Collaborative Construction of a Silver Nanocluster Fluorescent Probe Using the Pyridinium-Based Ionic Liquid [C4py][DCA].

A silver nanocluster fluorescent probe was synthesized by using the pyridinium-based ionic liquid [C4py][DCA] as the protective agent, AgNO3 as the precursor, and NaBH4 as the reducing agent. The presence of pyridine group enhanced the fluorescence intensity of Ag nanoclusters and facilitated the coordination interaction between Ag nanoclusters and AsO3 3-. Therefore, the collaborative construction of a silver nanocluster probe using the pyridinium-based ionic liquid [C4py][DCA] offered outstanding selectivity and sensitivity to detect AsO3 3- in water. More interestingly, the fluorescent probe quenched by AsO3 3- could be recovered with the addition of H2O2. This fluorescent probe provided a rapid and superior method for the detection of As(III) in the linear concentration range of 0-60 ppb with the lowest detection limit of 0.60 ppb. The mechanism of fluorescence quenching was a static quenching, considered to be due to electron migration between functional groups on the surface of Ag nanoclusters constructed with [C4py][DCA] and AsO3 3-.

Optimization of ultrasound-assisted extraction of poly-phenols from Ajuga ciliata Bunge and evaluation of antioxidant activities in vitro.

Effective extraction of natural antioxidants from cheap plant sources is still a problem. In this paper, an excellent method of ultrasound-assisted extraction of phenolic compounds from Ajuga ciliata Bunge was studied. The effects of four factors including ethanol volume fraction, ultrasonic time, ultrasonic temperature and material liquid ratio were discussed. After single factor experiments had been investigated, a 4-factor, 3-level Box-Behnken design experiment was used to obtain the model optimum conditions, which are shown as follows: ethanol volume fraction of 41%, liquid-solid ratio of 35:1 mL/g, ultrasonic temperature of 60 °C and ultrasonic time of 50 min. Under these conditions, the experimental productivity is 3.552 mg/g. The spectra of Fourier infrared and energy dispersive X-ray suggest that phenolic compounds exist in the extracts. Besides, free radical scavenging potentials of superoxide anion, hydroxyl and DPPH were measured to evaluate their antioxidant properties. This study proves that the ultrasonic-assisted extraction technique can extract phenolic compounds with antioxidant capacity from Ajuga ciliata Bunge.

Tetrathiomolybdate inhibits the reaction of cisplatin with human copper chaperone Atox1.

Cisplatin is a widely used anticancer drug in clinic, and ammonium tetrathiomolybdate ([(NH4)2MoS4], TM) is a copper chelator used in clinic for the treatment of Wilson's disease. Recently, TM has been found to enhance the therapeutic effect of cisplatin; however, the origin of this effect is not clear. Here we found that TM can inhibit the reaction of cisplatin with Cu-Atox1 and prevent the protein unfolding and aggregation induced by cisplatin. Although Ag(i) binds to Atox1 in a way similar to Cu(i)-Atox1, TM does not prevent the reaction of Ag-Atox1 with cisplatin. This result indicates that the formation of a Mo-centered trimeric protein cluster in the TM-Cu-Atox1 system plays a role in the inhibitory effect. This work provides new insights into the mechanism by which TM enhances the cytotoxic efficacy of cisplatin and helps to circumvent cisplatin resistance of tumor cells.

Synthesis of core-shell N-TiO2@CuO x with enhanced visible light photocatalytic performance.

In this paper, a core-shell N-TiO2@CuO x nanomaterial with increased visible light photocatalytic activity was successfully synthesized using a simple method. By synthesizing ammonium titanyl oxalate as a precursor, N-doped TiO2 can be prepared, then the core-shell structure of N-TiO2@CuO x with a catalyst loading of Cu on its surface was prepared using a precipitation method. It was characterized in detail using XRD, TEM, BET, XPS and H2-TPR, while its photocatalytic activity was evaluated using the probe reaction of the degradation of methyl orange. We found that the core-shell N-TiO2@CuO x nanomaterial can lessen the TiO2 energy band-gap width due to the N-doping, as well as remarkably improving the photo-degradation activity due to a certain loading of Cu on the surfaces of N-TiO2 supports. Therefore, a preparation method for a novel N, Cu co-doped TiO2 photocatalyst with a core-shell structure and efficient photocatalytic performance has been provided.

Conserved residues that modulate protein trans-splicing of Npu DnaE split intein.

The first crystal trans-structure of a naturally occurring split intein has been determined for the Npu (Nostoc punctiforme PCC73102) DnaE split intein. Guided by this structure, the residues NArg50 and CSer35, well conserved in DnaE split inteins, are identified to be critical in the trans-splicing of Npu DnaE split intein. An in vitro splicing assay demonstrates that NArg50 and CSer35 play synergistic roles in modulating its intein activity. The C-terminal CAsn36 exhibits two orientations of its side chain and interacts with both NArg50 and CSer35 through hydrogen bonding. These interactions likely facilitate the cyclization of asparagine in the course of protein splicing. The mutation of either residue reduces intein activity, and correlates with the low activity of the Ssp (Cyanobacterium synechocystis sp. strain PCC6803) DnaE split intein. On the other hand, NArg50 also forms a hydrogen bond with the highly conserved F-block CAsp17, thus influencing the N-S acyl shift during N-terminal cleavage. Sequence alignments show that residues NArg50 and CSer35 are rather conserved in those split inteins that lack a penultimate histidine residue. The conserved non-catalytic residues of split inteins modulate the efficiency of protein trans-splicing by hydrogen-bond interactions with the catalytic residues at the splice junction.

Mutual synergistic protein folding in split intein.

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with the concomitant ligation of the flanking polypeptides. Split inteins are expressed in two separated halves, and the recognition and association of two halves are the first crucial step for initiating trans-splicing. In the present study, we carried out the structural and thermodynamic analysis on the interaction of two halves of DnaE split intein from Synechocystis sp. PCC6803. Both isolated halves (IN and IC) are disordered and undergo conformational transition from disorder to order upon association. ITC (isothermal titration calorimetry) reveals that the highly favourable enthalpy change drives the association of the two halves, overcoming the unfavourable entropy change. The high flexibility of two fragments and the marked thermodynamic preference provide a robust association for the formation of the well-folded IN/IC complex, which is the basis for reconstituting the trans-splicing activity of DnaE split intein.

pK(a) coupling at the intein active site: implications for the coordination mechanism of protein splicing with a conserved aspartate.

Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pK(a) of 6.1, two units above the normal pK(a) of aspartate. The elevated pK(a) of D422 is coupled to the depressed pK(a) of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pK(a) to normal, while a D422G mutation increases the C1 pK(a) from 7.5 to 8.5. The pK(a) coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing.

Cisplatin inhibits protein splicing, suggesting inteins as therapeutic targets in mycobacteria.

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC(50) of ∼2 µM, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ∼40 µM, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.

Binding and inhibition of copper ions to RecA inteins from Mycobacterium tuberculosis.

Protein splicing is a unique post-translational process in which an intein excises itself from a precursor with the concomitant ligation of flanking sequences. The binding of zinc to intein inhibits protein splicing reversibly and EDTA relieves the inhibition. Copper was found to inhibit protein trans splicing; however, the recovery of intein splicing required both EDTA and TCEP, suggesting a different inhibition mechanism for copper compared to zinc. In this work, we have investigated the binding properties and inhibition effects of copper ions on the RecA intein from Mycobacterium tuberculosis. Both Cu(+) and Cu(2+) exhibited high binding affinity to inteins, while different binding sites were identified. Cu(2+) coordinates to Cys1, the key residue involved in the mechanism of protein splicing, however, Cu(+) does not coordinate to cysteine. An in vitro inhibition assay indicated that monovalent Cu(+) demonstrates reversible inhibition to protein splicing, and the inhibitory efficiency is comparable to Zn(2+). Redox reaction between Cu(2+) and cysteine in inteins were observed and the rate constants were determined. The results suggested a dual role for Cu(2+) in the inhibition of intein splicing: strong coordination of Cu(2+) to key residues (including Cys1) in the intein, and subsequent oxidation of Cys1, the residue required for the N-->S acyl shift step in protein splicing. A kinetic study suggested that the coordination could be the major cause of inhibition effect of Cu(2+) initially, whereas the redox reaction could play an additional role in inhibition at a later stage.

Metal ions binding to recA inteins from Mycobacterium tuberculosis.

Zinc has been found in the crystal structures of inteins and the zinc ion can inhibit intein splicing both in vitro and in vivo. The interactions between metal ions and three minimized recA inteins have been studied in this work. Isothermal titration calorimetry (ITC) results show that the zinc binding affinity to three inteins is in the order of DeltaI-SM > DeltaDeltaI(hh)-SM approximately DeltaDeltaI(hh)-CM, but is much weaker than to EDTA. These data explain the reversible inhibition and the presence of zinc only in the crystal structure of DeltaI-SM of recA intein. A positive correlation between binding constants and inhibition efficiency was observed upon the titration of different metal ions. Single-site binding modes were detected in all interactions, except DeltaDeltaI(hh)-CM which has two Zn sites. Zinc binding sites on DeltaDeltaI(hh)-CM were analyzed by NMR spectroscopy and ITC titration on inteins with chemical modifications. Results indicate that the Cys1 and His73 are the second zinc binding sites in DeltaDeltaI(hh)-CM. CD studies show the metal coordinations have negligible influence on protein structure. This work suggests that the mobility restriction of key residues from metal coordination is likely the key cause of metal inhibition of intein splicing.

1H, 13C, and 15N NMR assignments of an engineered intein based on Mycobacterium tuberculosis RecA.

The backbone and side chain resonance assignments of an engineered intein based on Mycobacterium tuberculosis RecA have been determined based on triple-resonance experiments with the uniformly [(13)C,(15)N]-labeled protein.