scholarly journals X-ray diffraction analysis andin vitrocharacterization of the UAM2 protein fromOryza sativa

2017 ◽  
Vol 73 (4) ◽  
pp. 241-245 ◽  
Author(s):  
Ditte Hededam Welner ◽  
Alex Yi-Lin Tsai ◽  
Andy M. DeGiovanni ◽  
Henrik Vibe Scheller ◽  
Paul D. Adams
Keyword(s):  
Metal Ion ◽  
Limited Proteolysis ◽  
Disulfide Bridge ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
Oligomeric State ◽  
X Ray ◽  
Functional Studies ◽  
Exclusion Chromatography

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein fromOryza sativa(OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

2014 ◽  
Vol 70 (10) ◽  
pp. 1362-1367 ◽  
Author(s):  
Emmanuel Nji ◽  
Dianfan Li ◽  
Declan A. Doyle ◽  
Martin Caffrey
Keyword(s):  
Metal Ion ◽  
Cell Volume Regulation ◽  
Ion Concentration ◽  
Size Exclusion ◽  
Substrate Selectivity ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP fromPseudomonas aeruginosawas recombinantly expressed inEscherichia coliand purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space groupP21, with unit-cell parametersa= 169.53,b= 169.53,c= 290.13 Å, γ = 120°.

Characterization of the sorbitol dehydrogenase SmoS from Sinorhizobium meliloti 1021

2021 ◽  
Vol 77 (3) ◽  
pp. 380-390
Author(s):  
MacLean G. Kohlmeier ◽  
Ben A. Bailey-Elkin ◽  
Brian L. Mark ◽  
Ivan J. Oresnik
Keyword(s):  
Sinorhizobium Meliloti ◽  
Model Organism ◽  
Time Frame ◽  
Industrial Applications ◽  
Size Exclusion ◽  
Ligand Docking ◽  
X Ray Diffraction ◽  
X Ray ◽  
Exclusion Chromatography

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol to fructose and tagatose, respectively, using NAD+ as a cofactor. The main objective of this study was to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon-optimized for heterologous expression in Escherichia coli BL21 (DE3) Gold cells and the protein was subsequently overexpressed and purified. Size-exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetramer. SmoS was crystallized, and crystals obtained in the absence of substrate diffracted to 2.1 Å resolution and those of a complex with sorbitol diffracted to 2.0 Å resolution. SmoS was characterized kinetically and shown to have a preference for sorbitol despite having a higher affinity for galactitol. Computational ligand-docking experiments suggest that tagatose binds the protein in a more energetically favourable complex than fructose, which is retained in the active site over a longer time frame following oxidation and reduces the rate of the reaction. These results supplement the inventory of biomolecules with potential for industrial applications and enhance the understanding of metabolism in the model organism S. meliloti.

scholarly journals Purification, crystallization and preliminary X-ray diffraction studies of UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) fromSynechococcussp. PCC 7942

2014 ◽  
Vol 70 (2) ◽  
pp. 203-205
Author(s):  
Asaithambi Killivalavan ◽  
Ningning Zhuang ◽  
Young Shik Park ◽  
Kon Ho Lee
Keyword(s):  
Size Exclusion ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
Dispersion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Multi Wavelength ◽  
Pcc 7942

A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacteriumSynechococcussp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method. The BGluT protein was purified by nickel-affinity and size-exclusion chromatography. It was then crystallized by the hanging-drop vapour-diffusion method using a well solution consisting of 0.1 Mbis-tris pH 5.5, 19%(w/v) polyethylene glycol 3350 with 4%(w/v) D(+)-galactose as an additive. X-ray diffraction data were collected to 1.99 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 171.35,b= 77.99,c = 53.77 Å, β = 90.27°.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeastSaccharomyces cerevisiae

2015 ◽  
Vol 71 (2) ◽  
pp. 243-246 ◽  
Author(s):  
Srinivasan Rengachari ◽  
Philipp Aschauer ◽  
Christian Sturm ◽  
Monika Oberer
Keyword(s):  
Crystal Form ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Monoglyceride Lipase ◽  
Exclusion Chromatography

The protein Yju3p is the orthologue of monoglyceride lipases in the yeastSaccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space groupP212121), with unit-cell parametersa= 77.2,b= 108.6,c= 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

scholarly journals Overproduction, crystallization and X-ray diffraction data analysis of ectoine synthase from the cold-adapted marine bacteriumSphingopyxis alaskensis

2015 ◽  
Vol 71 (8) ◽  
pp. 1027-1032 ◽  
Author(s):  
Stefanie Kobus ◽  
Nils Widderich ◽  
Astrid Hoeppner ◽  
Erhard Bremer ◽  
Sander H. J. Smits
Keyword(s):  
Ring Closure ◽  
Size Exclusion ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Key Enzyme ◽  
A Cell ◽  
Ectoine Biosynthesis

Ectoine biosynthetic genes (ectABC) are widely distributed in bacteria. Microorganisms that carry them make copious amounts of ectoine as a cell protectant in response to high-osmolarity challenges. Ectoine synthase (EctC; EC 4.2.1.108) is the key enzyme for the production of this compatible solute and mediates the last step of ectoine biosynthesis. It catalyzes the ring closure of the cyclic ectoine molecule. A codon-optimized version ofectCfromSphingopyxis alaskensis(Sa) was used for overproduction ofSaEctC protein carrying aStrep-tag II peptide at its carboxy-terminus. The recombinantSaEctC-Strep-tag II protein was purified to near-homogeneity fromEscherichia colicell extracts by affinity chromatography. Size-exclusion chromatography revealed that it is a dimer in solution. TheSaEctC-Strep-tag II protein was crystallized using the sitting-drop vapour-diffusion method and crystals that diffracted to 1.0 Å resolution were obtained.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of ArsH fromSynechocystissp. strain PCC 6803

2014 ◽  
Vol 70 (4) ◽  
pp. 497-500 ◽  
Author(s):  
Xiao Zhang ◽  
Xi-Mei Xue ◽  
Yu Yan ◽  
Jun Ye
Keyword(s):  
Sinorhizobium Meliloti ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Flavin Mononucleotide ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Pcc 6803

ArsH is an NADPH-dependent flavin mononucleotide reductase and is frequently encoded as part of anarsoperon. The function of thearsHgene remains to be characterized. Crystallization and structural studies may contribute to elucidating the specific biological function of ArsH associated with arsenic resistance. ArsH fromSynechocystissp. strain PCC 6803 was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 1.6 Å. The crystals belonged to the tetragonal space groupI4122, with unit-cell parametersa=b= 127.94,c= 65.86 Å and one molecule in the asymmetric unit. Size-exclusion chromatography and molecular-replacement results showed that the ArsH formed a tetramer. Further structural analysis and comparison with ArsH fromSinorhizobium melilotiwill provide information about the oligomerization of ArsH.

Toscana virusnucleoprotein oligomer organization observed in solution

2017 ◽  
Vol 73 (8) ◽  
pp. 650-659 ◽  
Author(s):  
Amal Baklouti ◽  
Adeline Goulet ◽  
Julie Lichière ◽  
Bruno Canard ◽  
Rémi N. Charrel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Size Exclusion ◽  
Integrative Approach ◽  
X Ray Diffraction ◽  
X Ray ◽  
Transmission Electron ◽  
Closed Ring ◽  
Rnp Complex ◽  
Exclusion Chromatography ◽  
Nucleoprotein N

Toscana virus(TOSV) is an arthropod-borne virus belonging to thePhlebovirusgenus within theBunyaviridaefamily. As in other bunyaviruses, the genome of TOSV is made up of three RNA segments. They are encapsidated by the nucleoprotein (N), which also plays an essential role in virus replication. To date, crystallographic structures of phlebovirus N have systematically revealed closed-ring organizations which do not fully match the filamentous organization of the ribonucleoprotein (RNP) complex observed by electron microscopy. In order to further bridge the gap between crystallographic data on N and observations of the RNP by electron microscopy, the structural organization of recombinant TOSV N was investigated by an integrative approach combining X-ray diffraction crystallography, transmission electron microscopy, small-angle X-ray scattering, size-exclusion chromatography and multi-angle laser light scattering. It was found that in solution TOSV N forms open oligomers consistent with the encapsidation mechanism of phlebovirus RNA.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 fromRalstonia eutrophaH16

2015 ◽  
Vol 71 (6) ◽  
pp. 758-762
Author(s):  
Jieun Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Ralstonia Eutropha ◽  
Size Exclusion ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Fourth Step

The gene product of A1887 fromRalstonia eutropha(ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA.ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purifiedReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, withKm,Vmaxandkcatvalues of 158 µM, 32 mM min−1and 5 × 106 s−1, respectively. TheReH16_A1887 protein was crystallized in 17% PEG 8K, 0.1 MHEPES pH 7.0 at 293 K and a complete data set was collected to 1.4 Å resolution. The crystal belonged to space groupP43212, with unit-cell parametersa=b= 129.52,c= 114.13 Å, α = β = γ = 90°. The asymmetric unit contained two molecules, with a solvent content of 58.9%.

scholarly journals Crystallographic studies of SarV, a global regulator fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1038-1041
Author(s):  
Yang Song ◽  
Fan Zhang ◽  
Xu Li ◽  
Jianye Zang ◽  
Xuan Zhang
Keyword(s):  
Heavy Atom ◽  
Size Exclusion ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
Global Regulator ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Heavy Atom Method

SarV, a member of the SarA protein family, is a global transcriptional regulator which has been reported to be involved in the regulation of autolysis inStaphylococcus aureus. In this study, SarV fromS. aureuswas successfully cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystals of SarV belonged to the monoclinic space groupP21, with unit-cell parametersa= 36.40,b= 119.64,c= 66.80 Å, α = γ = 90, β = 98.75°. The Matthews coefficient and the solvent content were estimated to be 2.57 Å3 Da−1and 52%, respectively, suggesting the presence of four molecules in the asymmetric unit. The results of size-exclusion chromatography (SEC) indicated thatS. aureusSarV exists as a homodimer in solution. Unfortunately, the structure cannot be solved by molecular replacement because of the low sequence identity ofS. aureusSarV to known structures. Further phase determination by selenomethionine single-wavelength anomalous dispersion (SAD) and the heavy-atom method is in progress.

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