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author:("auvinen, jha")
1.  Crystallization and preliminary X-ray analysis of Aspergillus oryzae catechol oxidase 
Catechol oxidase from A. oryzae was crystallized by the hanging-drop vapour-diffusion method.
Catechol oxidase is an enzyme that catalyzes the oxidation of o-diphenols to the corresponding o-quinones. It is a copper-containing enzyme with a binuclear copper active site. Here, the crystallization and multiple-wavelength anomalous dispersion data collection of catechol oxidase from the mould fungus Aspergillus oryzae are described. During the purification, three forms of the enzyme (39.3, 40.5 and 44.3 kDa) were obtained. A mixture of these three forms was initially crystallized and gave crystals that diffracted to 2.5 Å resolution and belonged to space group P3221, with unit-cell parameters a = b = 118.9, c = 84.5 Å, α = β = 90, γ = 120°. A preparation containing only the shorter form (39.3 kDa) produced crystals that diffracted to 2.9 Å resolution and belonged to space group P212121, with unit-cell parameters a = 51.8, b = 95.3, c = 139.5 Å, α = β = γ = 90°.
doi:10.1107/S1744309111010141
PMCID: PMC3107139  PMID: 21636908
catechol oxidases; Aspergillus oryzae; copper oxidases
2.  Crystallization and preliminary diffraction analysis of a β-galactosidase from Trichoderma reesei  
The crystallization and preliminary diffraction analysis of a β-galactosidase from T. reesei is described. The crystals diffracted to 1.2 Å resolution.
An extracellular β-galactosidase from Trichoderma reesei was crystallized from sodium cacodylate buffer using polyethylene glycol (PEG) as a precipant. Crystals grown by homogenous streak-seeding belonged to space group P1, with unit-cell parameters a = 67.3, b = 69.1, c = 81.5 Å, α = 109.1, β = 97.3, γ = 114.5°. The crystals diffracted to 1.8 Å resolution using a rotating-anode generator and to 1.2 Å resolution using a synchrotron source. On the basis of the Matthews coefficient (V M = 3.16 Å3 Da−1), one molecule is estimated to be present in the asymmetric unit. The aim of the determination of the crystal structure is to increase the understanding of this industrially significant enzyme.
doi:10.1107/S1744309109023926
PMCID: PMC2720328  PMID: 19652334
β-galactosidases; lactases; Trichoderma reesei
3.  Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris  
The crystallization and preliminary X-ray diffraction analysis of sarcosine dimethylglycine methyltransferase from H. halochoris is reported.
Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-­adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 Å resolution and the orthorhombic crystal diffracted to 1.8 Å resolution.
doi:10.1107/S1744309109026232
PMCID: PMC2720339  PMID: 19652345
sarcosine dimethylglycine methyltransferase; Halorhodospira halochoris; twinning
4.  Transient Dimers of Allergens 
PLoS ONE  2010;5(2):e9037.
Background
Allergen-mediated cross-linking of IgE antibodies bound to the FcεRI receptors on the mast cell surface is the key feature of the type I allergy. If an allergen is a homodimer, its allergenicity is enhanced because it would only need one type of antibody, instead of two, for cross-linking.
Methodology/Principal Findings
An analysis of 55 crystal structures of allergens showed that 80% of them exist in symmetric dimers or oligomers in crystals. The majority are transient dimers that are formed at high protein concentrations that are reached in cells by colocalization. Native mass spectrometric analysis showed that native allergens do indeed form transient dimers in solution, while hypoallergenic variants of them exist almost solely in the monomeric form. We created a monomeric Bos d 5 allergen and show that it has a reduced capability to induce histamine release.
Conclusions/Significance
The results suggest that dimerization would be a very common and essential feature for allergens. Thus, the preparation of purely monomeric variants of allergens could open up novel possibilities for specific immunotherapy.
doi:10.1371/journal.pone.0009037
PMCID: PMC2816702  PMID: 20140203
5.  Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen 
The high-resolution mass-spectrometric characterization, crystallization and X-ray diffraction studies of a recombinant IgE Fab fragment in complex with bovine β-lactoglobulin are reported.
A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT–ICR mass spectrometry and crystallized with bovine β-lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 Å resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 Å. The three-dimensional structure of the D1 Fab fragment–BLG complex will provide the first insight into IgE antibody–allergen interactions at the molecular level.
doi:10.1107/S174430910706160X
PMCID: PMC2373997  PMID: 18097096
antibodies; IgE; food allergens; mass spectrometry
6.  Preliminary X-ray analysis of cellobiohydrolase Cel7B from Melanocarpus albomyces  
The crystallization and preliminary X-ray diffraction analysis of cellobiohydrolase from M. albomyces is reported.
Cellobiohydrolases are enzymes that cleave off cellobiose units from cellulose chains in a processive manner. Melanocarpus albomyces Cel7B is a thermostable single-module cellobiohydrolase that has relatively low activity on small soluble substrates at room temperature. It belongs to glycoside hydrolase family 7, which includes endo-β-1,4-glucanases and cellobiohydrolases. Cel7B was crystallized using the hanging-drop vapour-diffusion method and streak-seeding. The crystals belonged to space group P21, with unit-cell parameters a = 50.9, b = 94.5, c = 189.8 Å, β = 90.0° and four monomers in the asymmetric unit. Analysis of the intensity statistics showed that the crystals were pseudo-merohedrally twinned, with a twinning fraction of 0.37. X-ray diffraction data were collected at 1.6 Å resolution using synchrotron radiation.
doi:10.1107/S1744309107034458
PMCID: PMC2376320  PMID: 17768346
cellulase; cellobiohydrolase; twinning
7.  The Contribution of Polystyrene Nanospheres towards the Crystallization of Proteins 
PLoS ONE  2009;4(1):e4198.
Background
Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins.
Methodology
The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT), and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times.
Conclusions
All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.
doi:10.1371/journal.pone.0004198
PMCID: PMC2615210  PMID: 19145241
8.  Characterization of the 3′ exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D 
Nucleic Acids Research  2004;32(8):2430-2440.
The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.
doi:10.1093/nar/gkh558
PMCID: PMC419447  PMID: 15121900

Results 1-8 (8)