Regenerating MXene by a Facile Chemical Treatment Method.

A popular substance in the MXene family, titanium carbide (Ti3C2Tx), has received substantial attention mainly due to its high metallic conductivity, easy solution processability, and environment friendliness. However, the poor oxygen resistance nature of MXene has prevented its practical applications from being realized. Despite significant attempts to improve the oxidative stability of MXenes, a comprehensive understanding of the oxidation mechanism is still elusive, thus leaving an optimal strategy for recycling oxidized MXene in question. Here, by developing a facile hydrofluoric acid (HF) post-treatment, we have unraveled the regeneration kinetics of the oxidized Ti3C2Tx. A systematic and extensive investigation using a combination of Raman spectroscopy, scanning electron microscopy, X-ray diffractometer, and X-ray photoelectron spectroscopy revealed that HF post-treatment is critical for restoring the structure/morphology and surface composition of MXene nanosheets. These are ascribed to the oxidizing agent removal kinetics, while the generation of amorphous carbon and Ti(III) in fluorinated derivatives provides efficient electrical conductivity. Our findings suggested that HF post-treatment is sufficient to evade and reduce the degradation process while maintaining the conductivity for a longer time, which will not only be economically advantageous but also a step forward for the rational design of Ti3C2Tx-based devices and functional coatings.

Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster.

U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

Bdp1 interacts with SNAPc bound to a U6, but not U1, snRNA gene promoter element to establish a stable protein-DNA complex.

In metazoans, U6 small nuclear RNA (snRNA) gene promoters utilize a proximal sequence element (PSE) recognized by the small nuclear RNA-activating protein complex (SNAPc). SNAPc interacts with the transcription factor TFIIIB, which consists of the subunits TBP, Brf1 (Brf2 in vertebrates), and Bdp1. Here, we show that, in Drosophila melanogaster, DmSNAPc directly recruits Bdp1 to the U6 promoter, and we identify an 87-residue region of Bdp1 involved in this interaction. Importantly, Bdp1 recruitment requires that DmSNAPc be bound to a U6 PSE rather than a U1 PSE. This is consistent with the concept that DmSNAPc adopts different conformations on U6 and U1 PSEs, which lead to the subsequent recruitment of distinct general transcription factors and RNA polymerases for U6 and U1 gene transcription.

Cutting-Processed Single-Wall Carbon Nanotubes with Additional Edge Sites for Supercapacitor Electrodes.

Carbon nanotubes are frequently selected for supercapacitors because of their major intrinsic properties of mechanical and chemical stability, in addition to their excellent electrical conductivity. However, electrodes using carbon nanotubes suffer from severe performance degradation by the phenomenon of re-stacking during fabrication, which hinders ion accessibility. In this study, short single-wall carbon nanotubes were further shortened by sonication-induced cutting to increase the proportion of edge sites. This longitudinally short structure preferentially exposes the active edge sites, leading to high capacitance during operation. Supercapacitors assembled using the shorter-cut nanotubes exhibit a 7-fold higher capacitance than those with pristine single-wall nanotubes while preserving other intrinsic properties of carbon nanotubes, including excellent cycle performance and rate capability. The unique structure suggests a design approach for achieving a high specific capacitance with those low-dimensional carbon materials that suffer from re-stacking during device fabrication.

Structural Insights into the Quaternary Catalytic Mechanism of Hexameric Human Quinolinate Phosphoribosyltransferase, a Key Enzyme in de novo NAD Biosynthesis.

Quinolinate phosphoribosyltransferase (QPRT) catalyses the production of nicotinic acid mononucleotide, a precursor of de novo biosynthesis of the ubiquitous coenzyme nicotinamide adenine dinucleotide. QPRT is also essential for maintaining the homeostasis of quinolinic acid in the brain, a possible neurotoxin causing various neurodegenerative diseases. Although QPRT has been extensively analysed, the molecular basis of the reaction catalysed by human QPRT remains unclear. Here, we present the crystal structures of hexameric human QPRT in the apo form and its complexes with reactant or product. We found that the interaction between dimeric subunits was dramatically altered during the reaction process by conformational changes of two flexible loops in the active site at the dimer-dimer interface. In addition, the N-terminal short helix α1 was identified as a critical hexamer stabilizer. The structural features, size distribution, heat aggregation and ITC studies of the full-length enzyme and the enzyme lacking helix α1 strongly suggest that human QPRT acts as a hexamer for cooperative reactant binding via three dimeric subunits and maintaining stability. Based on our comparison of human QPRT structures in the apo and complex forms, we propose a drug design strategy targeting malignant glioma.

Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide.

We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence. The crystal structure revealed that porcine QAPRTases have a hexameric structure that is similar to other eukaryotic QAPRTases, such as the human and yeast enzymes. However, the interaction between NAMN and porcine QAPRTase was different from the interaction found in prokaryotic enzymes, such as those of Helicobacter pylori and Mycobacterium tuberculosis. The crystal structure of porcine QAPRTase in complex with NAMN provides a structural framework for understanding the unique properties of the mammalian QAPRTase active site and designing new antibiotics that are selective for the QAPRTases of pathogenic bacteria, such as H. pylori and M. tuberculosis.

Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide.

Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Šresolution. The Ss-QAPRTase-NAMN cocrystals belonged to space group P321, with unit-cell parameters a=119.1, b=119.1, c=93.7 Å, γ=120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å3 Da(-1) and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit.

Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase.

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Šresolution. Native Hs-QPRTase crystals belonged to space group P2(1), with unit-cell parameters a=76.2, b=137.1, c=92.7 Å, ß=103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 Å3 Da(-1), which corresponds to a solvent content of 49.9%.

Crystallization and preliminary X-ray crystallographic analysis of MinE, the cell-division topological specificity factor from Helicobacter pylori.

Cell division in Gram-negative bacteria is driven by the formation of an FtsZ ring at the division site. MinE regulates the proper placement of the FtsZ ring at mid-cell by blocking the inhibitory action of the MinCD complex. Diffraction data were collected at 2.8 A resolution from a native crystal of full-length Helicobacter pylori MinE. The crystal belonged to space group P6(4). Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient was 2.58 A(3) Da(-1), which corresponds to a solvent content of 52.3%. For MAD phasing, a four-wavelength data set was collected at 3.0 A resolution.

Crystal structure of Helicobacter pylori MinE, a cell division topological specificity factor.

In Gram-negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid-cell division site. Its structure consists of an anti-MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long alpha-helix and two anti-parallel beta-strands, which mediate formation of a homodimeric alpha/beta structure. Here we report the crystal structure of full-length Helicobacter pylori MinE and redefine its TSD based on that structure. The N-terminal region of the TSD (residues 19-26), previously defined as part of the anti-MinCD domain, forms a beta-strand (betaA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti-parallel betaA-strands. Moreover, we observed serial dimer-dimer interactions within the crystal packing, resulting in the formation of a multimeric structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti-parallel beta-strand interactions.

Identification of SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 gene promoters.

The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In Drosophila melanogaster, the heterotrimeric DmSNAPc recognizes a 21-bp DNA sequence, the proximal sequence element A (PSEA), located approximately 40 to 60 bp upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II preinitiation complexes on U1 to U5 promoters but RNA polymerase III preinitiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc-PSEA complexes, we have developed a novel protocol that combines site-specific protein-DNA photo-cross-linking with site-specific chemical cleavage of the protein. This protocol has allowed us to map regions within each of the three DmSNAPc subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs.

Crystal structure of Rattus norvegicus Visfatin/PBEF/Nampt in complex with an FK866-based inhibitor.

Visfatin (Nampt/PBEF) plays a pivotal role in the salvage pathway for NAD(+) biosynthesis. Its potent inhibitor, FK866, causes cellular NAD(+) levels to decline, thereby inducing apoptosis in tumor cells. In an effort to improve the solubility and binding interactions of FK866, we designed and synthesized IS001, in which a ribose group is attached to the FK866 pyridyl ring. Here, we report the crystal structure of rat visfatin in complex with IS001. Like FK866, IS001 is positioned at the dimer interface, and all of the residues that interact with IS001 are involved in hydrophobic or pi-pi-stacking interactions. However, we were unable to detect any strong interactions between the added ribose ring of IS001 and visfatin, which implies that a bulkier modifying group is necessary for a tight interaction. This study provides additional structure-based information needed to optimize the design of visfatin inhibitors.

Overexpression and purification of the RyR1 pore-forming region.

Ryanodine receptor 1 (RyR1) is a large homotetrameric calcium channel that plays a pivotal role in skeletal muscle contraction. Sequence comparison and mutagenesis studies indicate that the pore architecture of RyR1, including the last two transmembrane helices and the luminal loop linking them, is similar to that of the bacterial KcsA K(+) channel. Here, we describe the overexpression and purification of the C-terminal polyhistidine-tagged RyR1 pore-forming region. The nonionic detergent lauryldimethylamine oxide (LDAO) was selected for solubilization of the protein based on its ability to extract the protein from the membrane and to maintain it in a monodisperse state. The protein was then purified using nickel-affinity chromatography and gel filtration. Gel filtration analysis confirmed that the RyR1 fragment containing the pore-forming region (amino acids 4829-5037) is sufficient to form a tetramer.

Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation.

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs that brings the N- and C-domains of PGK closer together. Here we present the 1.8A crystal structure of unliganded PGK from Thermus caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation.

Crystal structure of visfatin/pre-B cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anti-cancer agent FK-866.

Visfatin/pre-B cell colony-enhancing factor 1 (PBEF)/nicotinamide phosphoribosyltransferase (NAmPRTase) is a multifunctional protein having phosphoribosyltransferase, cytokine and adipokine activities. Originally isolated as a cytokine promoting the differentiation of B cell precursors, it was recently suggested to act as an insulin analog via the insulin receptor. Here, we describe the first crystal structure of visfatin in three different forms: apo and in complex with either nicotinamide mononucleotide (NMN) or the NAmPRTase inhibitor FK-866 which was developed as an anti-cancer agent, interferes with NAD biosynthesis, showing a particularly high specificity for NAmPRTase. The crystal structures of the complexes with either NMN or FK-866 show that the enzymatic active site of visfatin is optimized for nicotinamide binding and that the nicotinamide-binding site is important for inhibition by FK-866. Interestingly, visfatin mimics insulin signaling by binding to the insulin receptor with an affinity similar to that of insulin and does not share the binding site with insulin on the insulin receptor. To predict binding sites, the potential interaction patches of visfatin and the L1-CR-L2 domain of insulin receptor were generated and analyzed. Although the relationship between the insulin-mimetic property and the enzymatic function of visfatin has not been clearly established, our structures raise the intriguing possibility that the glucose metabolism and the NAD biosynthesis are linked by visfatin.

Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus.

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.