Assembly Mechanism of Mucin and von Willebrand Factor Polymers.

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

Yeast Ataxin-2 Forms an Intracellular Condensate Required for the Inhibition of TORC1 Signaling during Respiratory Growth.

Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.

Redox State Controls Phase Separation of the Yeast Ataxin-2 Protein via Reversible Oxidation of Its Methionine-Rich Low-Complexity Domain.

Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-ß polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.

Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains.

Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.

Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation.

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.

Physical modeling of nucleosome clustering in euchromatin resulting from interactions between epigenetic reader proteins.

Euchromatin is an accessible phase of genetic material containing genes that encode proteins with increased expression levels. The structure of euchromatin in vitro has been described as a 30-nm fiber formed from ordered nucleosome arrays. However, recent advances in microscopy have revealed an in vivo euchromatin architecture that is much more disordered, characterized by variable-length linker DNA and sporadic nucleosome clusters. In this work, we develop a theoretical model to elucidate factors contributing to the disordered in vivo architecture of euchromatin. We begin by developing a 1D model of nucleosome positioning that captures the interactions between bound epigenetic reader proteins to predict the distribution of DNA linker lengths between adjacent nucleosomes. We then use the predicted linker lengths to construct 3D chromatin configurations consistent with the physical properties of DNA within the nucleosome array, and we evaluate the distribution of nucleosome cluster sizes in those configurations. Our model reproduces experimental cluster-size distributions, which are dramatically influenced by the local pattern of epigenetic marks and the concentration of reader proteins. Based on our model, we attribute the disordered arrangement of euchromatin to the heterogeneous binding of reader proteins and subsequent short-range interactions between bound reader proteins on adjacent nucleosomes. By replicating experimental results with our physics-based model, we propose a mechanism for euchromatin organization in the nucleus that impacts gene regulation and the maintenance of epigenetic marks.

Development of broad modulus profile upon polymer-polymer interface formation between immiscible glassy-rubbery domains.

Interfaces of glassy materials such as thin films, blends, and composites create strong unidirectional gradients to the local heterogeneous dynamics that can be used to elucidate the length scales and mechanisms associated with the dynamic heterogeneity of glasses. We focus on bilayer films of two different polymers with very different glass transition temperatures ([Formula: see text]) where previous work has demonstrated a long-range (∼200 nm) profile in local [Formula: see text] is established between immiscible glassy and rubbery polymer domains when the polymer-polymer interface is formed to equilibrium. Here, we demonstrate that an equally long-ranged gradient in local modulus [Formula: see text] is established when the polymer-polymer interface ([Formula: see text]5 nm) is formed between domains of glassy polystyrene (PS) and rubbery poly(butadiene) (PB), consistent with previous reports of a broad [Formula: see text] profile in this system. A continuum physics model for the shear wave propagation caused by a quartz crystal microbalance across a PB/PS bilayer film is used to measure the viscoelastic properties of the bilayer during the evolution of the PB/PS interface showing the development of a broad gradient in local modulus [Formula: see text] spanning [Formula: see text]180 nm between the glassy and rubbery domains of PS and PB. We suggest these broad profiles in [Formula: see text] and [Formula: see text] arise from a coupling of the spectrum of vibrational modes across the polymer-polymer interface as a result of acoustic impedance matching of sound waves with [Formula: see text] nm during interface broadening that can then trigger density fluctuations in the neighboring domain.

Mechanically adaptive and deployable intracortical probes enable long-term neural electrophysiological recordings.

Flexible intracortical probes offer important opportunities for stable neural interfaces by reducing chronic immune responses, but their advances usually come with challenges of difficult implantation and limited recording span. Here, we reported a mechanically adaptive and deployable intracortical probe, which features a foldable fishbone-like structural design with branching electrodes on a temperature-responsive shape memory polymer (SMP) substrate. Leveraging the temperature-triggered soft-rigid phase transition and shape memory characteristic of SMP, this probe design enables direct insertion into brain tissue with minimal footprint in a folded configuration while automatically softening to reduce mechanical mismatches with brain tissue and deploying electrodes to a broader recording span under physiological conditions. Experimental and numerical studies on the material softening and structural folding-deploying behaviors provide insights into the design, fabrication, and operation of the intracortical probes. The chronically implanted neural probe in the rat cortex demonstrates that the proposed neural probe can reliably detect and track individual units for months with stable impedance and signal amplitude during long-term implantation. The work provides a tool for stable neural activity recording and creates engineering opportunities in basic neuroscience and clinical applications.

Organic nanoparticles with tunable size and rigidity by hyperbranching and cross-linking using microemulsion ATRP.

Unlike inorganic nanoparticles, organic nanoparticles (oNPs) offer the advantage of "interior tailorability," thereby enabling the controlled variation of physicochemical characteristics and functionalities, for example, by incorporation of diverse functional small molecules. In this study, a unique inimer-based microemulsion approach is presented to realize oNPs with enhanced control of chemical and mechanical properties by deliberate variation of the degree of hyperbranching or cross-linking. The use of anionic cosurfactants led to oNPs with superior uniformity. Benefitting from the high initiator concentration from inimer and preserved chain-end functionality during atom transfer radical polymerization (ATRP), the capability of oNPs as a multifunctional macroinitiator for the subsequent surface-initiated ATRP was demonstrated. This facilitated the synthesis of densely tethered poly(methyl methacrylate) brush oNPs. Detailed analysis revealed that exceptionally high grafting densities (~1 nm-2) were attributable to multilayer surface grafting from oNPs due to the hyperbranched macromolecular architecture. The ability to control functional attributes along with elastic properties renders this "bottom-up" synthetic strategy of macroinitiator-type oNPs a unique platform for realizing functional materials with a broad spectrum of applications.

Activity-driven chromatin organization during interphase: Compaction, segregation, and entanglement suppression.

In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension [Formula: see text] is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times ([Formula: see text]) longer than tens of minutes to be proportional to [Formula: see text]. We validate our results with hybrid molecular dynamics-Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation.

Modeling homologous chromosome recognition via nonspecific interactions.

In many organisms, most notably Drosophila, homologous chromosomes associate in somatic cells, a phenomenon known as somatic pairing, which takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, mediated by different proteins that bind to these different regions. Here, we use computational modeling to evaluate an alternative "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. In this model, buttons are nonuniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a nonhomolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. By simulating randomly generated nonuniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Mixed equilibrium/nonequilibrium effects govern surface mobility in polymer glasses.

Using angle-resolved X-ray photoelectron spectroscopy, sum-frequency generation vibrational spectroscopy, contact angle measurements, and molecular dynamics simulations, we verify that the glass transition temperature (Tg) of polymer glass is lower near the free surface. However, the experimental Tg-gradients showed a linear variation with depth (z) from the free surface, while the simulated equilibrium Tg-gradients exhibited a double exponential z-dependence. In typical simulations, Tg is determined based on the relaxation time of the system reaching a prescribed threshold value at equilibrium. Conversely, the experiments determined Tg by observing the unfreezing of molecular mobility during heating from a kinetically arrested, nonequilibrium glassy state. To investigate the impact of nonequilibrium effects on the Tg-gradient, we reduced the thermal annealing time in simulations, allowing the system to fall out of equilibrium. We observe a decrease in the relaxation time and the emergence of a modified z-dependence consistent with a linear Tg-gradient near the free surface. We further validate the impact of nonequilibrium effects by studying the dependence of the Tg on the heating/cooling rate for polymer films of varying thickness (h). Our experimental results reveal significant variations in the Tg-heating/cooling rate dependence with h below the bulk Tg, which are also observed in simulation when the simulated system is not equilibrated. We explain our findings by the reduction in mass density within the inner region of the system under nonequilibrium conditions, as observed in simulation, and recent research indicating a decrease in the local Tg of a polymer when placed next to a softer material.

A radical polymer membrane for simultaneous degradation of organic pollutants and water filtration.

Integrating reactive radicals into membranes that resemble biological membranes has always been a pursuit for simultaneous organics degradation and water filtration. In this research, we discovered that a radical polymer (RP) that can directly trigger the oxidative degradation of sulfamethozaxole (SMX). Mechanistic studies by experiment and density functional theory simulations revealed that peroxyl radicals are the reactive species, and the radicals could be regenerated in the presence of O2. Furthermore, an interpenetrating RP network membrane consisting of polyvinyl alcohol and the RP was fabricated to demonstrate the simultaneous filtration of large molecules in the model wastewater stream and the degradation of ~ 85% of SMX with a steady permeation flux. This study offers valuable insights into the mechanism of RP-triggered advanced oxidation processes and provides an energy-efficient solution for the degradation of organic compounds and water filtration in wastewater treatment.

Purely elastic turbulence in pressure-driven channel flows.

Solutions of long, flexible polymer molecules are complex fluids that simultaneously exhibit fluid-like and solid-like behavior. When subjected to an external flow, dilute polymer solutions exhibit elastic turbulence-a unique, chaotic flow state absent in Newtonian fluids, like water. Unlike its Newtonian counterpart, elastic turbulence is caused by polymer molecules stretching and aligning in the flow, and can occur at vanishing inertia. While experimental realizations of elastic turbulence are well-documented, there is currently no understanding of its mechanism. Here, we present large-scale direct numerical simulations of elastic turbulence in pressure-driven flows through straight channels. We demonstrate that the transition to elastic turbulence is sub-critical, giving rise to spot-like flow structures that, further away from the transition, eventually spread throughout the domain. We provide evidence that elastic turbulence is organized around unstable coherent states that are localized close to the channel midplane.

Motorized chain models of the ideal chromosome.

An array of motor proteins consumes chemical energy in setting up the architectures of chromosomes. Here, we explore how the structure of ideal polymer chains is influenced by two classes of motors. The first class which we call "swimming motors" acts to propel the chromatin fiber through three-dimensional space. They represent a caricature of motors such as RNA polymerases. Previously, they have often been described by adding a persistent flow onto Brownian diffusion of the chain. The second class of motors, which we call "grappling motors" caricatures the loop extrusion processes in which segments of chromatin fibers some distance apart are brought together. We analyze these models using a self-consistent variational phonon approximation to a many-body Master equation incorporating motor activities. We show that whether the swimming motors lead to contraction or expansion depends on the susceptibility of the motors, that is, how their activity depends on the forces they must exert. Grappling motors in contrast to swimming motors lead to long-ranged correlations that resemble those first suggested for fractal globules and that are consistent with the effective interactions inferred by energy landscape analyses of Hi-C data on the interphase chromosome.

Dynamics of membrane tubulation coupled with fission by a two-component module.

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes.

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

A molecular view of peptoid-induced acceleration of calcite growth.

The extensive deposits of calcium carbonate (CaCO3) generated by marine organisms constitute the largest and oldest carbon dioxide (CO2) reservoir. These organisms utilize macromolecules like peptides and proteins to facilitate the nucleation and growth of carbonate minerals, serving as an effective method for CO2 sequestration. However, the precise mechanisms behind this process remain elusive. In this study, we report the use of sequence-defined peptoids, a class of peptidomimetics, to achieve the accelerated calcite step growth kinetics with the molecular level mechanistic understanding. By designing peptoids with hydrophilic and hydrophobic blocks, we systematically investigated the acceleration in step growth rate of calcite crystals using in situ atomic force microscopy (AFM), varying peptoid sequences and concentrations, CaCO3 supersaturations, and the ratio of Ca2+/ HCO3-. Mechanistic studies using NMR, three-dimensional fast force mapping (3D FFM), and isothermal titration calorimetry (ITC) were conducted to reveal the interactions of peptoids with Ca2+ and HCO3- ions in solution, as well as the effect of peptoids on solvation and energetics of calcite crystal surface. Our results indicate the multiple roles of peptoid in facilitating HCO3- deprotonation, Ca2+ desolvation, and the disruption of interfacial hydration layers of the calcite surface, which collectively contribute to a peptoid-induced acceleration of calcite growth. These findings provide guidelines for future design of sequence-specific biomimetic polymers as crystallization promoters, offering potential applications in environmental remediation (such as CO2 sequestration), biomedical engineering, and energy storage where fast crystallization is preferred.

Unified understanding of the impact of semiflexibility, concentration, and molecular weight on macromolecular-scale ring diffusion.

Conformationally fluctuating, globally compact macromolecules such as polymeric rings, single-chain nanoparticles, microgels, and many-arm stars display complex dynamic behaviors due to their rich topological structure and intermolecular organization. Synthetic rings are hybrid objects with conformations that display both ideal random walk and compact globular features, which can serve as models of genomic DNA. To date, emphasis has been placed on the effect of ring molecular weight on their unusual behaviors. Here, we combine simulations and a microscopic force-level theory to build a unified understanding for how key aspects of ring dynamics depend on different tunable molecular properties including backbone rigidity, monomer concentration, degree of traditional entanglement, and molecular weight. Our large-scale molecular dynamics simulations of ring melts with very different backbone stiffnesses reveal unanticipated behaviors which agree well with our generalized theory. This includes a universal master curve for center-of-mass diffusion constants as a function of molecular weight scaled by a chemistry and thermodynamic state-dependent critical molecular weight that generalizes the concept of an entanglement cross-over for linear chains. The key physics is how backbone rigidity and monomer concentration induced changes of the entanglement length, interring packing, degree of interpenetration, and liquid compressibility slow down space-time dynamic-force correlations on macromolecular scales. A power law decay of the center-of-mass diffusion constant with inverse molecular weight squared is the first consequence, followed by an ultraslow activated hopping transport regime. Our results set the stage to address slow dynamics and kinetic arrest in different families of compact synthetic and biological polymeric systems.

Charge block-driven liquid-liquid phase separation - mechanism and biological roles.

Liquid-liquid phase separation (LLPS) has increasingly been found to play pivotal roles in a number of intracellular events and reactions, and has introduced a new paradigm in cell biology to explain protein-protein and enzyme-ligand interactions beyond conventional molecular and biochemical theories. LLPS is driven by the cumulative effects of weak and promiscuous interactions, including electrostatic, hydrophobic and cation-π interactions, among polypeptides containing intrinsically disordered regions (IDRs) and describes the macroscopic behaviours of IDR-containing proteins in an intracellular milieu. Recent studies have revealed that interactions between 'charge blocks' - clusters of like charges along the polypeptide chain - strongly induce LLPS and play fundamental roles in its spatiotemporal regulation. Introducing a new parameter, termed 'charge blockiness', into physicochemical models of disordered polypeptides has yielded a better understanding of how the intrinsic amino acid sequence of a polypeptide determines the spatiotemporal occurrence of LLPS within a cell. Charge blockiness might also explain why some post-translational modifications segregate within IDRs and how they regulate LLPS. In this Review, we summarise recent progress towards understanding the mechanism and biological roles of charge block-driven LLPS and discuss how this new characteristic parameter of polypeptides offers new possibilities in the fields of structural biology and cell biology.