Site Of 3C Research Articles

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system.

Baculovirus expression vector system (BEVS) has been recognized as a potent protein expression system in engineering valuable enzymes and vaccines. Various fusion tags facilitate protein purification, leaving the potential risk to influence the target protein's biological activity negatively. It is of great interest to consider removing the additional tags using site-specific proteases, such as human rhinoviruses (HRV) 3C protease. The current study validated the cleavage activity of 3C protease in Escherichia coli and silkworm-BEVS systems by mixing the cell or fat body lysates of 3C protein and 3C site containing target protein in vitro. Further verification has been performed in the fat body lysate from co-expression of both constructs, showing remarkable cleavage efficiency in vivo silkworm larvae. We also achieved the glutathione-S-transferase (GST) tag-cleaved product of the VP15 protein from the White spot syndrome virus after purification, suggesting that we successfully established a coinfection-based recognition-and-reaction BEVS platform for the tag-free protein engineering.

Rare-earth-free noncollinear metallic ferrimagnets Mn4-xZxN with compensation at room temperature

Compensated ferrimagnets show no net magnetization like antiferromagnets, but their transport and magneto-optic properties resemble those of ferromagnets, thereby creating opportunities for applications in high-frequency spintronics and low energy loss communications. Here we study the modification of the noncollinear triangular ferrimagnetic spin structure of Mn4N by a variety of metallic substitutions Z (Z = Cu — Ge and Ag — Sn) to achieve compensation at room temperature. The noncollinear frustrated 2.35µB moments of Mn on 3c sites of the (111) kagome planes tilt about 20° out-of-plane in Mn4N and are easily influenced by the substitutions on 1a sites, leading to an efficiency of compensation in Mn4-xZxN that increases gradually from group 11 (Cu, Ag) to group 14 (Ge, Sn) with increasing number of valence electrons. Elements from the 5th period are more efficient for compensation than those from the 4th period due to lattice expansion. The manganese site moments analyzed by constrained density functional theory are determined by Z, orbital hybridization, charge transfer and the tilt angle. The Ga compound with compensation at room temperature for x ≈ 0.26 is recommended for high-frequency spintronic applications.

First-principles investigation on the role of interstitial site preference on the hydrogen-induced disproportionation of ZrCo and its doped alloys

There are two phase structures involved in ZrCo hydrides (ZrCoHx). When x ≤ 1, the α-phase hydride is generated when hydrogen atoms occupy the 3c and 12i sites. When 1 < x ≤ 3, three interstitial sites of 4c2, 8f1, and 8e are occupied by H, and in turn the β-phase hydride is formed. There is a disproportionation reaction in β-phase hydrides during hydrogen discharging process to produce the ZrH2 phase with higher thermal stability, leading to inferior hydrogen storage performance. In this study, the influence of hydrogen storage capacity on thermodynamic and lattice stabilities of α- and β-phase hydrides for each occupancy position is investigated under the framework of the first-principles study. The results indicate that the binding energy in the 3c site is higher compared with the 12i site under the condition of identical hydrogen storage capacity. Similarly, the binding energy is the largest for the 8e site compared with the other two sites, indicating that there is the least energy released in the reaction process. Thus, the 8e site is proved as the most unfavorable site in β-phase ZrCo hydrides, which is due to its degraded thermodynamic stability. Also, comparisons of mechanical properties and total density of states for each site in two hydride phases are presented to demonstrate that compound lattice stability in the 8e site is the poorest, suggesting that it is more likely to produce disproportionation. Furthermore, the dependence of hydrogen storage performance of β-phase hydrides on Ti/Rh doping is examined as well. It is discovered that there is improved thermodynamic stability and lattice stability in the 8e site for Zr0.875Ti0.125Co after Zr is partially substituted by Ti, which significantly enhances the disproportionation resistance. In contrast, when Co is partially replaced by Rh, there is a deterioration in the thermodynamic stability of ZrCo0.875Rh0.125 in the 8e site, but its lattice stability is somewhat improved.

Extended Chemical Flexibility of Cubic Anti-Perovskite Lithium Battery Cathode Materials.

Novel bichalcogenides with the general composition (Li2TM)ChO (TM = Mn, Co; Ch = S, Se) were synthesized by single-step solid-state reactions. These compounds possess cubic anti-perovskite crystal structure with Pm3̅ m symmetry; TM and Li are disordered on the crystallographic site 3c. According to Goldschmidt tolerance factor calculations, the available space at the 3c site is too large for Li+ and TM2+ ions. As cathode materials, all title compounds perform less prominent in lithium-ion battery setups in comparison to the already known TM = Fe homologue; e.g., (Li2Co)SO has a charge density of about 70 mAh g-1 at a low charge rate. Nevertheless, the title compounds extend the chemical flexibility of the anti-perovskites, revealing their outstanding chemical optimization potential as lithium battery cathode material.

Structure stability and magnetic properties of RIn3−xTx (R = Gd, Pr,T = Co, Fe, Mn)

The syntheses and crystal structures and magnetic properties of novel RIn3−xTx (R = Gd，Pr；T = Fe，Co，Mn；x = 0–0.3) intermetallic compounds in rare earth-In-3d transition metal ternary system have been systematically investigated. It reveals that RIn3−xTx crystallizes in cubic AuCu3 type structure with a space group of Pm$\bar 3$m and Z = 1. The 1a and 3c crystal positions are occupied by R and In atoms, respectively. The 3d transition metals substitute partly for In and prefer to occupy the 3c site. The lattice parameters and unit cell volumes decrease with increasing the content of 3d transition metal in RIn3−xTx intermetallic compounds. The magnetic properties of RIn3−xTx are sensitive to T content. With increasing T content, GdIn3−xTx alloys show the paramagnetic, mixture of ferromagnetic and paramagnetic and ferromagnetic behavior. T doping into RIn3 induces the presence of ferromagnetic phase in GdIn3−xTx, which is totally different from those of the pure binary RIn3.

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D.

Enterovirus 71 (EV71) can cause hand-foot-and-mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remains poorly understood. Here, we report that EV71 induces degradation of gasdermin D (GSDMD), an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1 to 193 (GSDMD1-193). However, unlike the N-terminal fragment produced by caspase-1 cleavage, this fragment fails to trigger cell death or inhibit EV71 replication. Importantly, a T239D or F240D substitution abrogates the activity of GSDMD consisting of amino acids 1 to 275 (GSDMD1-275). This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response.IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated interleukin-1β (IL-1β) secretion. In this process, the N-terminal domain of p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection downregulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N-terminal fragment disrupted for inducing cell pyroptosis. Notably, GSDMD1-275 (p30) inhibits EV71 replication whereas GSDMD1-193 does not. These results reveal a new strategy for EV71 to evade the antiviral response.

Cyanohydrin as an Anchoring Group for Potent and Selective Inhibitors of Enterovirus 71 3C Protease

Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S,2S,2'S,5S)-16 and (1R,2S,2'S,5S)-16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result of the specific interactions between the cyanohydrin moiety and the catalytic site of 3C(pro). Cyanohydrin as an anchoring group with high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors.

Adsorption of sarin on MgO nanotubes: Role of doped and defect sites

Sarin is a highly toxic organophosphorus chemical warfare agent which has been employed in various wars and terrorist attacks. Due to an urgent need of finding safe methods to decompose this toxic nerve agent, the research on decomposition of sarin gains importance. In the present work, the decomposition of sarin molecule on MgO nanotube and Ti-doped MgO nanotube has been investigated. For this purpose, the structural and electronic characteristics of nanotubes are first examined. It is seen that although doping with Ti modifies the properties of the nanotube, adsorption of sarin on both kinds of nanotubes presents similar characteristics. Adsorption is found to be more favorable at low-coordinated sites, i.e., the 3c site is preferred over 4c. Five kinds of surface defect sites have been considered i.e., O4c, O4c2−, Mg4c, Mg4c2+ and (MgO)4c. Adsorption of sarin on various defect sites produces different products. In two of the cases, the neutral oxygen defect and MgO defect, the molecule breaks completely into fragments and is destructively decomposed. Hence, our study proposes a new metal oxide system that might destructively adsorb chemical warfare agents and highlights the need for further exploration of untested metal oxide systems.

Variation of hydrogen occupation in LaNi 4.78Sn 0.22D x along the P– C isotherms studied by in situ neutron powder diffraction

We performed in situ neutron powder diffraction measurements of LaNi 4.78Sn 0.22D x along the absorption and desorption pressure–composition ( P– C) isotherms using 90° banks of a TOF diffractometer. Structure of the hydride phase for various hydrogen contents was analyzed by the Rietveld method. Variation of the hydrogen occupation and the lattice parameters for the hydride phase was investigated. The full hydride LaNi 4.78Sn 0.22D 6.1 has a P6 mm structure with four hydrogen sites; 3c sites (O[La 2Ni 4]), 6e 1 sites (T[La 2Ni 2]), 6e 2 sites (T[La 1Ni 3]) and 2b sites (T[Ni 4]) with occupancies of 0.98(2), 0.34(1), 0.168(9) and 0.24(2), respectively. The 3c site was fully occupied regardless of the total hydrogen content. Occupancies of the other three sites changed depending on the hydrogen content, with hysteresis between absorption and desorption in the two-phase region. The hydrogen occupation and the lattice parameters for the hydride phase varied even in the two-phase region during desorption.

Evidence for emission and transfer of energy from excited edge sites of MgO smokes by photoluminescence experiments

New features are observed in photoluminescence spectra of MgO smokes produced in controlled conditions, an experiment inspired by previous measurements on cleaved MgO(1 0 0). Upon photoexcitation at 5.17–5.45 eV, most luminescence spectra observed can be decomposed into two bands, peaking at 3.82–3.86 eV (the highest energy ever recorded in such experiment) and 3.20 eV, respectively. It is assumed that the 3.82–3.86 eV band is related to the luminescence at four-coordinated (4C) defect-free edge sites and that the 3.20 eV band arises from the excitation transfer from 4C edge sites towards less-coordinated 3C sites. A minor contribution to the 3.20 eV band observed upon excitation with 4.6 eV photons is assigned to a direct excitation of 3C sites. Within these assumptions, water chemisorption–desorption is suggested to produce less-coordinated sites (the 3.20 eV band increases in intensity) at the expense of 4C sites (the 3.84 eV band decreases). The huge sensitivity of the 3.84 eV/3.20 eV branching ratio to the formation of defects on 4C edges is suggested to explain the lack of consensus about the photoluminescence of MgO.

Crystal structure and hydrogen occupation of LaNi4.9Al0.1Dx (5.0 ≤ x ≤ 6.1) on the desorption isotherm studied by in situ neutron powder diffraction

We performed in situ neutron powder diffraction measurements of LaNi4.9Al0.1Dx along the desorption P–C isotherm using 90° banks of a TOF diffractometer. Structure of the hydride phase for various hydrogen contents was determined by Rietveld refinement. Variation of hydrogen site occupation in the hydride phase was clearly observed. The full hydride LaNi4.9Al0.1D6.1 has a P6mm structure with four hydrogen sites; 3c sites (O[La2Ni4]) with occupancy of 0.95(2) and three T sites, 6e1 (T[La2Ni2]), 6e2 (T[La1Ni3]) and 2b (T[Ni4]), with occupancies of 0.324(6), 0.195(6) and 0.22(1), respectively. In the two-phase region, the hydride phase has the limiting composition LaNi4.9Al0.1D5.0. Decreased hydrogen content from x = 6.1 to 5.0 in the hydride phase involved decrease of occupation in all sites, but behavior of the decrease depended on the sites. The 3c and 6e1 sites are preferably occupied in the limiting composition, whereas extra occupation caused by hydrogen solution in the hydride single-phase region is most preferable in the 6e2 sites.

Optical investigations and Raman scattering characterisation of carbon bonding in hard amorphous hydrogenated carbon films

Hydrogenated amorphous carbon films were prepared by plasma enhanced chemical vapour deposition (PECVD) of methane–argon (5%) gas mixtures at low pressure, in a dual electron cyclotron resonance (ECR)-rf glow discharge. Optical transmission spectroscopy, Raman spectroscopy, elastic recoil detection analysis (ERDA) and infra-red (IR) absorption spectra were combined to examine the relationship between the local microstructure (CH and CC bonds) and the optical properties at different negative bias voltage. The amount of bonded H, obtained by IR spectra, is higher in series deposited at low bias voltage, in qualitative agreement with the ERDA results. In all cases, most of the incorporated H is bonded to sp 3C sites, with a predominance of CH 3 methyl groups. These results are also consistent with the Raman spectroscopy measurements. The positions, widths, and relative intensities of the two characteristic features, the D and G peaks are found to vary systematically with deposition conditions and film properties. The series prepared at high bias voltage shows a higher disorder (D) over the graphitic (G) band ratio, which also indicates a structure with a high disorder form of Csp 2 sites.

Hydrogen incorporation and its structural effect on a-C:H films deposited by magnetron sputtering

Films of a-C:H have been prepared by dc magnetron sputtering from a graphite target with various ratios R of flow rates, or pressures, of H to Ar plus H in the chamber. In the analysis of the infrared (IR) spectra of a set of films with R varying from 2.5 × 10 −5 to 0.9, features characteristic of vibrational modes associated with H bonded in a variety of configurations, namely sp 2 and sp 3 sites involving single-, double- or (in the case of sp 3 sites) triple-bonded H, are detected. The relative fractions of the different sites do not change with R up to about 0.1 but, for larger R, the fraction of sp 3C sites bonded to a single H atom increases and the other configurations decrease. At the same time the linewidths of the peaks contributing to the overall spectra increase with R, which we suggest is due to increased stress in the films.

Further Requirements for Cleavage by the Murine Coronavirus 3C-like Proteinase: Identification of a Cleavage Site within ORF1b

The coronavirus mouse hepatitis virus strain A59 (MHV-A59) encodes a 3C-like proteinase (3CLpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp1a and pp1ab. In this study we demonstrate that the Q939↓S940 peptide bond, located between the polymerase and Zn-finger regions of pp1ab (the POL↓Zn site), is processed by the 3CLpro, albeit inefficiently. Mutagenesis of the POL↓Zn site, as well as the previously identified HD1↓3C site in the 1a region of pp1a and pp1ab, demonstrated that the amino acid residues at the P2 and P1 positions of the cleavage site, occupied by L and Q, respectively, were important determinants of 3CLpro substrate specificity. Finally, a direct comparison of the 3CLpro-mediated cleavages at the HD1↓3C and POL↓Zn sites was made by determining the rate constants using synthetic peptides. The results show that while a larger polypeptide substrate carrying the HD1↓3C site was processed more efficiently than a polypeptide substrate carrying the POL↓Zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. This indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase.

Refined X-ray crystallographic structure of the poliovirus 3C gene product

The X-ray crystallographic structure of the recombinant poliovirus 3C gene product (Mahoney strain) has been determined by single isomorphous replacement and non-crystallographic symmetry averaging and refined at 2.1 Å resolution. Poliovirus 3C is comprised of two six-stranded antiparallel β-barrel domains and is structurally similar to the chymotrypsin-like serine proteinases. The shallow active site cleft is located at the junction of the two β-barrel domains and contains a His40, Glu71, Cys147 catalytic triad. The polypeptide loop preceding Cys147 is flexible and likely undergoes a conformational change upon substrate binding. The specificity pockets for poliovirus 3C are well-defined and modeling studies account for the known substrate specificity of this proteinase. Poliovirus 3C also participates in the formation of the viral replicative initiation complex where it specifically recognizes and binds the RNA stem-loop structure in the 5′ non-translated region of its own genome. The RNA recognition site of 3C is located on the opposite side of the molecule in relation to its proteolytic active site and is centered about the conserved KFRDIR sequence of the domain linker. The recognition site is well-defined and also includes residues from the amino and carboxy-terminal helices. The two molecules in the asymmetric unit are related by an approximate 2-fold, non-crystallographic symmetry and form an intermolecular antiparallel β-sheet at their interface.

Cluster quantum-chemical study of the chemisorption of methane on a lithium-promoted magnesium oxide doped by zinc oxide

The reaction of a methane molecule with a lithium-doped magnesium oxide catalyst ( Li MgO ) containing small amounts of Zn 2+ cations (Zn/Li/MgO) was theoretically studied using a modified MINDO/3 method and applying a supermolecular approach. The surface of magnesium oxide (MgO) was modelled by a Mg 32O 32 four-layer molecular cluster containing all types of structural defects i.e., low-coordinated magnesium and oxygen ions (Mg 2+ LC and O 2− LC) of various faces, edges, corners etc. Molecular clusters of lithium-promoted magnesia ( Li MgO ) were simulated by isomorphic substitution of Mg 2+ LC by Li + LC; the excess negative charge of the cluster was compensated by a proton connected to an O 2− 3C site. For Zn-doped Li MgO or MgO an isomorphic substitution of Mg 2+ LC by Zn 2+ LC was assumed. The calculations indicate that for Zn/Li/MgO or Zn MgO the substitution of a threefold coordinated magnesium cation by zinc is more favourable by energetics than for four- and five-fold coordinated Mg cations. The computational results are used to interpret the experimentally observed increase of C 2+ hydrocarbons selectivity in the oxidative coupling of methane when doping a NaOH-promoted CaO catalyst with minor amounts of Zn 2+ cations.

Synthesis of N‐octylmonoaza‐15‐crown‐5 having a dodecylphosphoric acid functional group and the binding ability toward alkali metal cations

AbstractA new N‐octylmonoaza‐15‐crown‐5 having an alkylphosphoric acid functional group (3c) was synthesized. It was revealed that 3c selectively transported sodium ion under neutral source phase/acidic receiving phase condition, and selectively transported lithium ion under basic source phase/acidic receiving phase condition. From an ir and 13C nmr spectral study of the lithium hydroxide and sodium thiocyanate complexes of 3c, it is suggested that 3c does not incorporate cations into the three dimensional cavity using the crown ring and the phosphoric acid site, but that the crown and the phosphoric acid sites act on the cations independently; only the crown ether site of 3c significantly coordinates to the cations under neutral source phase condition, and the phosphate anion is mainly employed under basic conditions.

Electronic structures and magnetic properties of iron nitrides

To investigate the effect of nitrogen atoms on the magnetic properties of iron atoms, band calculations were carried out for Fe 4N and Fe 16N 2 by the Linearized Muffin Tin Orbitals (LMTO) method within the framework of the Local-Spin-Density (LSD) approximation. It was found that the magnetic moments of iron atoms near to nitrogen atoms decrease on the 3c sites of Fe 4N and on the 4e sites of Fe 16N 2, while the iron atoms far from nitrogen atoms have giant magnetic moments on the 1a sites of Fe 4N and on the 4d sites of Fe 16N 2.

Biologically active protease 3C of human rhinovirus 1A is expressed from a cloned cDNA segment in Escherichia coli

We produced the putative protease 3C of human rhinovirus 1A (HRV-1 A), a minor group rhinovirus, by Escherichia coli expression of a segment of HRV-1A cDNA coding for 3A, 3B, 3C, and parts of 2C and 3D (Δ2C3AB CΔ3D). The protease 3C was expected to be processed by intramolecular Q-G cleavages from the virus-specific precursor polypeptide. While the N-terminal 3B-3C site was correctly cleaved, the C-terminal Q-G site of 3C was not processed. Western blotting with a site-specific polyclonal antipeptide antibody showed that not the mature 3C polypeptide, but the 3C-containing precursor 3CΔ3D was the only rhinovirus-specific protein. Mature 3C was obtained by introducing two stop codons at positions glycine-1 and glutamine-2 of 3D by site-specific mutagenesis. This mutant produced the mature 3C protease of HRV-1A. In contrast to poliovirus, the mature 3C protease of HRV-1A is a minor peptide in virus-infected HeLa cells. The 3C protein can be detected only by Western blotting with a polyclonal antipeptide 3C antibody but not by radiolabeling the viral polypeptide.

Analysis of putative active site residues of the poliovirus 3C protease

It was recently suggested that the picornavirus 3C proteases are homologous to the chymotrypsin-like serine proteases. The two structural models proposed differ in one of the postulated active site residues, Glu/Asp 71 or Asp 85. We changed Glu 71 of the poliovirus type 1 protease to Asp or Gln and Asp 85 to Glu by oligonucleotide-directed site-specific mutagenesis of an infectious cDNA, and attempted to recover virus after transfection. Both Glu 71 changes were lethal for the virus and proteolytic activity was abolished in vitro with the exception of the primary cleavage event at the P2/P3 junction. In contrast, the Asp 85 → Glu virus was viable. This mutant was temperature-sensitive for growth at 39° and exhibited a minute plaque phenotype at permissive temperature. This defect correlated with low levels of viral-specific RNA and protein syntheses and slow virus growth. Proteolytic processing at the COOH-terminus of 3C was impaired, reducing the production of mature 3C and the viral replicase 3D. In addition, 3C-mediated cleavage events within the P2 region of the polyprotein seemed to occur rather inefficiently. 3C-specific processing within P1 and elsewhere within P3 was unaffected. We suggest that Asp 85 does not form part of the active site of 3C, but could be important for the specific recognition of cleavage sites within P2.