Structural and biochemical investigation into stable FGF2 mutants with novel mutation sites and hydrophobic replacements for surface-exposed cysteines.

Fibroblast growth factor 2 (FGF2) is an attractive biomaterial for pharmaceuticals and functional cosmetics. To improve the thermo-stability of FGF2, we designed two mutants harboring four-point mutations: FGF2-M1 (D28E/C78L/C96I/S137P) and FGF2-M2 (D28E/C78I/C96I/S137P) through bioinformatics, molecular thermodynamics, and molecular modeling. The D28E mutation reduced fragmentation of the FGF2 wild type during preparation, and the substitution of a whale-specific amino acid, S137P, enhanced the thermal stability of FGF2.

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).

Expression and purification of intracrine human FGF 11 and study of its FGFR-dependent biological activity.

Fibroblast growth factor 11 (FGF11) is one of intracrine FGFs (iFGFs), which function within cells. Unlike canonical FGFs, FGF11 remains intracellularly and plays biological roles in FGF receptor (FGFR)-independent manner. Here, we established an expression system of recombinant FGF11 proteins in E.

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.

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.

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.

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

ACC-1 is a plasmid-encoded class C β-lactamase identified in clinical isolates of , , , and 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.

FGF11 influences 3T3-L1 preadipocyte differentiation by modulating the expression of PPARγ regulators.

Fibroblast growth factor 11 (FGF11) is a member of the intracellular fibroblast growth factor superfamily. Here, we identified FGF11 as a novel mediator of adipogenesis. During 3T3-L1 adipocyte differentiation, the expression of FGF11 decreased at the mitotic clonal expansion stage and increased at the terminal differentiation stage.

Structural and mechanistic characterization of an archaeal-like chaperonin from a thermophilic bacterium.

The chaperonins (CPNs) are megadalton sized hollow complexes with two cavities that open and close to encapsulate non-native proteins. CPNs are assigned to two sequence-related groups that have distinct allosteric mechanisms. In Group I CPNs a detachable co-chaperone, GroES, closes the chambers whereas in Group II a built-in lid closes the chambers.

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

Nucleotides were effective in inhibiting the class C β-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a value of 16.2 μM.

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/β 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.

Purification, crystallization, and preliminary X-ray crystallographic analysis of the Group III chaperonin from Carboxydothermus hydrogenoformans.

Chaperonins (CPNs) are megadalton sized ATP-dependent nanomachines that facilitate protein folding through complex cycles of complex allosteric articulation. They consist of two back-to-back stacked multisubunit rings. CPNs are usually classified into Group I and Group II.

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

Endo-β-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold-adapted β-mannanase that has the lowest optimum temperature (30°C) of all known β-mannanases. Here, we report the apo- and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other β-mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site.

Purification, crystallization and preliminary X-ray crystallographic analysis of the UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) from Acinetobacter baumannii.

The emergence and global spread of multidrug-resistant Acinetobacter baumannii strains are major threats to public health. Inhibition of peptidoglycan biosynthesis is an effective strategy for the development of antibiotics. The ATP-dependent UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) that is responsible for the last step of peptidoglycan biosynthesis is a validated target for the development of antibiotics.

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 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.

Expression at 279 K, purification, crystallization and preliminary X-ray crystallographic analysis of a novel cold-active β-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 β-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.

Structural basis for the β-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 β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-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 β-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 β-lactam antibiotics.

In-house zinc SAD phasing at Cu Kα edge.

De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source.

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.

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.