Cavity-dumped mode-locked Alexandrite laser oscillator with 100 mJ pulses stabilized by using a double trigger system.

We report a mode-locked Alexandrite single pulse laser with cavity dumping. Mode locking was achieved by using an AOM and an EOM was used for Q-switching and cavity dumping. The instability of the single pulse laser energy output was reduced down to a tenth of that of the conventional single trigger system by introducing a novel double trigger system. The single pulse laser energy and pulse width were 100 mJ and 475 ps in multiple mode and 12.5 mJ and 275 ps in single mode, obtained without a laser amplifier.

The Analyses of Cetacean Virus-Responsive Genes Reveal Evolutionary Marks in Mucosal Immunity-Associated Genes.

Viruses are the most common and abundant organisms in the marine environment. To better understand how cetaceans have adapted to this virus-rich environment, we compared cetacean virus-responsive genes to those from terrestrial mammals. We identified virus-responsive gene sequences in seven species of cetaceans, which we compared with orthologous sequences in seven terrestrial mammals. As a result of evolution analysis using the branch model and the branch-site model, 21 genes were selected using at least one model. IFN-ε, an antiviral cytokine expressed at mucous membranes, and its receptor IFNAR1 contain cetacean-specific amino acid substitutions that might change the interaction between the two proteins and lead to regulation of the immune system against viruses. Cetacean-specific amino acid substitutions in IL-6, IL-27, and the signal transducer and activator of transcription (STAT)1 are also predicted to alter the mucosal immune response of cetaceans. Since mucosal membranes are the first line of defense against the external environment and are involved in immune tolerance, our analysis of cetacean virus-responsive genes suggests that genes with cetacean-specific mutations in mucosal immunity-related genes play an important role in the protection and/or regulation of immune responses against viruses.

α-Poly-L-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes.

α-Poly-L-lysine (PLL) has been used for various purposes such as cell attachment, immunization, and molecular delivery, and is known to be cytotoxic to several cell lines. Here, we studied the effect of PLL on the adipogenesis of 3T3-L1 cells and investigated the underlying mechanism. Differentiation media containing PLL with a molecular weight (MW) greater than 4 kDa enhanced lipid droplet formation and increased adipogenic marker levels, indicating an increase in adipocyte differentiation. PLL with a molecular weight between 30 and 70 kDa was more effective than PLL of other sizes in 3T3-L1 cell differentiation. Moreover, PLL induced 3T3-L1 adipogenesis in insulin-free adipocyte differentiation medium. Incubation with insulin and PLL exhibited greater adipogenesis than insulin treatment only even at a high concentration. PLL stimulated insulin signaling and augmented the signaling pathway when it was added with insulin. While PLL did not activate the glucocorticoid receptor, which is phosphorylated by dexamethasone (DEX), it showed a positive effect on the cAMP signal pathway when preadipocytes were treated with PLL and 3-isobutyl-1-methylxanthine (IBMX). Consistent with these results, incubation with PLL and DEX without IBMX induced adipocyte differentiation. We also observed that the mitotic clonal expansion phase was the critical stage in adipogenesis for inducing the effects of PLL. These results suggest that PLL functions as an adipogenic inducer in 3T3-L1 preadipocytes and PLL has a direct effect on insulin signaling, one of the main regulatory pathways.

Structural Insights into Catalytic Relevances of Substrate Poses in ACC-1.

ACC-1 is a plasmid-encoded class C ß-lactamase identified in clinical isolates of Klebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, and Escherichia coli ACC-1-producing bacteria are susceptible to cefoxitin, whereas they are resistant to oxyimino cephalosporins. Here, we depict crystal structures of apo ACC-1, adenylylated ACC-1, and acylated ACC-1 complexed with cefotaxime and cefoxitin. ACC-1 has noteworthy structural alterations in the R2 loop, the Ω loop, and the Phe119 loop located along the active-site rim. The adenylate covalently bonded to the nucleophilic serine reveals a tetrahedral phosphorus mimicking the deacylation transition state. Cefotaxime in ACC-1 has a proper conformation for the substrate-assisted catalysis in that its C-4 carboxylate and N-5 nitrogen are adequately located to facilitate the deacylation reaction. In contrast, cefoxitin in ACC-1 has a distinct conformation, in which those functional groups cannot contribute to catalysis. Furthermore, the orientation of the deacylating water relative to the acyl carbonyl group in ACC-1 is unfavorable for nucleophilic attack.

GMP and IMP Are Competitive Inhibitors of CMY-10, an Extended-Spectrum Class C ß-Lactamase.

Nucleotides were effective in inhibiting the class C ß-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a Ki value of 16.2 µM. The crystal structure of CMY-10 complexed with GMP or IMP revealed that nucleotides fit into the R2 subsite of the active site with a unique vertical binding mode where the phosphate group at one terminus is deeply bound in the subsite and the base at the other terminus faces the solvent.

Structural and mechanistic insights into the inhibition of class C ß-lactamases through the adenylylation of the nucleophilic serine.

Objectives: : Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C ß-lactamases. Methods: : The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5'-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates. Results: : The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies ( k inact / K I ) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M -1 s -1 , respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria. Conclusions: : Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C ß-lactamase inhibitors on the basis of the nucleotide scaffold.

Crystal structure of EstSRT1, a family VIII carboxylesterase displaying hydrolytic activity toward oxyimino cephalosporins.

EstSRT1 is a family VIII carboxylesterase that hydrolyzes oxyimino third- and fourth-generation cephalosporins, first-generation cephalosporins and ester substrates. According to the crystal structure of EstSRT1 (2.0-Å resolution), this protein contains a large α/ß domain and a small α-helical domain and harbors three catalytic residues (Ser71, Lys74, and Tyr160) in the cavity at the domain interface, similarly to other family VIII carboxylesterases. Comparison of the structures of EstSRT1 and EstU1, a family VIII carboxylesterase with no hydrolytic activity toward bulky oxyimino cephalosporins, revealed that EstSRT1 has a smaller active site, despite its extended substrate range. The B-factors of the active site segments that could potentially contact with the oxyimino groups and the R2 side chains of oxyimino cephalosporins are higher in EstSRT1 than in EstU1, thus suggesting the role of the active site's structural flexibility in the extension of EstSRT1's substrate spectrum.

Structure-based investigation into the functional roles of the extended loop and substrate-recognition sites in an endo-ß-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus.

Endo-ß-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold-adapted ß-mannanase that has the lowest optimum temperature (30°C) of all known ß-mannanases. Here, we report the apo- and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other ß-mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site. Structural and mutational analyses suggest that this extended loop is linked to the cold-adapted enzymatic activity. From the CaMan-M5 complex structure, we defined the mannose-recognition subsites and observed unreported M5 binding site on the surface of CaMan.

Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber.

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine-lysine catalytic dyad. We report the 2.0-Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three-tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight- and weak-binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP-driven protein unfolding and translocation. The bowl-shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.

ATP-binding mode including a carbamoylated lysine and two Mg(2+) ions, and substrate-binding mode in Acinetobacter baumannii MurF.

MurF adds d-Ala-d-Ala dipeptide to UDP-N-acetylmuramyl-l-Ala-γ-d-Glu-m-DAP (or l-Lys) in an ATP-dependent manner, which is the last step in the biosynthesis of monomeric precursor of peptidoglycan. Here we report crystal structures of two MurF-ATP complexes: the MurF-ATP complex and the MurF-ATP-UDP complex. The ATP-binding mode revealed by the crystal structure of the MurF-ATP complex confirms the previous biochemical demonstration that a carbamoylated lysine and two Mg(2+) ions are required for enzyme activity of MurF. The UDP-MurF interactions observed in the crystal structure of the MurF-ATP-UDP complex depict the characteristic substrate-binding mode of MurF. The emergence and dissemination of multidrug-resistant Acinetobacter baumannii strains are great threats to public health. Therefore, the structural information on A. baumannii MurF as a validated target for drug discovery will provide a framework to develop antibacterial agents against multidrug-resistant A. baumannii infections as well as to understand the reaction mechanism of MurF.

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur.

Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M- and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes (znuA, rpmF2) more sensitively than others (rpmG2, SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5×10(-16) M and 7.9×10(-16) M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.

YajL, prokaryotic homolog of parkinsonism-associated protein DJ-1, functions as a covalent chaperone for thiol proteome.

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.

Structural basis for the ß-lactamase activity of EstU1, a family VIII carboxylesterase.

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used ß-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a ß-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C ß-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of ß-lactam antibiotics. This result explains the weak ß-lactamase activity of EstU1 compared with class C ß-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum.

The crystal structure of a novel phosphopantothenate synthetase from the hyperthermophilic archaea, Thermococcus onnurineus NA1.

Pantothenate is the essential precursor of coenzyme A (CoA), a fundamental cofactor in all aspects of metabolism. In bacteria and eukaryotes, pantothenate synthetase (PS) catalyzes the last step in the pantothenate biosynthetic pathway, and pantothenate kinase (PanK) phosphorylates pantothenate for its entry into the CoA biosynthetic pathway. However, genes encoding PS and PanK have not been identified in archaeal genomes. Recently, a comparative genomic analysis and the identification and characterization of two novel archaea-specific enzymes show that archaeal pantoate kinase (PoK) and phosphopantothenate synthetase (PPS) represent counterparts to the PS/PanK pathway in bacteria and eukaryotes. The TON1374 protein from Thermococcus onnurineus NA1 is a PPS, that shares 54% sequence identity with the first reported archaeal PPS candidate, MM2281, from Methanosarcina mazei and 91% sequence identity with TK1686, the PPS from Thermococcus kodakarensis. Here, we report the apo and ATP-complex structures of TON1374 and discuss the substrate-binding mode and reaction mechanism.

FrsA functions as a cofactor-independent decarboxylase to control metabolic flux.

The interaction between fermentation-respiration switch (FrsA) protein and glucose-specific enzyme IIA(Glc) increases glucose fermentation under oxygen-limited conditions. We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues required for catalysis as well as the structural basis for the activation by d-IIA(Glc).

Expression at 279†K, purification, crystallization and preliminary X-ray crystallographic analysis of a novel cold-active ß-1,4-D-mannanase from the Antarctic springtail Cryptopygus antarcticus.

The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type ß-1,4-D-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris-HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan-mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5.

Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency.

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

Experimental phasing using zinc anomalous scattering.

Zinc is a suitable metal for anomalous dispersion phasing methods in protein crystallography. Structure determination using zinc anomalous scattering has been almost exclusively limited to proteins with intrinsically bound zinc(s). Here, it is reported that multiple zinc ions can easily be charged onto the surface of proteins with no intrinsic zinc-binding site by using zinc-containing solutions. Zn derivatization of protein surfaces appears to be a largely unnoticed but promising method of protein structure determination.

Cultivation at 6-10°C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli.

Protein expression in Escherichia coli at 15-25°C is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6-10°C without cold adapted chaperon systems. By using E. coli Rosetta-gami2(DE3), we obtained 1.8 and 0.9mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1l culture grown at 6 and 10°C, respectively. Cultivation at 10°C also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21(DE3). Consequently, E. coli cultivation at 6-10°C is an effective strategy for overcoming a major hurdle of the inclusion body formation.

Crystallization and preliminary X-ray crystallographic analysis of the putative NADP(H)-dependent oxidoreductase YncB from Vibrio vulnificus.

The yncB gene product from Vibrio vulnificus, which belongs to the medium-chain dehydrogenase/reductase (MDR) superfamily, was crystallized using the microbatch crystallization method at 295 K. Diffraction data sets were collected using synchrotron radiation. Crystals of selenomethionine-substituted YncB protein belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 90.52, b = 91.56, c = 104.79 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 57%. Crystals of the YncB-NADP(H) complex belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 90.14, c = 105.61 Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 56.42%.