Physical Chemistry Chemical Physics

Nanoplastic contamination is increasingly recognized as a biochemical stressor, yet the molecular determinants governing how polymer fragment size dictates protein-level interactions remain poorly understood. Here, we combine molecular docking and all-atom molecular dynamics simulations to characterize the interaction of polyethylene (PE) oligomers of increasing length (n = 3, 5, 7, and 14 repeat units) with human catalase, a tetrameric heme enzyme central to cellular antioxidant defense. Single-chain simulations reveal that short PE fragments (3-, 5-, and 7-mer) migrate from an initial domain-connector pose toward a heme-proximal, previously characterized inhibitor-binding site, whereas the 14-mer remains kinetically confined to a peripheral surface site, implicating short-chain PE as a potential catalase inhibitor. In multichain corona systems, backbone dynamics show a non-monotonic, chain-length-dependent destabilization, with the 3- and 5-mer inducing the greatest structural perturbation and the 14-mer best preserving native-like dynamics despite forming an extensive, percolating surface network across all four subunits. MM-PBSA free energy decomposition identifies van der Waals interactions as the dominant thermodynamic driver of binding, while electrostatic surface mapping indicates that cationic surface patches instead govern long-range spatial targeting of PE chains. These findings establish a framework linking nanoplastic fragment size to protein-level toxicity, with direct implications for assessing the biochemical risk of environmentally weathered polyethylene.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family in the human body and the major targets of FDA-approved medications. Unlike some other physiological proteins, GPCRs are highly plastic and often exist as collections of conformational ensembles in the environment to exert their function. Therefore, understanding how a GPCR adopts different conformational ensembles in response to ligands and signaling partners is essential for drug development. 19F-NMR has emerged as one of the most promising tools in this regard; however, a well-established 19F-labeling system is still lacking, including an effective 19F probe that meets both environmental susceptibility and NMR sensitivity requirements, even though progress has been made dramatically in recent years. Thus, this manuscript reports a novel conformational probe (4-(bromomethyl)-ethoxy-2-(trifluoromethyl)benzene, BTMB) that offers paramount benefits for future GPCR conformational dynamics studies. To rationalize the mechanism behind BTMB's superiority for future probe development, we performed MD simulations, referenced to the current state-of-the-art probe, BTFMA. Our study suggests that the spatial proximity of aromatic residues significantly influences the chemical shift dispersion and linewidths of related 19F-probe-profiled conformational states, as well as the rigidity and compactness of -CF3 in the target protein.

Lipid nanoparticles (LNPs) are a leading drug delivery platform for mRNA or other nucleic acid-based therapeutics. LNP platforms are typically composed of ionizable aminolipids, phospholipids, sterols, and lipids tagged with polyethylene glycol. The ionizable aminolipids are typically cationic at low pH, allowing them to encapsulate negatively charged mRNA. The pH at which the ionizable head groups change charge states, the apparent pKa, is crucial to understand the characteristics and formulation of LNP. However, this is difficult to measure as the apparent pKa differs from the pKa of the aminolipids in bulk solution due to the complex chemical and charge environment. In this study, we use molecular dynamics (MD) simulations with alchemical free energy perturbation to calculate the pKa shift as a function of lipid composition, treating LNP-like and vesicle-like environments. Our results show that the pH-dependent structure of the systems affects the trends in pKa shifts in complex ways, driven by factors including surface electrostatics and lipid internalization. Our all-atom simulation protocol allows us to efficiently estimate the apparent pKa of LNP-like models formed with novel ionizable aminolipids and estimate how it will change with composition.

Whether the homologous parallel triplex (HP-triplex, formed by dsDNA and a homologous ssDNA) acts as an intermediate prior to DNA recombination remains controversy, owing to the intrinsic instability and lake of experimental data. Here, we report a HP-triplex that is stable at least at 35 oC under protein-free conditions. Two complementary circular ssDNAs were first hybridized at a 1:1 ratio to form an LR-chimera containing both Z-DNA and B-DNA. Then the circular homologous ssDNA (T-rich) was found to easily hybridize with this LR-chimera to form the HP-triplex (strand ratio of 2:1), involving both left- and right-handed triplex parts under the topological constraint. The third strand locates in the major groove and forms hydrogen bonds with both bases in each base pair of dsDNA without obvious strand exchange. Our data indicate that the third-strand ssDNA may first bind to the B-DNA region, followed by association with the Z-DNA region to assemble the full triplex structure. Structural simulations and analyses reveal that topological constraint imposed by the circular architecture raises the energy barrier for strand displacement, thereby stabilizing this triplex. In summary, the dynamic HP triplex can be stabilized via a Z B junction structure that mimics the in vivo one formed by negative supercoiling. These findings demonstrate that HP-triplex may act as an intermediate for DNA recombination, anti-gene strategy, and other interactions between dsDNA and homologous ssDNA or RNA.

Japanese Encephalitis Virus (JEV) belongs to the Flavivirus family, and the RNA dependent RNA polymerase (RdRp) domain located at the C terminus of non structural protein 5 (NS5) regulates de novo viral genome synthesis. The conserved priming loop inserted in the thumb domain of RDRP initiates de novo genome synthesis. Interestingly, the reported replication process initiates with an interaction between NS5 (methyltransferase/RDRP) and NS3 (protease/helicase), and inhibiting this interaction directly correlates with the inhibition of viral replication. The function of the priming loop in the context of the NS5 NS3 interaction is not yet known. In this study, we studied the priming loop function in the NS5 NS3 interaction and viral replication. Using a structure based drug design approach, we screened the Maybridge compound library against the RdRp priming loop and identified 15 candidate compounds based on binding energy. Among them, DSHS00151 showed significant dose dependent inhibition of the NS5 NS3 interaction with an IC50 of 3.5 micromolar in the mammalian two hybrid assay, inhibited viral infectivity with an IC50 of 2.54 micromolar, and reduced viral RNA load with an IC50 of 2.9 micromolar, compared to CD10712. Molecular Dynamics (MD) simulations revealed that DSHS00151 exhibits stronger and more stable binding with the priming loop residues (790 to 812) compared to CD10712. The molecular mechanism suggested by the docking of the NS5 and NS3 proteins showed that the priming loop tends to rotate toward the NS3 protein to stabilize the complex, and compound binding restricts this loop rotation, thus compromising the stability of the NS5 and NS3 complex and affecting viral replication. Overall, our study validated the function of the NS5 priming loop as an allosteric site, and compounds binding to this allosteric site belong to the Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI) class of inhibitors, which disrupt the NS5 and NS3 interaction and could be developed as novel anti JEV therapeutics.

Nanoplastic interactions with cell membranes can influence particle uptake, translocation, and biological effects, making their molecular description important for assessing the environmental and biological consequences of plastic pollution. However, molecular simulations generally represent nanoplastics using monodisperse polymer chains, whereas nanoplastics formed by environmental degradation are polydisperse. Here, we construct 4 nm polyethylene terephthalate (PET), polyethylene (PE), and polystyrene (PS) nanoparticles with lognormal molecular-weight distributions reported for degraded polymers, and with melt-like chain packing and entanglement. Using Martini coarse-grained molecular dynamics, we simulate their interactions for 10 microseconds with three lipid bilayers of increasing complexity. PE and PS insert rapidly and remain largely intact, whereas PET remains surface-associated and progressively releases individual chains. Fluid, polyunsaturated lipid environments undergo greater deformation and promote substantially more chain release, up to 56% of the chains for PET and 43% for PS. Release begins with the shortest chains and progressively extends to longer chains, while semicrystalline PE remains largely intact. In contrast, a size-matched monodisperse PS particle releases no chains and minimally perturbs the membrane. These results identify the molecular-weight distribution as a key determinant of nanoplastic-membrane interactions, and indicate that uniform model particles underestimate the release of polymer chains into biological membranes.

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays a fundamental role in regulating cellular proliferation, survival, and differentiation. EGFR's kinase domain catalyses the autophosphorylation that drives downstream signalling and is a known hotspot for cancer-associated mutations. Such substitutions often enhance receptor activation, driving oncogenesis and therapeutic resistance, with major implications for prognosis and treatment choice; consequently, a detailed understanding of this domain's functional dynamics, and how mutations reshape them, is essential. Here, we apply dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to probe how changes at the ATP-binding site are transmitted throughout the kinase domain, ultimately shaping the behaviour of distal regions in the wild-type protein and in two of the most clinically observed non-small cell lung cancer variants, L858R and L858R+T790M. D-NEMD reveals previously uncharacterised communication networks linking the ATP pocket to key functional motifs, including the P-loop, C-helix, hinge region, activation loop, and F-helix, showing the intricate allosteric connectivity within this domain. Notably, numerous cancer-related mutation sites, despite being distant from the ATP site, map onto or lie close to these communication pathways, indicating that D-NEMD simulations can identify functionally relevant allosteric positions and potential mutation sites in EGFR. Our simulations further reveal that the two variants rewire EGFR's internal signal propagation network in distinct ways: L858R diminishes the transmission of structural changes to distal regions of the C-lobe, whereas introducing T790M in addition to L858R partially restores wild-type-like behaviour through compensatory rerouting of early-stage dynamical pathways.

Aromatic side chains carry the optical and redox chemistry of peptides, and lengthening one is widely expected to extend its conjugation and narrow its HOMO-LUMO gap. We test that expectation with plane-wave density functional theory on 22 isolated neutral peptides NH2-(X)n-COOH (X = Phe, Tyr, Trp, Cys; n = 1 to 5, and n = 10 for Cys and Trp). The gap does narrow, at the same rate for all three aromatic residues, i.e., -0.17 to -0.18 eV per residue, even though benzene, phenol and indole are very different chromophores. Residue-resolved projections reveal why: nothing delocalises. Every aromatic frontier orbital is 97 to 100 % confined to a single residue and the HOMO participation number saturates at one ring. Each residue instead keeps its own local gap: for Trp it is invariant to within 0.03 eV over n = 2 to 10, while over that same span the global gap falls by 1.19 eV. The global gap is therefore an extreme-value functional of the residue site energies, gap(n) =<g_f>-{Delta}(n), whose alignment penalty accounts for the entire effect: the polar backbone generates a monotonic internal electrostatic ramp that leaves the two gap edges at opposite ends of the chain, 32 [A] apart. We set out the resulting design rules for peptide-based bio-organic semiconductors.

The development of NIR fluorescent dyes with favorable optical properties is of great importance especially for applications within the field of optical imaging. In this study, we report the design, synthesis and photophysical characterization of novel BODIPY-Xanthene hybrid dyes obtained via Knoevenagel condensation between an asymmetrical BODIPY core and the functionalized xanthene aldehydes intermediates. The synthesized dyes ex-hibit absorption (667 nm) and emission bands (720-722 nm) within the NIR region. Detailed UV-Vis and fluo-rescence studies were performed and molar extinction coefficients and stokes shifts also calculated. Moreover, findings highlighting the influence of structural modifications on the optical behavior of the hybrid dyes and their potential utility in optical imaging applications are reported.

Plasma membranes (PM) have been traditionally studied by fluorescent probes by either labelling lipid or protein parts. Based on our MemGraft concept, we report a fluorescent probe based on solvatochromic dye Nile Red (MemGraft-NR) for sensing lipid organization in the immediate surrounding of proteins, i.e. at the protein-lipid interface. Two sequentially attached MemGraft scaffolds are required to ensure robust grafting of Nile Red to PM. In contrast to classical lipid-bound membrane probes, MemGraft-NR exhibits high resistance to the presence of serum and fixation with permeabilization. Two-color confocal imaging of MemGraft-NR in live cells reveals higher polarity at the protein-lipid interface compared to lipid-bound probe NR12A. Surprisingly, MemGraft-NR displays higher sensitivity to variation of cholesterol content in PM compared to NR12A, suggesting that protein-lipid interface is particularly impacted by changes in the lipid composition. MemGraft-NR also enabled in situ monitoring of lipid exchange and permeabilization of PM, something impossible for classical solvatochromic PM probes. Unexpectedly, MemGraft-NR reveals a dramatic decrease in the local polarity in early endosomes, suggesting that endosomal negative curvature provides strong impact on organization of the protein-lipid interface. The developed probe opens a new way to study biological membranes by sensing lipid surrounding of membrane proteins.

Interpreting single-molecule force spectroscopy (SMFS) data from optical tweezers with steered molecular dynamics (SMD) is hindered by a severe timescale mismatch: SMD trajectories span nanoseconds to microseconds, while SMFS experiments unfold over milliseconds to seconds, forcing SMD to pull six to nine orders of magnitude faster than experiment and systematically distorting rupture forces, molecular pathways, and kinetic intermediates. We introduce force-probe coupled steered molecular dynamics (fp-SMD), which explicitly propagates the force probe (the optically trapped bead) alongside the biomolecule within a single classical Hamiltonian description of the coupled molecule-linker-bead system. A Predict-Correct Trajectory Propagation (PCTP) scheme resolves the resulting computational bottleneck by decoupling the fast molecular relaxation from the slow, macroscopic bead motion, using metadynamics and transition-state theory to identify the kinetically resolved molecular intermediate reached at each macroscopic time step. On the cholesterol-{beta}-cyclodextrin ({beta}-CD) host-guest complex, fp-SMD reproduces the experimental rupture-force histogram directly at matched conditions, without extrapolation from standard SMD. fp-SMD further resolves how applied force reshapes the molecular escape pathway itself--cholesterol dissociates via a high-angle route at low force and a direct, small-angle route at high force--providing, to our knowledge, the first direct structural evidence for force-induced pathway switching within small host-guest systems believed to underlie catch-slip bond behavior. fp-SMD/PCTP thus offers a general route to simulating force-probe-coupled biomolecular mechanics at experimentally relevant timescales.

Stimulator of interferon genes (STING) provides an informative model system for developing methods to extract and compare residue-pair fluctuation patterns from molecular dynamics (MD) trajectories. We combined a one-dimensional convolutional autoencoder trained on apo STING with temporal mean pooling, Mahalanobis-distance clustering, and residue-pair similarity networks to compare apo, ligand-bound, and SAVI-associated multi-mutant datasets using 25-dimensional latent descriptors. Networks constructed from clusters capturing state-dependent fluctuation patterns showed lid-dominated organization in the ligand-bound dataset and a1b1-enriched, structurally distributed relationships involving residue 281 in the multi-mutant dataset. These findings identify contrasting residue-pair fluctuation signatures in the analyzed trajectories and provide an interpretable framework for generating hypotheses about ligand-associated and mutation-associated dynamic allostery.

Peptide-brush polymers prepared via graft-through polymerization of peptide-based olefinic monomers form Protein-Like Polymers (PLPs) with demonstrated utility as chemical biology tools for modulating intracellular protein--protein interactions and are in development as a emerging class of therapeutic. This favorable bioactivity has been achieved in part by mimicking some of the structural and functional features of proteins, such as their dynamic, globular solution-phase structures, ability to insert into lipid membranes and enter cells, and tendency to enforce -helicity in peptide side chains. However, because structure leads to function, insight into the structure of these proteomimetic polymers, particularly their overall tertiary structure and the extent to which this influences the brush peptide structure, is incomplete. To further this understanding, we prepared a set of brush polymers incorporating the trpzip2 peptide sequence, commonly used as a model system for protein folding. We examine these polymers via circular dichroism spectroscopy, infrared spectroscopy, and small-angle X-ray scattering, coupled with insights from molecular dynamics simulations. Here, a loss of the {beta}-hairpin structure of the monomer is observed regardless of polymer composition, and simulations corroborate that the polymer backbone destabilizes local secondary structure, restricting peptide motion and altering the accessible free-energy landscape. This study provides insight into design rules governing these synthetic polymer-based proteomimetics.

Amyloid {beta}-protein (A{beta}), which is known for its role in Alzheimer's disease (AD), is also involved in several physiological functions. Of the two predominant isoforms, A{beta}40 and A{beta}42, the latter is more aggregation-prone and more active in health and disease. In the brain, A{beta} interacts with free lipids, which may affect its assembly pathways. Here, thioflavin T (ThT) fluorescence spectroscopy is combined with molecular dynamics (MD) to investigate A{beta}40 and A{beta}42 co-assembly with free dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (CHOL) lipids. ThT measurements at several protein-to-lipid (PL) ratios show isoform- and lipid composition-dependent kinetics of A{beta} fibril formation with clear isoform-dependent lipid effects at a PL ratio of 1:5. Early co-assembly stages of six A{beta}40/A{beta}42 with DMPC and DMPC/CHOL lipids at this PL ratio are then examined by MD. MD results indicate that lipids alter the secondary structure more and interact more avidly with A{beta}40 than with A{beta}42. In turn, A{beta}40 increases the planar order of lipid bicelles more than A{beta}42. Unlike A{beta}40, A{beta}42 decreases the nematic order of CHOL. In DMPC/CHOL lipids, A{beta}42 but not A{beta}40 exhibits two A{beta}-lipid interaction modes associated with mutually exclusive A{beta}42-lipid and A{beta}42-A{beta}42 interactions. Our MD findings provide a plausible explanation for an experimentally observed increase in the lag time for A{beta}40 fibril formation in DMPC lipids and even more so in DMPC/CHOL lipids that contrasts with much smaller effects of lipids on A{beta}42 fibril formation and offer additional atomistic-level insights into A{beta} co-assembly with lipids amenable to experimental testing.

Liquid-liquid phase separation (LLPS) of biomacromolecules has been shown to influence a large variety of biological processes via the formation of membraneless organelles. Aberrant LLPS, however, has been implicated in a number of oncological and neurodegenerative disorders. Proteins related to Alzheimer's, Parkinson's diseases and other major amyloidoses have all been shown to form these condensates. Interestingly, some amyloidogenic proteins were demonstrated to form heterotypic droplets, where both molecules coexisted within the same condensate and affected each other's aggregation. While such cross-interactions have been reported numerous times under non-LLPS conditions, the heterotypic droplet assemblies were only shown for a very limited number of amyloidogenic proteins. In this work, we examine heterotypic LLPS of prion proteins and the pro-inflammatory S100A9, whose cross-interaction under non-LLPS conditions we confirmed in a previous work. The results show that this interaction under condensation-promoting conditions enhances their aggregation, leading to the formation of a higher concentration of amyloid structures. These findings suggest that the pro-inflammatory S100A9 may have a critical role in promoting prion protein aggregation via the assembly of heterotypic condensates under high molecular crowding conditions.

Reactive metal-oxygen intermediates are central to metalloenzyme catalysis, yet their low populations and short lifetimes make vibrational characterization under aqueous reaction conditions challenging. Here, we establish shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) as a powerful vibrational approach for visualizing reaction-associated metal-ligand signatures of reactive metalloenzyme states. Across the heme and non-heme iron enzymes TauD, VioC, CYP119, and TqaLNc, isotope-sensitive features provide vibrational information on reactive coordination states. In TauD and VioC, isotope-sensitive features provide vibrational characterization of Fe(IV)=O intermediates. In CYP119, an isotope-sensitive Fe(IV)=O vibration is assigned to P450 Compound I generated by m-CPBA, including under lauric-acid turnover conditions. Most notably, in TqaLNc, combined 18O- and 15N-sensitive vibrational responses support formation of the proposed (R)HN-Fe(IV)=O coordination state and the dynamic-coordination mechanism underlying selective C-N bond formation. These results demonstrate that SHINERS can serve as a powerful technique for the in situ vibrational visualization of reactive intermediates and their coordination states in metalloenzyme catalysis.

Cell membranes are decorated by dense, polymeric macromolecules such as proteins with bulky intrinsically disordered regions, glycosylated proteins, and glycopolymers. Experiments have illustrated that the curvature of cellular membranes, which plays a role in diverse biological processes, can be regulated by the local density of these membrane-tethered polymers and their response to environmental changes. However, the mechanisms underlying the feedback between solution conditions, membrane curvature, and the local density and molecular weight of membrane-tethered polymers remain largely unexplored. In this work, using a combination of theory and experiment, we show that the ionic strength of the surrounding solution and the degree of ionization both play important roles in regulating the conformation of the membrane-tethered polymers, which in turn influences their membrane curvature generation capabilities. Specifically, we show that for negatively charged, disordered proteins and glycans, the thickness of the polymer brush decreases with ionic strength but increases with grafting density and degree of ionization, resulting in enhanced sensitivity to membrane curvature and an amplified ability to drive membrane curvature at lower ionic concentrations. Our findings reveal a mechanism by which membrane-tethered polymers generate membrane curvature to minimize free energy, with the extent of curvature generation modulated by the polymer grafting density and layer thickness, as well as by the ionic strength of the surrounding solution. The implications of these findings are broad, suggesting that perturbations to biopolymer charge, such as sialylation of glycans and phosphorylation of disordered proteins, could actively modulate membrane curvature induction in diverse cellular processes from endocytosis to cell motility.

Nanopore sensors have transformed single-molecule analysis, enabling real-time detection of biomolecules with unprecedented resolution. Understanding and modelling noise in nanopore sensing is essential to unlocking their full analytical potential. Although mechanistic models of nanopore noise exist, a rigorous statistical framework that characterises noise structure across diverse experimental conditions is still lacking. Here, we present a comprehensive empirical analysis of nanopore noise using twelve diverse experimental conditions acquired with different nanopores, analytes, and electronic recording systems. By employing multiple, mutually reinforcing statistical methods, we establish that nanopore noise is well described by a composite fractional model comprising multiple fractional Gaussian noise and fractional Brownian motion components. This finding is supported by confirmation of Gaussianity, rigorous interpretation of second-order exponents, multifractal analysis, and evidence that transient deviations are attributable to deterministic or experimental artefacts rather than alternative stochastic mechanisms. We further resolve quasi-deterministic structures, such as baseline trends and structural breaks, and demonstrate their separability from the underlying stochastic profile. Collectively, these results provide the first formal statistical validation of a composite fractional model for nanopore noise and establish a principled basis for noise reconstruction, realistic simulation, and statistically grounded algorithm design for nanopore sensing applications. This study provides the foundation for the creation of high-fidelity performance evaluation datasets for signal processing, and the controlled generation and augmentation of data for machine learning while maintaining statistical fidelity to experimental conditions.

In the enzymatic toolbox of depollution, hydrolytic dehalogenases are of particular interest for the bioremediation of organohalogenated compounds. To better identify and characterize these enzymes, it is important to develop appropriate sensing methods. Fluorescent probes for dehalogenase detection have been developed in the past years, however, to this date none were available for application using the common FITC parameters (i.e. excitation laser at 488 nm). To address this limitation, we herein report a new fluorescent probe, COU4-Cl, excitable in the violet (405 nm) and blue (488 nm) wavelengths adapted for the detection and characterization of haloalkane dehalogenases. Its applicability is discussed and evaluated in comparison to the previously described COU2-Cl probe only excitable in the violet (405 nm). This study shows the advantages and complementarity of the two probes, and further demonstrate their application for the discrimination of dehalogenase activity in whole cell assays.

Defective interfering particles (DIPs) represent promising antivirals against influenza A virus (IAV), yet strategies to further enhance their antiviral activity remain largely unexplored. Here, we engineered a next-generation OP7 DIP by truncating the viral interferon (IFN) antagonist non-structural protein 1 (NS1) to enhance IFN-mediated antiviral activity. A previously validated mathematical multiscale model predicted that loss of NS1 function would enhance antiviral activity, providing a mechanistic rationale for this design. We engineered OP7-trNS1 DIP, established a cell culture-based production process yielding infectious virus-free material, and evaluated its antiviral activity in human lung epithelial cells in vitro. OP7-trNS1 inhibited IAV replication by approx. two orders of magnitude more effectively than the parental OP7 DIP while inducing a stronger IFN response, consistent with the model predictions. Together, these findings demonstrate that reducing viral IFN antagonism is a viable strategy for rationally enhancing DIP antiviral activity and establish OP7-trNS1 as a promising candidate for further preclinical development.