of the assembly environment with multi-scale optical and structural probes, enabling direct correlation between morphological evolution and excitonic functionality during supramolecular growth. Using this approach, we track in real time the formation of double-walled nanotubes (DWNTs) from the amphiphilic cyanine dye C8S3, a synthetic analogue of the light-harvesting chlorosomes in green sulfur bacteria. The results show that the outer nanotube structures first, while the inner nanotube follows with a delay, ultimately giving rise to electronically coupled coaxial architectures. While the principal excitonic signatures and morphological motifs emerge within minutes of self-assembly, axial elongation and orientational refinement continue over tens of hours through a nucleation-elongation mechanism. Notably, suppressing local concentration gradients through more efficient mixing abolishes DWNT formation, establishing spatial heterogeneity as a key parameter governing hierarchical self-assembly. By linking structural evolution with excitonic functionality in real time, this combined platform provides a framework for dissecting non-equilibrium pathways in supramolecular materials.
S. Krishnaswamy, Alexey V. Kuevda, M. Stuart et al.· Nanoscale· 0 citations
The formation of microcompartments is a form of self-assembly that governs the transition from chaotic mixtures of molecules to complex systems with functional states. Despite recent advances in systems chemistry, how microcompartments favor the formation of their constituents and become functional remains largely unexplored. Here, we show that combinatorial libraries based on the dynamic covalent imine bond provide a suitable model to investigate selection mechanisms in micellar formation, leading to the emergence of catalysis and structural changes in the assembly toward more organized structures. Systematic studies reveal that micelles protect their imine-based surfactants from hydrolysis through the microenvironments they generate, thereby stabilizing their own components via a self-selection process. Remarkably, this mechanism also promotes the self-sorting of reaction networks, which further reinforces the robustness of the selection. Increasing the molecular diversity enables the emergence of complex behaviors within the assembly, such as mimicking hydrolase activity through the sorting of catalytic groups and changing the structure of the parent micelle itself. This study thus provides guidelines for probing the emergence of microcompartments by elucidating key selection rules that may be extended to a wider range of microcompartments.
Tanguy Rieu, F. Esteve, F. Mihoubi et al.· Journal of the American Chem...· 0 citations
The formation of biomolecular condensates has been linked to a broad range of essential cellular processes, including immune signaling cascades, mRNA transport, and autophagy. Conversely, phase-separated condensates have also been associated with aberrant protein misfolding and subsequent aggregation in neurodegenerative disease-related processes. Protein phase separation and phase transitions involve the formation of heterogeneous and dynamic assemblies that can evolve into gel-like or semi-crystalline states, which are challenging to characterize using high-resolution structural biology techniques. Solid-state nuclear magnetic resonance (NMR) spectroscopy now offers a broad arsenal of methods to probe the structural and dynamic features of viscous condensates, elastic solids, coacervates and rigid protein assemblies. This review provides an overview of solid-state NMR approaches that are readily applicable and discusses potential methodological developments to investigate protein condensates and coacervates as well as to study protein phase separation and phase transitions.
Seamoon Deb, Erick J Dufourc, Birgit Habenstein et al.· Biophysical Chemistry· 0 citations
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.
Bikash R. Sahoo, B. Krishnarjuna, Thirupathi Ravula et al.· Journal of Colloid and Inter...· 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