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Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.
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Transporte Activo de Núcleo Celular , VIH-1/química , ARN Mensajero/química , ARN Viral/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Células HEK293 , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Poro Nuclear/metabolismo , Conformación de Ácido Nucleico , Pliegue del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Protein crystallization plays a central role in structural biology. Despite this, the process of crystallization remains poorly understood and highly empirical, with crystal contacts, lattice packing arrangements and space group preferences being largely unpredictable. Programming protein crystallization through precisely engineered side-chain-side-chain interactions across protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach for designing three-dimensional protein crystals with prespecified lattice architectures at atomic accuracy that hierarchically constrains the overall number of degrees of freedom of the system. We design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into >100 µm three-dimensional crystals. The structures of these crystals are nearly identical to the computational design models, closely corresponding in both overall architecture and the specific protein-protein interactions. The dimensions of the crystal unit cell can be systematically redesigned while retaining the space group symmetry and overall architecture, and the crystals are extremely porous and highly stable. Our approach enables the computational design of protein crystals with high accuracy, and the designed protein crystals, which have both structural and assembly information encoded in their primary sequences, provide a powerful platform for biological materials engineering.
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Proteínas , Proteínas/química , CristalizaciónRESUMEN
Use of metal-selective ligands in solvent extraction is instrumental in extraction of critical materials and recycling, yet, diluent effects on extraction performance are not well understood. Experimental and empirical solvent parameters have been proposed to correlate with extraction performance, but are often inadequate predictors. We follow the hypothesis that the diluents' primary influence on extraction efficiency is whether or not it hinders assembly of the bulky extracting ligands into a geometry necessary for metal complexation. This behavior is readily accessible with molecular dynamics (MD), where the atomistic description of molecules can be applied to arbitrary extractant-solvent molecules and their mixtures. Several simulated quantities are considered, from both pairwise and graph theoretical analyses, and compared to experimental distribution ratio data for americium extraction by TODGA in a series of inert, non-interacting diluents. These simple properties, especially the formation of closed triplets corresponding to the 3 : 1 ligand : metal stoichiometric solvate, suggest a potential predictive power of this approach. This methodology provides a path forward to comprehensively understand and predict diluent effects in more complex systems involving different extracting ligands and multi-component diluent mixtures.
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Structure at the nanoscale in the organic phase of liquid-liquid extraction systems is often tied to separation performance. However, the weak interactions that drive extractant assembly lead to poorly defined structures that are challenging to identify. Here, we investigate the mechanism of water extraction for a malonamide extractant commonly applied to f-element separations. We measure extractant concentration fluctuations in the organic phase with small angle X-ray scattering (SAXS) before and after contact with water at fine increments of extractant concentration, finding no qualitative changes upon water uptake that might suggest significant nanoscopic reorganization of the solution. The critical composition for maximum fluctuation intensity is consistent with small water-extractant adducts. The extractant concentration dependence of water extraction is consistent with a power law close to unity in the low concentration regime, suggesting the formation of 1 : 1 water-extractant adducts as the primary extraction mechanism at low concentration. At higher extractant concentrations, the power law slope increases slightly, which we find is consistent with activity effects modeled using Flory-Huggins theory without introduction of additional extractant-water species. Molecular dynamics simulations are consistent with these findings. The decrease in interfacial tension with increasing extractant concentration shows a narrow plateau region, but it is not correlated with any change in fluctuation or water extraction trends, further suggesting no supramolecular organization such as reverse micellization. This study suggests that water extraction in this system is particularly simple: it relies on a single mechanism at all extractant concentrations, and only slightly enhances the concentration fluctuations characteristic of the dry binary extractant/diluent mixture.
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Electrochemical approaches provide unique means to fabricate thin films of metal-organic frameworks (MOFs). However, the kinetics of electrochemical MOF deposition has not been quantitatively investigated so far. In this study, we report the first in situ measurements of electrochemical MOF growth using transmission synchrotron X-ray scattering. Electrochemical cells based on poly(lactic acid) with two windows were fabricated using fused-deposition modeling. The resulting three-dimensional (3D)-printed cells, the surface of which was coated with paraffin wax to prevent solvent percolation through the polymer material, were used to monitor the cathodic growth of zeolitic imidazolate framework-8 (ZIF-8) on graphite in a methanol solution containing ZnCl2 and 2-methylimidazole (Hmim) at different cathodic potentials. The resulting time-resolved X-ray diffraction data showed a gradual increase in crystal size with negligible changes in crystal orientation during the cathodic ZIF-8 deposition. More importantly, the time-resolved data provided a means to quantitatively assess the kinetics of the cathodic ZIF-8 growth using the Gualtieri model, revealing that the cathodic potential and Hmim concentration affected crystal growth kinetics but not nucleation kinetics. These ZIF-8 samples exhibited changes in X-ray diffraction patterns after being washed with methanol and dried in air, indicating that in situ measurements are essential to investigate mechanisms behind MOF electrodeposition.
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Extractant aggregation in liquid-liquid extraction organic phases impacts extraction energetics and is related to the deleterious efficiency-limiting liquid-liquid phase transition known as third phase formation. Using small angle X-ray scattering, we find that structural heterogeneities across a wide range of compositions in binary mixtures of malonamide extractants and alkane diluents are well described by Ornstein-Zernike scattering. This suggests that structure in these simplified organic phases originates from the critical point associated with the liquid-liquid phase transition. To confirm this, we measure the temperature dependence of the organic phase structure, finding critical exponents consistent with the 3D Ising model. Molecular dynamics simulations were also consistent with this mechanism for extractant aggregation. Due to the absence of water or any other polar solutes required to form reverse-micellar-like nanostructures, these fluctuations are inherent to the binary extractant/diluent mixture. We also show how the molecular structure of the extractant and diluent modulate these critical concentration fluctuations by shifting the critical temperature: critical fluctuations are suppressed by increasing extractant alkyl tail lengths or decreasing diluent alkyl chain lengths. This is consistent with how extractant and diluent molecular structure are known to impact metal and acid loading capacity in many-component LLE organic phases, suggesting phase behavior of practical systems may be effectively studied in simplified organic phases. Overall, the explicit connection between molecular structure, aggregation and phase behavior demonstrated here will enable the design of more efficient separations processes.
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Nanostructured materials with high surface area and low coordinated atoms present distinct intrinsic properties from their bulk counterparts. However, nanomaterials' nucleation/growth mechanism during the synthesis process and the changes of the nanomaterials in the working state are still not thoroughly studied. As two indispensable methods, X-ray absorption spectroscopy (XAS) provides nanomaterials' electronic structure and coordination environment, while small-angle X-ray scattering (SAXS) offers structural properties and morphology information. A combination of in situ/operando XAS and SAXS provides high temporal and spatial resolution to monitor the evolution of nanomaterials. This review gives a brief introduction to in situ/operando SAXS/XAS cells. In addition, the application of in situ/operando XAS and SAXS in preparing nanomaterials and studying changes of working nanomaterials are summarized.
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Nanoestructuras , Dispersión del Ángulo Pequeño , Espectroscopía de Absorción de Rayos X , Difracción de Rayos XRESUMEN
All cells use organized lipid compartments to facilitate specific biological functions. Membrane-bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid-based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)-modified phospholipids that form stable covalent linkages with an O6 -methylguanine DNA methyltransferase (SNAP-tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid-aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP-tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid-based artificial organelles.
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Gotas Lipídicas , O(6)-Metilguanina-ADN Metiltransferasa , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Fosfolípidos , ProteínasRESUMEN
Liquid-liquid extraction is an essential chemical separation technique where polar solutes are extracted from an aqueous phase into a nonpolar organic solvent by amphiphilic extractant molecules. A fundamental limitation to the efficiency of this important technology is third phase formation, wherein the organic phase splits upon sufficient loading of polar solutes. The nanoscale drivers of phase splitting are challenging to understand in the complex hierarchically structured organic phases. In this study, we demonstrate that the organic phase structure and phase behavior are fundamentally connected in a way than can be understood with critical phenomena theory. For a series of binary mixtures of trialkyl phosphate extractants with linear alkane diluents, we combine small angle x-ray scattering and molecular dynamics simulations to demonstrate how the organic phase mesostructure over a wide range of compositions is dominated by critical concentration fluctuations associated with the critical point of the third phase formation phase transition. These findings reconcile many longstanding inconsistencies in the literature where small angle scattering features, also consistent with such critical fluctuations, were interpreted as reverse micellar-like particles. Overall, this study shows how the organic phase mesostructure and phase behavior are intrinsically linked, deepening our understanding of both and providing a new framework for using molecular structure and thermodynamic variables to control mesostructure and phase behavior in liquid-liquid extraction.
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The microscopic molecular structure and dynamics of a new deep eutectic solvent (DES) composed of an ionic liquid (1-hexyl-3-methylimidazolium chloride) and an amide (trifluoroacetamide) at various molar ratios were investigated using linear and non-linear infrared spectroscopy with a vibrational probe. The use of the ionic liquid allows us to investigate the changes that the system undergoes with the addition of the amide or, equivalently, the changes from an ionic liquid to a DES. Our studies revealed that the vibrational probe in the DES senses a very similar local environment irrespective of the cation chemical structure. In addition, the amide also appears to perceive the same molecular environment. The concentration dependence studies also showed that the amide changes from being isolated from other amides in the ionic liquid environment to an environment where the amide-amide interactions are favored. In the case of the vibrational probe, the addition of the amide produced significant changes in the slow dynamics associated with the making and breaking of the ionic cages but did not affect the rattling-in-cage motions perceived by it. Furthermore, the concentration dependence of slow dynamics showed two regimes which are linked to the changes in the overall structure of the solution. These observations are interpreted in the context of a nanoscopic heterogeneous environment in the DES which, according to the observed dynamical regimes, appears at very large concentrations of the amide (molar ratio of greater than 1:1) since for lower amide molar ratios, the amide appears to be not segregated from the ionic liquid. This proposed molecular picture is supported by small angle x-ray scattering experiments.
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Localized surface plasmon resonances (LSPR) of nanostructures can be tuned by controlling their morphology, local dielectric environment, and free carrier concentration. We report the colloidal synthesis of an â¼3 tungsten-oxygen (W-O) layer thick (â¼1 nm), two-dimensional (2D) WO3-x nanoplatelets (NPLs) (x ≈ 0.55-1.03), which display tunable near-infrared LSPR properties and additionally high free electron density (Ne) that arises predominantly from the large shape factor of 2D NPLs. Importantly, the W to O composition ratios inferred from their LSPR measurements show much higher percentage of oxygen vacancies than those determined by X-ray diffraction analysis, suggesting that the aspect ratio of ultrathin WO3-x NPLs is the key to producing an unprecedentedly large Ne, although synthesis temperature is also an independent factor. We find that NPL formation is kinetically controlled, whereas thermodynamic parameter manipulation leads to Ne values as high as 4.13 × 1022 cm-3, which is close to that of plasmonic noble metals, and thus our oxide-based nanostructures can be considered as quasi-metallic. The unique structural properties of 2D nanomaterials along with the high Ne of WO3-x NPLs provide an attractive alternative to plasmonic noble metal nanostructures for various plasmon-driven energy conversions and design of photochromic nanodevices.
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Understanding the origins of enhanced reactivity of supported, subnanometer in size, metal oxide clusters is challenging due to the scarcity of methods capable to extract atomic-level information from the experimental data. Due to both the sensitivity of X-ray absorption near edge structure (XANES) spectroscopy to the local geometry around metal ions and reliability of theoretical spectroscopy codes for modeling XANES spectra, supervised machine learning approach has become a powerful tool for extracting structural information from the experimental spectra. Here, we present the application of this method to grazing incidence XANES spectra of size-selective Cu oxide clusters on flat support, measured in operando conditions of the methanation reaction. We demonstrate that the convolution neural network can be trained on theoretical spectra and utilized to "invert" experimental XANES data to obtain structural descriptors-the Cu-Cu coordination numbers. As a result, we were able to distinguish between different structural motifs (Cu2O-like and CuO-like) of Cu oxide clusters, transforming in reaction conditions, and reliably evaluate average cluster sizes, with important implications for the understanding of structure, composition, and function relationships in catalysis.
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DEAD-box proteins are nonprocessive RNA helicases and can function as RNA chaperones, but the mechanisms of their chaperone activity remain incompletely understood. The Neurospora crassa DEAD-box protein CYT-19 is a mitochondrial RNA chaperone that promotes group I intron splicing and has been shown to resolve misfolded group I intron structures, allowing them to refold. Building on previous results, here we use a series of tertiary contact mutants of the Tetrahymena group I intron ribozyme to demonstrate that the efficiency of CYT-19-mediated unfolding of the ribozyme is tightly linked to global RNA tertiary stability. Efficient unfolding of destabilized ribozyme variants is accompanied by increased ATPase activity of CYT-19, suggesting that destabilized ribozymes provide more productive interaction opportunities. The strongest ATPase stimulation occurs with a ribozyme that lacks all five tertiary contacts and does not form a compact structure, and small-angle X-ray scattering indicates that ATPase activity tracks with ribozyme compactness. Further, deletion of three helices that are prominently exposed in the folded structure decreases the ATPase stimulation by the folded ribozyme. Together, these results lead to a model in which CYT-19, and likely related DEAD-box proteins, rearranges complex RNA structures by preferentially interacting with and unwinding exposed RNA secondary structure. Importantly, this mechanism could bias DEAD-box proteins to act on misfolded RNAs and ribonucleoproteins, which are likely to be less compact and more dynamic than their native counterparts.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas Fúngicas/metabolismo , Intrones/genética , Conformación de Ácido Nucleico , ARN/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Activación Enzimática/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Magnesio/farmacología , Modelos Moleculares , Neurospora crassa/enzimología , Pliegue de Proteína/efectos de los fármacos , ARN/metabolismo , ARN Catalítico/química , ARN Catalítico/metabolismo , Tetrahymena/metabolismoRESUMEN
A typical type of core-shell polyoxometalates can be obtained through the Keggin-type polyoxometalate-templated growth of a layer of spherical shell structure of {Mo72Fe30}. Small-angle X-ray scattering is used to study the structural features and stability of the core-shell structures in aqueous solutions. Time-resolved small-angle X-ray scattering is applied to monitor the synthetic reactions, and a three-stage formation mechanism is proposed to describe the synthesis of the core-shell polyoxometalates based on the monitoring results. New protocols have been developed by fitting the X-ray data with custom physical models, which provide more convincing, objective, and completed data interpretation. Quasi-elastic and inelastic neutron scattering are used to probe the dynamics of water molecules in the core-shell structures, and two different types of water molecules, the confined and structured water, are observed. These water molecules play an important role in bridging core and shell structures and stabilizing the cluster structures.
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Understanding the formation mechanism of giant molecular clusters is essential for rational design and synthesis of cluster-based nanomaterials with required morphologies and functionalities. Here, typical synthetic reactions of a 2.9 nm spherical molybdenum oxide cluster, {Mo132} (formula: [Mo(VI)72Mo(V)60O372(CH3COO)30(H2O)72](42-)), with systematically varied reaction parameters have been fully explored to determine the morphologies and concentration of products, reduction of metal centers, and chemical environments of the organic ligands. The growth of these clusters shows a typical sigmoid curve, suggesting a general multistep self-assembly mechanism for the formation of giant molecular clusters. The reaction starts with a lag phase period when partial Mo(VI) centers of molybdate precursors are reduced to form {Mo(V)2(acetate)} structures under the coordination effect of the acetate groups. Once the concentration of {Mo(V)2(acetate)} reaches a critical value, it triggers the co-assembly of Mo(V) and Mo(VI) species into the giant clusters.
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A novel setup for containment-free time-resolved experiments at a free-hanging drop is reported. Within a dead-time of 100â ms a drop of mixed reactant solutions is formed and the time evolution of a reaction can be followed from thereon by various techniques. As an example, a small-angle X-ray scattering study on the formation mechanism of EDTA-stabilized CdS both at a synchrotron and a laboratory X-ray source is presented here. While the evolution can be followed with one drop only at a synchrotron source, a stroboscopic mode with many drops is preferable for the laboratory source.
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Materials-binding peptides represent a unique avenue towards controlling the shape and size of nanoparticles (NPs) grown under aqueous conditions. Here, employing a bionanocombinatorics approach, two such materials-binding peptides were linked at either end of a photoswitchable spacer, forming a multi-domain materials-binding molecule to control the in situ synthesis and organization of Ag and Au NPs under ambient conditions. These multi-domain molecules retained the peptides' ability to nucleate, grow, and stabilize Ag and Au NPs in aqueous media. Disordered co-assemblies of the two nanomaterials were observed by TEM imaging of dried samples after sequential growth of the two metals, and showed a clustering behavior that was not typically observed without both metals and the linker molecules. While TEM evidence suggested the formation of AuNP/AgNP assemblies upon drying, SAXS analysis indicated that no extended assemblies existed in solution, suggesting that sample drying plays an important role in facilitating NP clustering. Molecular simulations and experimental data revealed tunable materials-binding based upon the isomerization state of the photoswitchable unit and metal employed. This work is a first step in generating externally actuated biomolecules with specific material-binding properties that could be used as the building blocks to achieve multi-material switchable NP assemblies.
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The activation of CO2 and its hydrogenation to methanol are of much interest as a way to utilize captured CO2. Here, we investigate the use of size-selected Cu4 clusters supported on Al2O3 thin films for CO2 reduction in the presence of hydrogen. The catalytic activity was measured under near-atmospheric reaction conditions with a low CO2 partial pressure, and the oxidation state of the clusters was investigated by in situ grazing incidence X-ray absorption spectroscopy. The results indicate that size-selected Cu4 clusters are the most active low-pressure catalyst for catalytic CO2 conversion to CH3OH. Density functional theory calculations reveal that Cu4 clusters have a low activation barrier for conversion of CO2 to CH3OH. This study suggests that small Cu clusters may be excellent and efficient catalysts for the recycling of released CO2.
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Understanding the structure-property relationships and the phenomena responsible for ion conduction is one of the keys in the design of novel ionomers with improved properties. In this report, the morphology and the mechanism of ion exchange in a model anion exchange membrane (AEM), poly(vinyl benzyl trimethyl ammonium bromide)-block-poly(methylbutylene) ([PVBTMA][Br]-b-PMB), is investigated with small angle X-ray scattering, high-resolution thermogravimetry, modulated differential scanning calorimetry, dynamic mechanical analysis, and broadband electrical spectroscopy. The hyper-morphology of the material consists of hydrophilic domains characterized by stacked sides of [PVBTMA][Br] which are sandwiched between "spaghetti-like" hydrophobic cylindrical parallel domains of the PMB block. The most important interactions in the hydrophilic domains occur between the dipoles of ammonium bromide ion pairs in the side chains of adjacent chains. A reordering of the ion pair dipoles is responsible for a disorder-order transition (Tδ) at high temperature, observed here for the first time in AEMs, which results in a dramatic decrease of the ionic conductivity. The overall mechanism of long range charge transfer, deduced from a congruent picture of all of the results, involves two distinct ion conduction pathways. In these pathways, hydration and the motion of the ionic side groups are crucial to the conductivity of the AEM. Unlike the typical perfluorinated sulfonated proton-conducting polymer, the segmental motion of the backbone is negligible.
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We demonstrate that the true hydroxide conductivity in an e-beam grafted poly(ethylene-co-tetrafluoroethylene) [ETFE] anion exchange membrane (AEM) is as high as 132 mS cm(-1) at 80 °C and 95% RH, comparable to a proton exchange membrane, but with very much less water present in the film. To understand this behaviour we studied ion transport of hydroxide, carbonate, bicarbonate and chloride, as well as water uptake and distribution. Water uptake of the AEM in water vapor is an order of magnitude lower than when submerged in liquid water. In addition (19)F pulse field gradient spin echo NMR indicates that there is little tortuosity in the ionic pathways through the film. A complete analysis of the IR spectrum of the AEM and the analyses of water absorption using FT-IR led to conclusion that the fluorinated backbone chains do not interact with water and that two types of water domains exist within the membrane. The reduction in conductivity was measured during exposure of the OH(-) form of the AEM to air at 95% RH and was seen to be much slower than the reaction of CO2 with OH(-) as the amount of water in the film determines its ionic conductivity and at relative wet RHs its re-organization is slow.