Molecular-level Insights Research Articles

Multiscale simulations of TDP-43 full-length uncover molecular determinants of its phase separation beyond N/C-terminal oligomerization.

TAR DNA-binding protein 43 (TDP-43) is a multidomain protein involved in the regulation of RNA processing, and its aggregates have been observed in neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 is composed of a well-folded N-terminal domain (NTD), two RNA recognition motifs (RRM1/2), an intrinsically disordered C-terminal domain (CTD), and short flexible linkers between folded domains. Recently, TDP-43 has been shown to undergo liquid-liquid phase separation (LLPS) to form membraneless compartments referred to as biomolecular condensates. While NTD oligomerization was shown to promote the phase separation of full-length TDP-43 strongly, CTD can also undergo LLPS on its own. However, the complete picture of domain-level intermolecular interactions of full-length TDP-43 within the biological condensates has remained unclear due to the challenges associated with its purification. Here, we utilize a multiscale simulation approach to get molecular-level insights into the role of interdomain interactions in TDP-43 phase separation. Atomistic, single-chain simulations show that interdomain contacts are mediated by electrostatic interactions. Our coarse-grained, condensed phase simulations also reveal that electrostatic interactions involving RRM/NTD-linker modulate the phase separation of TDP-43, in addition to the NTD/CTD-oligomerization. These findings should pave the way for a greater understanding of molecular determinants of TDP-43 phase separation.

Direct Probe of Electrochemical Pseudocapacitive pH Jump at a Graphene Electrode.

Molecular-level insight into interfacial water at a buried electrode interface is essential in electrochemistry, but spectroscopic probing of the interface remains challenging. Here, using surface-specific heterodyne-detected sum-frequency generation (HD-SFG) spectroscopy, we directly access the interfacial water in contact with the graphene electrode supported on calcium fluoride (CaF2 ). We find phase transition-like variations of the HD-SFG spectra vs. applied potentials, which arises not from the charging/discharging of graphene but from the charging/discharging of the CaF2 substrate through the pseudocapacitive process. The potential-dependent spectra are nearly identical to the pH-dependent spectra, evidencing that the pseudocapacitive behavior is associated with a substantial local pH change induced by water dissociation between the CaF2 and graphene. Our work evidences the local molecular-level effects of pseudocapacitive charging at an electrode/aqueous electrolyte interface.

Modulating Catalytic Activity and Stability of Atomically Precise Gold Nanoclusters as Peroxidase Mimics via Ligand Engineering.

Metal nanoclusters (NCs), composed of a metal core and protecting ligands, show promising potentials as enzyme mimics for producing fuels, pharmaceuticals, and valuable chemicals, etc. Herein, we explore the critical role of ligands in modulating the peroxidase mimic activity and stability of Au NCs. A series of Au15(SR)13 NCs with various thiolate ligands [SR = N-acetyl-l-cysteine (NAC), 3-mercaptopropionic acid (MPA), or 3-mercapto-2-methylpropanoic acid (MMPA)] are utilized as model catalysts. It is found that Au15(NAC)13 shows higher structural stability than Au15(MMPA)13 and Au15(MPA)13 against external stimuli (e.g., pH, oxidants, and temperature) because of the intramolecular hydrogen bonds. More importantly, detailed enzymatic kinetics data show that the catalytic activity of Au15(NAC)13 is about 4.3 and 2.7 times higher than the catalytic activity of Au15(MMPA)13 and Au15(MPA)13, respectively. Density functional theory (DFT) calculations reveal that the Au atoms on the motif of Au NCs should be the active centers, whereas the superior peroxidase mimic activity of Au15(NAC)13 should originate from the emptier orbitals of Au atoms because of the electron-withdrawing effect of acetyl amino group in NAC. This work demonstrates the ligand-engineered electronic structure and functionality of atomically precise metal NCs, which afford molecular and atomic level insights for artificial enzyme design.

Molecular features underlying phase separation of protein-nucleic acid condensates revealed by a new coarse-grained DNA model.

Recent advances in residue-level coarse-grained (CG) computational models for proteins have enabled molecular-level insights into the properties of protein condensates. However, most of the existing CG nucleic acid models include anisotropic potentials between bases that are computationally demanding and are not fully compatible with the existing CG protein models. Here, we present a new CG DNA model that utilizes isotropic potentials between bases and can be used for investigating protein-DNA complexes and nucleosome arrays, thereby enabling a mechanistic understanding of how molecular information is propagated up at the genome level in a computationally tractable manner. To demonstrate the suitability of our model to facilitate large-scale simulations with a molecular resolution, we simulate nucleosomes (mono, di, etc.) to generate equilibrium conformational ensembles. The simulation data provide essential insights into the role of chromatin in the liquid-liquid phase separation of HP1α protein. We find an extensive network of interactions between HP1α, DNA, and flexible histone tails in the condensates formed by these different biomolecules. The findings of this work illuminate the complex molecular framework that contributes to heterochromatin regulation and function.

Protein-protein interactions at the tight junctions interface.

The epithelial and endothelial tissues are the physiological barriers against chemical and biological insurgents by constituting the tight junctions. Tight junctions are specialized adhesion constructs formed by the assembly of claudin family proteins. Membrane-embedded claudin proteins are the functional determinants of the tight junctions. In the paracellular space between two adjacent cells, claudins form size and charge selective pores that impede the free diffusion of these solutes. The vital role of tight junctions in various tissue barriers such as the blood-brain barrier, renal barrier, and gut barrier are currently active research fields, yet a complete molecular understanding of the tight junctions is elusive. Here we have employed multiscale molecular dynamics simulations to gain insight into the underpinnings of claudin assembly at tight junctions. From these simulations, a clear picture of how the membrane milieu influences the tight junction's assembly is evident. Both cholesterol and ceramide lipids limit claudin-claudin interaction in the lipid bilayer due to reduced fluidity. Further, we demonstrated that claudins preferentially assemble into distinct dimers that are the primary building blocks of the paracellular pores. The molecular-level insights gained from this work are critical in unraveling the physiological roles of tight junctions.

Response of Sulfonated Polystyrene Melts to Nonlinear Elongation Flows

Ionizable polymers form dynamic networks with domains controlled by two distinct energy scales, ionic interactions and van der Waals forces; both evolve under elongational flows during their processing into viable materials. A molecular level insight of their nonlinear response, paramount to controlling their structure, is attained by fully atomistic molecular dynamics simulations of a model ionizable polymer, polystyrene sulfonate. As a function of increasing elongational flow rate, the systems display an initial elastic response, followed by an ionic fraction-dependent strain hardening, stress overshoot, and eventually strain-thinning. As the sulfonation fraction increases, the chain elongation becomes more heterogeneous. Flow-driven ionic assembly dynamics that continuously break and reform control the response of the system.

Observing Nonpreferential Absorption of Linear and Cyclic Carbonate on the Silicon Electrode.

Silicon is reported to be a promising anode material due to its high storage capacity and excellent energy conversion rate. Molecular-level insight into the interaction between silicon electrodes and electrolyte solutions is essential for understanding the formation of a stable solid electrolyte interphase (SEI), but it is yet to be explored. In this study, we apply femtosecond sum frequency generation vibrational spectroscopy to investigate the initial adsorption of various pure and mixed electrolyte molecules on the silicon anode surface by monitoring the SFG signals from the carbonyl group of electrolyte molecules. When the silicon comes in contact with a pure carbonate solution, the linear carbonates of diethyl carbonate and ethyl methyl carbonate adopt two conformations with opposite C═O orientations on the silicon interface while the cyclic carbonates of ethylene carbonate and propylene carbonate almost adopt one conformation with C═O bonds pointing toward the silicon electrode. When the silicon comes in contact with the mixed linear and cyclic carbonate solutions, the total SFG intensity from the mixed solutions is approximately 2∼5 times weaker than those of pure cyclic carbonates. The C═O bonds of cyclic carbonates point toward the silicon electrode, while the C═O bonds of linear carbonates face toward the bulk solution at the silicon/mixed solution interface. No preferential absorption behaviors of the linear and cyclic carbonate electrolytes on the silicon electrode are observed. Such findings may help to understand the mechanism by which the SEI formed on the silicon anode is unstable.

Molecular-Level Insight into Charge Carrier Transport and Speciation in Solid Polymer Electrolytes by Chemically Tuning Both Polymer and Lithium Salt.

The advent of Li-metal batteries has seen progress toward studies focused on the chemical modification of solid polymer electrolytes, involving tuning either polymer or Li salt properties to enhance the overall cell performance. This study encompasses chemically modifying simultaneously both polymer matrix and lithium salt by assessing ion coordination environments, ion transport mechanisms, and molecular speciation. First, commercially used lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) salt is taken as a reference, where F atoms become partially substituted by one or two H atoms in the -CF3 moieties of LiTFSI. These substitutions lead to the formation of lithium(difluoromethanesulfonyl)(trifluoromethanesulfonyl)imide (LiDFTFSI) and lithium bis(difluoromethanesulfonyl)imide (LiDFSI) salts. Both lithium salts promote anion immobilization and increase the lithium transference number. Second, we show that exchanging archetypal poly(ethylene oxide) (PEO) with poly(ε-caprolactone) (PCL) significantly changes charge carrier speciation. Studying the ionic structures of these polymer/Li salt combinations (LiTFSI, LiDFTFSI or LiDFSI with PEO or PCL) by combining molecular dynamics simulations and a range of experimental techniques, we provide atomistic insights to understand the solvation structure and synergistic effects that impact macroscopic properties, such as Li+ conductivity and transference number.

Efficiently Light-Driven Nonoxidative Coupling of Methane on Ag/NaTaO3: A Case for Molecular-Level Understanding of the Coupling Mechanism

Metal-decorated oxide semiconductors are overwhelming photocatalysts for nonoxidative coupling of methane (NOCM). However, the overall NOCM mechanism remains an unopened black box, which hinders the design of high-performance catalysts. Herein, we systematically studied a series of noble metal (Ag, Au, Pt, Pd, Cu, and Ni)-decorated oxides (NaTaO3, CaTiO3, LiNbO3, and TiO2) for NOCM. We proposed that the active sites for H abstraction and C–C coupling of CH4 are spatially separated. Specifically, NaTaO3 only completes the initial H abstraction of CH4 activation, while metal nanoparticles are responsible for the final C–C coupling. Noble metals dominate NOCM by significantly decreasing the energy barrier of CH4 dissociation and promoting C–C coupling. Among various metals, Ag is preferential for the weak adsorption of ·CH3 intermediates and subsequent metal-induced C–C coupling. This contributes to Ag/NaTaO3 the highest C2H6 yield of 194 μmol g–1 h–1 and stoichiometric H2 with 11.2% quantum efficiency. This work provides a molecular-level insight into the CH4 coupling mechanism on metal-decorated photocatalysts.

Critical impacts of interfacial water on C–H activation in photocatalytic methane conversion

On-site and on-demand photocatalytic methane conversion under ambient conditions is one of the urgent global challenges for the sustainable use of ubiquitous methane resources. However, the lack of microscopic knowledge on its reaction mechanism prevents the development of engineering strategies for methane photocatalysis. Combining real-time mass spectrometry and operando infrared absorption spectroscopy with ab initio molecular dynamics simulations, here we report key molecular-level insights into photocatalytic green utilization of methane. Activation of the robust C–H bond of methane is hardly induced by the direct interaction with photogenerated holes trapped at the surface of photocatalyst; instead, the C–H activation is significantly promoted by the photoactivated interfacial water species. The interfacial water hydrates and properly stabilizes hydrocarbon radical intermediates, thereby suppressing their overstabilization. Owing to these water-assisted effects, the photocatalytic conversion rates of methane under wet conditions are dramatically improved by typically more than 30 times at ambient temperatures (~300 K) and pressures (~1 atm) in comparison to those under dry conditions. This study sheds new light on the role of interfacial water and provides a firm basis for design strategies for non-thermal heterogeneous catalysis of methane under ambient conditions.

Cycloaddition of CO2 to Epichlorohydrin over Pyridine, Vinylpyridine, and Poly(vinylpyridine): The Influence of Steric Crowding on the Reaction Mechanism

The cycloaddition of CO2 with epoxides is a 100% atom economical reaction that utilizes CO2 and produces valuable cyclic carbonate products. Organic bases are attractive catalysts for this reaction because of their green characteristics, such as low cost and low toxicity. However, further catalyst development is required to achieve acceptable yields at mild temperatures and pressures. This work aims to establish catalyst structure–performance relationships to accelerate the rational design of high performance organic base catalysts for the cycloaddition reaction. Toward this end, we investigate the influence of steric crowding at the ring nitrogen atom of a series of pyridine-based catalysts on the reaction mechanism and catalyst performance using batch reactor measurements at a mild temperature (57 °C) and atmospheric pressure under solventless conditions combined with in situ infrared spectroscopic characterization of the catalysts. We show that steric crowding at the ring nitrogen atom has a significant influence on the reaction mechanism and catalyst performance. Catalysts with unhindered access to the ring nitrogen atom, such as pyridine, 4-vinylpyridine, 3-vinylpyridine, and poly(4-vinylpyridine), are completely transformed during the cycloaddition reaction by quaternization from the epoxide reactant, producing zwitterionic pyridinium-epoxide adducts and ammonium salts that precipitate out of solution. Catalysts with hindered access to the ring nitrogen atom, such as 2-vinylpyridine and poly(2-vinylpyridine), resist quaternization from the epoxide reactant due to the high degree of steric hindrance at the ring nitrogen atom. The poly(2-vinylpyridine) catalyst in particular displayed high stability, high product selectivity, and high activity for cyclic carbonate synthesis. These molecular-level insights have significant implications for the rational design of active, stable, and metal-free catalysts for cyclic carbonate synthesis by tuning the steric crowding at the active site.

SO2 enhances aerosol formation from anthropogenic volatile organic compound ozonolysis by producing sulfur-containing compounds

Abstract. Sulfur dioxide (SO2) can affect aerosol formation in the atmosphere, but the underlying mechanisms remain unclear. Here, we investigate aerosol formation and composition from the ozonolysis of cyclooctene with and without SO2 addition in a smog chamber. Liquid chromatography equipped with high-resolution tandem mass spectrometry measurements indicates that monomer carboxylic acids and corresponding dimers with acid anhydride and aldol structures are important components in particles formed in the absence of SO2. A 9.4–12.6-times increase in particle maximum number concentration is observed in the presence of 14–192 ppb SO2. This increase is largely attributed to sulfuric acid (H2SO4) formation from the reactions of stabilized Criegee intermediates with SO2. In addition, a number of organosulfates (OSs) are detected in the presence of SO2, which are likely products formed from the heterogeneous reactions of oxygenated species with H2SO4. The molecular structures of OSs are also identified based on tandem mass spectrometry analysis. It should be noted that some of these OSs have been found in previous field studies but were classified as compounds from unknown sources or of unknown structures. The observed OSs are less volatile than their precursors and are therefore more effective contributors to particle formation and growth, partially leading to the increase in particle volume concentration under SO2-presence conditions. Our results provide an in-depth molecular-level insight into how SO2 alters particle formation and composition.

Unveiling the mechanism and origin of stereocontrol in dinuclear-zinc-catalyzed reductive desymmetrization of malonic esters

Mechanism of catalytic desymmetrization of malonic esters have been explored, which provides molecular-level insights on how the ligand-casted chiral scaffolding can be intentionally applied to differentiate symmetrical elements in desymmetrization.

Role of mutations in a chemoenzymatic enantiodivergent C(sp3)-H insertion: exploring the mechanism and origin of stereoselectivity.

New-to-nature enzymes have emerged as powerful catalysts in recent years for streamlining various stereoselective organic transformations. While synthetic strategies employing engineered enzymes have witnessed proliferating success, there is limited clarity on the mechanistic front and more so when considering molecular-level insights into the role of selected mutations, dramatically escalating catalytic competency and selectivity. We have investigated the mechanism and correlation between mutations and exquisite stereoselectivity of a lactone carbene insertion into the C(sp3)-H bond of substituted aniline, catalyzed by two mutants of a cytochrome P450 variant, "P411" (engineered through directed evolution) in which the axial cysteine has been mutated to serine, utilizing various computational tools. The pivotal role of S264 and L/R328 mutations in the active site has been delineated computationally using two cluster models, thus rationalizing the enantiodivergence. This report provides much-needed insights into the origin of enantiodivergence, furnishing a mechanistic framework for understanding the anchoring effects of H-bond donor residues with the lactone ring. This study is expected to have important implications in the rational design of stereodivergent enzymes and toward successful in silico enzyme designing.

Structure Determination of SH2-Phosphopeptide Complexes by X-Ray Crystallography: The Example of p120RasGAP.

The p120RasGAP protein contains two Src homology 2 (SH2) domains, each with phosphotyrosine-binding activity. We describe the crystallization of the isolated and purified p120RasGAP SH2 domains with phosphopeptides derived from a binding partner protein, p190RhoGAP. Purified recombinant SH2 domain protein is mixed with synthetic phosphopeptide at a stoichiometric ratio to form the complex in vitro. Crystallization is then achieved by the hanging drop vapor diffusion method over specific reservoir solutions that yield single macromolecular co-crystals containing SH2 domain protein and phosphopeptide. This protocol yields suitable crystals for X-ray diffraction studies, and our recent X-ray crystallography studies of the two SH2 domains of p120RasGAP demonstrate that the N-terminal SH2 domain binds phosphopeptide in a canonical interaction. In contrast, the C-terminal SH2 domain binds phosphopeptide via a unique atypical binding mode. The crystallographic studies for p120RasGAP illustrate that although the three-dimensional structure of SH2 domains and the molecular details of their binding to phosphotyrosine peptides are well defined, careful structural analysis can continue to yield new molecular-level insights.

Revealing the interaction between peptide drugs and permeation enhancers in the presence of intestinal bile salts.

Permeability enhancer-based formulations offer a promising approach to enhance the oral bioavailability of peptides. We used all-atom molecular dynamics simulations to investigate the interaction between two permeability enhancers (sodium caprate, and SNAC), and four different peptides (octreotide, hexarelin, degarelix, and insulin), in the presence of taurocholate, an intestinal bile salt. The permeability enhancers exhibited distinct effects on peptide release based on their properties, promoting hydrophobic peptide release while inhibiting water-soluble peptide release. Lowering peptide concentrations in the simulations reduced peptide-peptide interactions but increased their interactions with the enhancers and taurocholates. Introducing peptides randomly with enhancer and taurocholate molecules yielded dynamic molecular aggregation, and reduced peptide-peptide interactions and hydrogen bond formation compared to peptide-only systems. The simulations provided insights into molecular-level interactions, highlighting the specific contacts between peptide residues responsible for aggregation, and the interactions between peptide residues and permeability enhancers/taurocholates that are crucial within the mixed colloids. Therefore, our results can provide insights into how modifications of these critical contacts can be made to alter drug release profiles from peptide-only or mixed peptide-PE-taurocholate aggregates. To further probe the molecular nature of permeability enhancers and peptide interactions, we also analyzed insulin secondary structures using Fourier transform infrared spectroscopy. The presence of SNAC led to an increase in β-sheet formation in insulin. In contrast, both in the absence and presence of caprate, α-helices, and random structures dominated. These molecular-level insights can guide the design of improved permeability enhancer-based dosage forms, allowing for precise control of peptide release profiles near the intended absorption site.

Differential membrane curvature induced by distinct protein conformers.

Membrane topology changes are associated with various cellular processes and are modulated by synergistic effects between lipid composition and membrane-associated proteins. However, how protein shape or conformational dynamics couples to membrane molecular properties remains unclear. In this work, we aim to investigate this coupling behavior using the curvature-inducing protein caveolin-1. We considered distinct protein conformers of the helical hairpin protein corresponding to different protein shapes, such as the wedge and the banana-shaped conformers. The different protein conformers were simulated in a coarse-grain representation in the presence of cholesterol-sphingomyelin rich membrane. We observed that membrane curvature is dependent on protein shape and is the lowest for the wedge conformer and maximal for the banana conformer. The differences in the net stress between the two membrane leaflets, calculated from the lateral pressure profile distributions in lipid bilayers for different protein conformers, show a similar trend. In conjunction, we show that cholesterol and sphingomyelin clustering in the membrane is modulated by protein shape. Overall, our results provide molecular-level insights into the coupling between membrane topology, protein shape and lipid clustering in cell membranes.

Molecular-level insight into the low-k properties and regulatory mechanisms of polybenzoxazines

Polybenzoxazine PCBZ-aptmds has a dielectric constant of 2.62 at 1 MHz. The simulated dielectric constant is in good agreement with the experimental value. Furthermore, the dielectric constant highly correlates to the volume polarizability.

Molecular-level insights into a tripolyphosphate and pyrophosphate templated membrane assembly.

Templated assembly of small molecules into nano-structural architectures has been used extensively by nature throughout its evolution. These systems have also been studied in artificial systems to design a phosphate templated assembly. However, it is yet to be investigated how the molecules interact among themselves at the molecular level and whether the phosphate templated assembly has any role in the formation of prebiotic protocellular membranes. Here, we report the prebiotic synthesis of choline-based cationic amphiphiles (-N+Me3) and the templated assembly of these amphiphiles with tripolyphosphate (TPP) and pyrophosphate (PPi). SEM, TEM, FLIM, DLS, fluorescence, and encapsulation studies suggest that the number of phosphate units in the phosphate backbone controls the formation and size of the protocell vesicles. Isothermal titration calorimetry, turbidimetric studies, and NMR experiments suggest that the cationic amphiphile forms a 3 : 1 catanionic complex with TPP and a 2 : 1 catanionic complex with PPi. The templated catanionic complex further self-assembles into vesicles, and the structure of the complex guides the size of the assembly. The size-controlling ability of the phosphate backbone might have been utilized in the prebiotic era to support the dynamics and tunability of protocellular membrane compartments.

Processive binding mechanism of Cel9G from Clostridium cellulovorans: molecular dynamics and free energy landscape investigations.

The degradation of recalcitrant polysaccharides such as cellulose and chitin requires the synergistic functionality of processive glycosidase (GH) cocktails. Understanding the fundamental phenomenon of processivity is of biological and economic importance for the conversion of biomass into biofuel. In this work, cellulase family 9 from Clostridium cellulovorans (Cel9G), which is a processive endoglucanase, was used to elucidate the processive binding mechanism with respect to polysaccharides, since it exhibits a multimodular crystallographic structure. Metadynamics and molecular dynamics simulations were performed to explore the dynamics of cellulose chain binding to Cel9G via processive motion. The processive movement of the cellulose chain towards the catalytic domain may exhibit several local minima, which are related to strong CH/π interactions between the sugar rings and the aromatic residues distributed at the active site. For the binding of the G6 and G12 molecules, the energy barriers were determined to be 4.8 and 7.4 kcal mol-1, respectively. Based on the site-directed mutagenesis simulations of Y520A, it was found that the existence of Y520 is critical for processive binding. It is likely that Y520 and H125/Y416 form two anchor points to facilitate processive binding to polysaccharides. More importantly, the straight-line morphology of the substrate could be observed after the formation of the so-called slide mode, which is different from the V-shaped Michaelis complex structure revealed by quantum mechanics/molecular mechanics simulations. This indicates that an additional step, namely, catalytic activation, probably exists between processive binding and the hydrolysis reaction. Finally, a four-step catalytic cycle was proposed for Cel9G. Our work provides novel molecular-level insights into the structure-function relationship for the processive enzyme Cel9G and should aid the development of improved GH cocktails for the efficient cleavage of glycosidic linkages.