scholarly journals Structure of the Prx6-subfamily 1-Cys peroxiredoxin from Sulfolobus islandicus

2019 ◽  
Vol 75 (6) ◽  
pp. 428-434 ◽  
Author(s):  
Sander Stroobants ◽  
Inge Van Molle ◽  
Queen Saidi ◽  
Karl Jonckheere ◽  
Dominique Maes ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Quaternary Structure ◽  
Size Exclusion ◽  
Molecular Replacement ◽  
Oligomeric State ◽  
Data Set ◽  
Cell Parameters ◽  
Aeropyrum Pernix ◽  
Sulfolobus Islandicus ◽  
Exclusion Chromatography

Aerobic thermoacidophilic archaea belonging to the genus Sulfolobus harbor peroxiredoxins, thiol-dependent peroxidases that assist in protecting the cells from oxidative damage. Here, the crystal structure of the 1-Cys peroxiredoxin from Sulfolobus islandicus, named 1-Cys SiPrx, is presented. A 2.75 Å resolution data set was collected from a crystal belonging to space group P212121, with unit-cell parameters a = 86.8, b = 159.1, c = 189.3 Å, α = β = γ = 90°. The structure was solved by molecular replacement using the homologous Aeropyrum pernix peroxiredoxin (ApPrx) structure as a search model. In the crystal structure, 1-Cys SiPrx assembles into a ring-shaped decamer composed of five homodimers. This quaternary structure corresponds to the oligomeric state of the protein in solution, as observed by size-exclusion chromatography. 1-Cys SiPrx harbors only a single cysteine, which is the peroxidatic cysteine, and lacks both of the cysteines that are highly conserved in the C-terminal arm domain in other archaeal Prx6-subfamily proteins such as ApPrx and that are involved in the association of dimers into higher-molecular-weight decamers and dodecamers. It is thus concluded that the Sulfolobus Prx6-subfamily protein undergoes decamerization independently of arm-domain cysteines.

scholarly journals Crystallization and preliminary crystallographic analysis of poly(3-hydroxybutyrate) depolymerase fromBacillus thuringiensis

2014 ◽  
Vol 70 (10) ◽  
pp. 1421-1423 ◽  
Author(s):  
Yung-Lin Wang ◽  
Yi-Ting Lin ◽  
Chia-Lin Chen ◽  
Gwo-Chyuan Shaw ◽  
Shwu-Huey Liaw
Keyword(s):  
Crystal Structure ◽  
Crystallographic Analysis ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Data Set ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Potential Applications ◽  
Polymer Biodegradation ◽  
Key Enzyme

Poly[(R)-3-hydroxybutyrate] (PHB) is a microbial biopolymer that has been commercialized as biodegradable plastics. The key enzyme for the degradation is PHB depolymerase (PhaZ). A new intracellular PhaZ fromBacillus thuringiensis(BtPhaZ) has been screened for potential applications in polymer biodegradation. Recombinant BtPhaZ was crystallized using 25% polyethylene glycol 3350, 0.2 Mammonium acetate, 0.1 Mbis-tris pH 6.5 at 288 K. The crystals belonged to space groupP1, with unit-cell parametersa= 42.97,b= 83.23,c= 85.50 Å, α = 73.45, β = 82.83, γ = 83.49°. An X-ray diffraction data set was collected to 1.42 Å resolution with anRmergeof 6.4%. Unexpectedly, a molecular-replacement solution was obtained using the crystal structure ofStreptomyces lividanschloroperoxidase as a template, which shares 24% sequence identity to BtPhaZ. This is the first crystal structure of an intracellular poly(3-hydroxybutyrate) depolymerase.

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 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.

scholarly journals The Oligomeric State of the Plasma Membrane H+-ATPase from Kluyveromyces lactis

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 958 ◽  
Author(s):  
Yadira Ruiz-Granados ◽  
Valentín De La Cruz-Torres ◽  
José Sampedro
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Quaternary Structure ◽  
Kluyveromyces Lactis ◽  
Size Exclusion ◽  
Oligomeric State ◽  
Aggregation State ◽  
Activated State ◽  
Exclusion Chromatography ◽  
Native Electrophoresis

The plasma membrane H+-ATPase was purified from the yeast K. lactis. The oligomeric state of the H+-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H+-ATPase hexamer in both MASs as the sole/main oligomeric state—in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H+-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H+-ATPase. It is concluded that the quaternary structure of the H+-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

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.

scholarly journals The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent-solubilized state

2006 ◽  
Vol 395 (2) ◽  
pp. 345-353 ◽  
Author(s):  
Stéphanie Ravaud ◽  
Marie-Ange Do Cao ◽  
Marie Jidenko ◽  
Christine Ebel ◽  
Marc Le Maire ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Size Exclusion ◽  
Supramolecular Organization ◽  
Oligomeric State ◽  
Atp Binding Cassette Transporter ◽  
Bound Lipids ◽  
Exclusion Chromatography ◽  
Stokes Radius

BmrA from Bacillus subtilis is a half-size ABC (ATP-binding cassette) transporter involved in multidrug resistance. Although its supramolecular organization has been investigated after reconstitution in a lipid bilayer environment, and shows a dimeric and possibly a tetrameric form, the precise quaternary structure in a detergent-solubilized state has never been addressed. In the present study, BmrA was purified from Escherichia coli membranes using an optimized purification protocol and different detergents. Furthermore, the ATPase activity of BmrA and the quantity of bound lipids and detergent were determined, and the oligomeric state was analysed using SEC (size-exclusion chromatography) and analytical ultracentrifugation. The activity and the quaternary structure of BmrA appeared to be strongly influenced by the type and concentration of the detergent used. SEC data showed that BmrA could be purified in a functional form in 0.05 and 0.01% DDM (n-dodecyl-β-D-maltoside) and was homogeneous and monodisperse with an Rs (Stokes radius) of 5.6 nm that is compatible with a dimer structure. Sedimentation-velocity and equilibrium experiments unequivocally supported that BmrA purified in DDM is a dimer and excluded the presence of other oligomeric states. These observations, which are discussed in relation to results obtained in proteoliposomes, also constitute an important first step towards crystallographic studies of BmrA structure.

scholarly journals Crystallization and preliminary X-ray characterization of the full-length bacteriophytochrome from the plant pathogenXanthomonas campestrispv.campestris

2014 ◽  
Vol 70 (12) ◽  
pp. 1636-1639 ◽  
Author(s):  
Sebastián Klinke ◽  
Lisandro H. Otero ◽  
Jimena Rinaldi ◽  
Santiago Sosa ◽  
Beatriz G. Guimarães ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Data Bank ◽  
Full Length ◽  
Size Exclusion ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Exclusion Chromatography ◽  
Output Module

Phytochromes give rise to the largest photosensor family known to date. However, they are underrepresented in the Protein Data Bank. Plant, cyanobacterial, fungal and bacterial phytochromes share a canonical architecture consisting of an N-terminal photosensory module (PAS2–GAF–PHY domains) and a C-terminal variable output module. The bacteriumXanthomonas campestrispv.campestris, a worldwide agricultural pathogen, codes for a single bacteriophytochrome (XccBphP) that has this canonical architecture, bearing a C-terminal PAS9 domain as the output module. Full-length XccBphP was cloned, expressed and purified to homogeneity by nickel–NTA affinity and size-exclusion chromatography and was then crystallized at room temperature bound to its cofactor biliverdin. A complete native X-ray diffraction data set was collected to a maximum resolution of 3.25 Å. The crystals belonged to space groupP43212, with unit-cell parametersa=b= 103.94,c= 344.57 Å and a dimer in the asymmetric unit. Refinement is underway after solving the structure by molecular replacement.

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°.

scholarly journals Structural characterization of a novel monotreme-specific protein with antimicrobial activity from the milk of the platypus

2018 ◽  
Vol 74 (1) ◽  
pp. 39-45 ◽  
Author(s):  
Janet Newman ◽  
Julie A. Sharp ◽  
Ashwantha Kumar Enjapoori ◽  
John Bentley ◽  
Kevin R. Nicholas ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Antimicrobial Activity ◽  
Mammalian Cells ◽  
Unit Cell ◽  
Protein Crystal ◽  
Unit Cell Parameters ◽  
Flag Epitope ◽  
Data Set ◽  
Space Group ◽  
Cell Parameters

Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus (Ornithorhynchus anatinus) MLP was determined at 1.82 Å resolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being observed (P1,C2 andP21). A 1.82 Å resolution native data set was collected from a crystal in space groupP1, with unit-cell parametersa= 51.2,b= 59.7,c= 63.1 Å, α = 80.15, β = 82.98, γ = 89.27°. The structure was solved by SAD phasing using a protein crystal derivatized with mercury in space groupC2, with unit-cell parametersa= 92.7,b = 73.2,c= 56.5 Å, β = 90.28°. MLP comprises a monomer of 12 helices and two short β-strands, with much of the N-terminus composed of loop regions. The crystal structure of MLP reveals no three-dimensional similarity to any known structures and reveals a heretofore unseen fold, supporting the idea that monotremes may be a rich source for the identification of novel proteins. It is hypothesized that MLP in monotreme milk has evolved to specifically support the unusual lactation strategy of this lineage and may have played a central role in the evolution of these mammals.

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