Self-catalyzed Process Research Articles

Ligation of multiple protein domains using orthogonal inteins with non-native splice junctions.

Protein splicing is a self-catalyzed process in which an internal protein domain (the intein) is excised from its flanking sequences, linking them together with a canonical peptide bond. Trans-inteins are separated in two different precursor polypeptide chains that must assemble to catalytically self-excise and ligate the corresponding flanking exteins to join even when expressed separately either in vitro or in vivo. They are very interesting to construct full proteins from separate domains because their common small size favors chemical synthesis approaches. Therefore, trans-inteins have multiple applications such as protein modification and purification, structural characterization of protein domains or production of intein-based biosensors, among others. For many of these applications, when using more than one trans-intein, orthogonality between them is a critical issue to ensure the proper ligation of the exteins. Here, we confirm the orthogonality (lack of cross-reactivity) of four different trans- or split inteins, gp41-1, gp41-8, IMPDH-1 and NrdJ-1 both in vivo and in vitro, and built different constructs that allow for the sequential fusion of up to four protein fragments into one final spliced product. We have characterized the splicing efficiency of these constructs. All harbor non-native extein residues at the splice junction between the trans-intein and the neighboring exteins, except for the essential Ser + 1. Our results show that it is possible to ligate four different protein domains using inteins gp41-1, IMPDH-1 and NrdJ-1 with non-native extein residues to obtain a final four-domain spliced product with a not negligible yield that keeps its native sequence.

Facile synthesis of amine hybrid silica aerogel globule via wet casting assisted self-catalysed sol-gel process for CO2 capture including direct air capture

Amine hybrid silica aerogel globule (AHSAG) for CO2 capture including direct air capture (DAC) was firstly synthesized via wet casting assisted self-catalyzed sol-gel process. AHSAG has abundant interpenetrating macropores, which favors diffusion of CO2 into internal amine sites and can enhance CO2 adsorption kinetics and capacity. The optimal CO2 adsorption capacity of AHSAG with dry 100% and 400 ppm CO2 are 3.82 (30 °C) and 1.86 mmol/g (55 °C), respectively. Corresponding amine efficiencies are 0.478 and 0.233. The effect of temperature on CO2 adsorption capacity is attributed to simultaneous enhancement of mass transfer kinetics and adsorption thermodynamics. The highest CO2 adsorption rate of AHSAG is achieved at 70 and 55 °C with 100% and 400 ppm CO2, respectively. The trend of CO2 adsorption rate with temperature is attributed to the synergistic effect of diffusion kinetics and desorption. Based on experimental CO2 adsorption kinetics, adsorption mechanism in 400 ppm CO2 was estimated with different diffusion models. Both film diffusion resistance and intraparticle diffusion resistance are involved in CO2 capture of AHSAG from air under given condition to control the overall adsorption process. Three kinetics models were used to fit the experimental data of AHSAG for DAC, and the Avrami model showed the best fitting within the whole adsorption period. Comparing with its state-of-the-art counterparts, AHSAG offers significant advantages for practical DAC, such as high adsorption capacity and amine efficiency, fast adsorption kinetics, and excellent cyclic stability.

Hydrophobic and elastic silica-based aerogels with organic and inorganic in-situ hybrid structure for cryogenic insulation

Silica aerogels have potential to be used as alternatives in the cryogenic applications, but are still limited by the complex preparation process and high cost. In this work, a simple self-catalyzed gelling process and ambient drying method were utilized to prepare hydrophobic and low-density silica-based gels (HSAs) with organic and inorganic in-situ hybrid structure using methyltrimethoxysilane and bis[3-(triethoxysilyl)propyl]amine (BTPA) as precursors. The self-catalyzed gelling strategy is ascribed to the amine groups from BTPA. HSAs show a reversible deformation of 14 % and high failure strength up to 1.6 MPa even at the low temperature of −50 °C. Benefiting from the low density and high specific surface area, the thermal conductivity of the silica-based aerogels at −50 °C is as low as 21.8 mW·m−1·K−1. The HSAs are potential as ideal cryogenic insulation materials in a low-temperature and humid environment.

Probing the Stoichiometry Dependence of Enzyme-Catalyzed Junction Zone Network Formation in Aiyu Pectin Gel via a Reaction Kinetics Model.

We investigate the enzymatic self-catalyzed gelation process in aiyu gel, a natural ion crosslinked polysaccharide gel. The gelation process depends on the concentration ratio (Rmax) of the crosslinking calcium ions and all galacturonic acid binding sites. The physical gel network formation relies on the assembly of calcium-polysaccharide crosslink bonds. The crosslinks are initially transient and through break-up/rebinding gradually re-organizing into long, stable junction zones. Our previous study formulated a reaction kinetics model to describe enzymatic activation, crosslinker binding, and crosslink microstructural reorganization, in order to model the complex growth of elasticity. In this study, we extend the theory for the time-dependent profile of complex moduli and examine the interplay of enzyme conversion, crosslink formation, and crosslink re-organization. The adjusted model captures how the gelation and structural rearrangement characteristic times vary with the polymer and calcium concentrations. Furthermore, we find that calcium ions act as both crosslinkers and dopants in the excess calcium ion scenario and the binding dynamics is determined by Rmax. This study provides perspectives on the dynamic binding behaviors of aiyu pectin gel system and the theoretical approach can be generalized to enzyme-catalyzed ionic gel systems.

Transformation of postsynthesized F-MOF to Fe/N/F-tridoped carbon nanotubes as oxygen reduction catalysts for high power density Zn-air batteries

The oxygen reduction reaction (ORR), an important process in Zn-air batteries (ZABs), shows sluggish reaction kinetics, which significantly impairs the further improvement of battery performance. Thus, rationally designing cathodic catalysts for ZABs has drawn sufficient attention. We herein synthesize and characterize Fe/N/F-tridoped CNTs (FeNFCs) by annealing the postsynthesized trifluoroacetic anhydride-modified Fe-MIL-88B-NH 2 nanocrystals with melamine at high temperature in a N 2 atmosphere. Benefiting from the Fe/N/F element doping, high specific surface area, and CNT structure, the FeNFC800 catalyst prepared at 800°C exhibits a preferable half-wave potential of 0.829 V vs . RHE. The Zn-air battery equipped with FeNFC800 shows a high open-circuit voltage of 1.47 V, a gratifying peak power density of 196 mW/cm 2 , and extraordinary long-term stability, outperforming the benchmark 20% Pt/C. Fe/N/F-tridoped carbon nanotubes were grown from the postsynthesized F-Fe-MIL-88B-NH 2 microcrystals through a self-catalyzed process. The synergistic effect of Fe, N, F doping and the carbon nanotube structure enhanced the ORR catalytic performance. A Zn-air battery assembled with FeNFC800 shows a high peak power density of 196 mW/cm 2 , outperforming the benchmark 20% Pt/C.

Fullerene-Derived Carbon Nanotubes and Their Electrocatalytic Properties in Oxygen Reduction and Zn-Air Batteries.

Carbon-based materials with superior electrochemical performances have been prepared from fullerenes by releasing their intrinsic advantages such as pentagon defects and π-electron carbons. To the best of our knowledge, fullerene-derived carbon nanotubes (CNTs) and their electrochemical behavior have not been experimentally investigated. In this work, in situ growth of CNT composites from fullerene is realized via a self-catalyzed process by employing an Fe-decorated fullerene (ferrocenylpyrrolidine C60) as the precursor and NH3 as the pyrolysis atmosphere. The results show that the in situ Fe doping in fullerene, the self-assembly of fullerene molecules, the pyrolysis temperature, and the NH3 flow play essential roles in the generation of CNTs. The as-prepared MN7-10/3 CNT composite exhibits efficient oxygen reduction performance with E1/2 = 0.82 V and Eon = 1.02 V vs the RHE. The flexible solid-state Zn-air battery constructed based on MN7-10/3 exhibits a superior power density (109.3 mW cm-2 at 180.9 mA cm-2) and long-term durability (the voltage remains at 95.6% of the initial value after discharging for 5000 s) compared with the benchmark Pt/C catalyst. The transformation of the Fe-decorated fullerene to CNTs reveals a new function of fullerenes and demonstrates a new solid-state synthetic method for CNTs.

Water Promotes the Oxidation of SO2 by O2 over Carbonaceous Aerosols.

Severe haze episodes typically occur with concurrent high relative humidity. Here, the vital role of water in promoting the oxidation of SO2 by O2 on carbonaceous soot surfaces was identified at the atomic level by first-principles calculations. Water molecules can dissociate into surface hydroxyl groups through a self-catalyzed process under ambient conditions. The surface hydroxyl groups, acting as facilitators, can significantly accelerate the conversion of SO2 to SO3 (precursor of particulate sulfate) over soot aerosols by reducing the reaction barriers. Specifically, the hydroxyl groups activate the reactants and stabilize the transition states and products through hydrogen-bonding interactions, making the reactions both thermodynamically and kinetically more favorable at room temperature. The findings indicate that atmospheric humidity plays an important role in enhancing the atmospheric oxidation capacity, thus exacerbating SO2 oxidation and severe haze development. Also, this study unravels a mechanism of surface hydroxyl-assisted O2 and H2O dissociation over metal-free carbocatalysts under normal conditions.

Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing.

Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS.

Tuning the nanostructures and optical properties of undoped and N-doped ZnO by supercritical fluid treatment

Treatment of ZnO films in a supercritical fluid (SCF) has been reported to improve the performance of devices in which the treated ZnO films are incorporated; however, the mechanism of this improvement remains unclear. In this paper, we study the transformation of the surface morphologies and emission properties of ZnO films before and after SCF treatment, establishing the relationship between the treated and untreated structures and thereby enabling tuning of the catalytic or opto-electronic performance of ZnO films or ZnO-film-based devices. Both undoped and N-doped ZnO nanostructures generated by SCF treatment of films are investigated using techniques to characterize their surface morphology (scanning electron microscopy (SEM) and atomic force microscopy (AFM)) as well as room-temperature photoluminescence (RT-PL) spectroscopy. The water-mixed supercritical CO2 (W-SCCO2) technology was found to form nanostructures in ZnO films through a self-catalyzed process enabled by the Zn-rich conditions in the ZnO films. The W-SCCO2 was also found to promote the inhibition of defect luminescence by introducing -OH groups onto the films. Two models are proposed to explain the effects of the treatment with W-SCCO2. This work demonstrates that the W-SCCO2 technology can be used as an effective tool for the nanodesign and property enhancement of functional metal oxides.

Adsorption of NH3with Different Coverages on Single-Walled ZnO Nanotube: DFT and QTAIM Study

NH3 adsorption with different coverages on single-walled armchair and zigzag ZnO nanotubes (ZnONT) has been studied via periodic computational simulations at the all-electron B3LYP level. In order to fully characterize the molecules-surface interaction, infrared (IR) spectra were calculated for the first time. A rigorous analysis of the electron density in the bonding region, according to the quantum theory of atoms in molecules, was performed. NH3 molecules physisorb without dissociation via a self-catalyzed process. Although the nanotubes undergo sensitive lattice deformations with low coverages, its fundamental electronic properties were not modified. Owing to these analyses, the ZnONTs can be applicable as NH3 gas sensor.

Solution synthesis of conveyor-like MnSe nanostructured architectures with an unusual core/shell magnetic structure

We report for the first time one-dimensional (1D) wurtzite (WZ) MnSe nanoconveyors with a single-crystalline configuration fabricated by a solution-processed colloidal method. High-resolution transmission electron microscopy (HRTEM) measurements show that the stem of MnSe nanoconveyors grows along the [100] direction, while the teeth grow along the [0001] direction. We find that the initial WZ nanobelts with the [100] growth direction are crucial to the formation of nanoconveyors, whereas the teeth are a result of a self-catalyzed growth process induced by the Mn-terminated (0001) surface. The magnetic measurements suggest that 1D WZ MnSe nanoconveyors consist of an antiferromagnetic core and a ferromagnetic shell below the blocking temperature. Furthermore, the hysteresis measurements indicate that these nanoconveyors have 300 Oe coercive fields, which is attributed to the high surface-to-volume ratio of the nanoconveyors. This facile solution-based strategy can be anticipated to synthesize WZ metal chalcogenide nanomaterials with 1D hierarchical structures, for potential applications from spintronics to photocatalysis.

Mechanistic Insights into Asymmetric C–H Insertion Cooperatively Catalyzed by a Dirhodium(II) Complex and Chiral Phosphoric Acid

The reaction mechanism of methyl 2-diazo-2-phenylacetate (DIA) with 1-phenylpyrrolidine (An) cooperatively catalyzed by a dirhodium(II) complex and chiral spirophosphoric acid (SPA) has been studied with the aid of DFT methods. The results show that the nucleophilic attack of An at the in situ generated carbenoid is preferred to give a metal-associated enol intermediate. In addition, a stronger interaction between the dirhodium(II) complex and molecules such as the enol and chiral spirophosphoric acid is better for the asymmetric C–H insertion. This is due to the fact that a stronger interaction can restrict the chiral spirophosphoric acid around the dirhodium(II) complex. In this case, the dissociated enol can be captured by chiral spirophosphoric acid immediately to give the major S-enantiomer product. Otherwise, the dimerization of enol molecules will occur followed by a self-catalyzed process to give the racemic C–H insertion product. This finding should be important for the design of other related du...

Even stressed cells are individuals: second messengers of free radicals in pathophysiology of cancer

Pathophysiological processes associated with disturbances in cell and tissue oxidative homeostasis, are associated with self-catalyzed process of lipid peroxidation. The end products of lipid peroxidation are reactive aldehydes such as 4-hydroxy-2-nonenal (HNE), acting as “second messengers of free radicals.” Although reactive aldehydes were first recognized only as cytotoxic, new evidence has come to light, related to their cell growth regulatory functions achieved through cell signaling. The variable appearance of HNE in several organs indicates that its mode of action might be related to an individual cell stress adaptation. The underlying mechanism could be that specific mutations and epigenetic changes on one hand interfere with hormesis on the other. The precise role of oxidative stress and lipid peroxidation in these processes still needs more clarification at molecular level. Finally, an individual approach to each patient, based on the individual cell response to stress, opens a new possibility of integrative medicine in cancer treatment and strongly supports modern concepts of personalized medicine.

Intramolecular Disulfide Bond between Catalytic Cysteines in an Intein Precursor

Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.

Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi

Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 Å and a heavy atom root mean square deviation of 0.93 Å. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra β-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.

Self-Catalyzed Degradable Cationic Polymer for Release of DNA

The controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. This self-catalyzed hydrolysis process of PDMAEA forms poly(acrylic acid) and N,N'-dimethylamino ethyl ethanol, both of which have little or no toxicity to cells, and offers the advantage of little or no toxicity to off-target cells and tissues. We found that PDMAEA makes an ideal component of a delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then has the capacity to release the DNA inside the cells after ~10 h. The PDMAEA formed large nanoparticle complexes with oligo DNA of ~400 nm that protected the oligo DNA from DNase in serum. The nanoparticle complexes showed no toxicity for all molecular weights at a nitrogen/phosphorus (N/P) ratio of 10. Only the higher molecular weight polymers at very high N/P ratios of 200 showed significant levels of cytotoxicity. These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.

Self-Aggregation Processes of 1,6-Diphenyl-1,3,5-Hexatriene in Water/Ethanol Mixtures with High Water Percentages

This work describes the behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in ethanol/water mixtures. The dependence of DPH photophysical properties (absorption and fluorescence emission) on the water percentage in ethanol indicates that DPH undergoes self-aggregation processes in solvent conditions above a critical water content. Evidence such as an additional absorption band, Beer's law deviation, kinetic behavior, and other experimental results obtained from temperature variation and surfactant addition demonstrated the presence of several types of DPH aggregates. Resonance light scattering measurements proved that the aggregate grew in water-rich media by a self-catalyzed process.

Self-Catalyzed Degradation of Linear Cationic Poly(2-dimethylaminoethyl acrylate) in Water

We report the synthesis of a low cytotoxic polycation that maintains its cationic strength for well over a few hours then degrades into a benign polymer with nontoxic byproducts. Well-defined poly(2-dimethylaminoethyl acrylate) (PDMAEA) of five different molecular weights prepared using reversible addition-fragmentation chain transfer (RAFT) "living" radical polymerization degrades slowly over 200 h (∼8 days). As this degradation is independent of both the polymer molecular weight and solution pH, it is consistent with a self-catalyzed hydrolysis process without the need for an internal or external degradation trigger. In addition, the polymer shows little or no cytotoxicity to HeLa cells for the molecular weights of 5600 and below, even at very high polymer concentrations (equivalent to a nitrogen/phosphorus ratio of 200). Therefore, at sufficiently low molecular weights this polymer has the essential attributes (i.e., ability to autodegradable and low toxicity) for a delivery carrier suitable for DNA or siRNA.

An auto-catalytic surface for conformational replication of amyloid fibrils--genesis of an amyloid world?

Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65°C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.

Room-temperature ferromagnetic/ferroelectric BiFeO3 synthesized by a self-catalyzed fast reaction process

Bismuth ferrite BiFeO3 has attracted a great deal of interest because of its multiferroic properties. However, BiFeO3 synthesized by conventional methods in the forms of single crystals, ceramics or thin films only exhibit ferroelectricity and antiferromagnetic order at room temperature, with weak ferromagnetism appearing at very low temperatures. To fully explore the potential of multiferroism in such applications as new memory devices, it is necessary to synthesize materials that show ferromagnetic order at room temperature as well, which will a priori allow for magnetoelectric coupling. In this paper, we report a new synthetic technique for the synthesis of BiFeO3 that exhibits unusual ferromagnetic properties. This method involves a low temperature fast solid state reaction based on tartaric acid. The mechanism of the reaction deduced from thermogravimetric analysis (TGA) and differential thermal analysis (TGA) suggests that a self-catalyzed process in the presence of iron and bismuth oxides triggers the oxidation of tartaric acid at low temperature and gives out a large amount of heat, which, in turn, leads to the formation of BiFeO3. The BiFeO3 synthesized in this way is ferromagnetic. The origin of the unusual ferromagnetism is supposed to be associated with point defects of oxygen vacancies generated during the self-catalyzed extremely fast exothermic reaction, which suppress the spin circular cycloid in BiFeO3. Ferroelectric hysteresis loops are displayed in the BiFeO3 samples. The presence of room temperature ferromagnetic and ferroelectric orders makes BiFeO3 a truly multiferroic material potentially interesting in such applications as magnetoelectric devices.