Biomedical subjects

Ingrid Zegers

Publications and source records attributed to Ingrid Zegers.

8 recordsLinked to original sources

PubMed2006

Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study.

A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 A, and a data set was collected to 2.76 A.

Acetyltransferases↗

PubMed2006

Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase.

The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 A. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-L-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer.

Bacillus subtilis↗

PubMed2005

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of Ss-LrpB, a transcription regulator from Sulfolobus solfataricus.

Ss-LrpB from Sulfolobus solfataricus P2 belongs to the bacterial/archaeal superfamily of Lrp-like (leucine-responsive regulatory protein-like) transcription regulators. The N-terminal DNA-binding domain contains a HTH motif and the C-terminal domain contains an alphabeta-sandwich (betaalphabetabetaalphabeta fold). The C-terminal domain was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 59.35, b = 74.86, c = 38.08 A and a data set was collected to 2.0 A resolution. Structure determination using a selenomethionine derivative is under way.

Amino Acid Motifs↗

PubMed2005

Protein crystallization in hydrogel beads.

The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4 kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0 kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein-agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead.

Alginates↗

PubMed2003

Purification of an oxidation-sensitive enzyme, pI258 arsenate reductase from Staphylococcus aureus.

Arsenate reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (K(a)=1.1 mM) and potassium was stabilizing ArsC (DeltaT(m)=+6.2 degrees C). Also due to the use of these salt ions, the final yield of the purification had improved with a factor of four, i.e. 73 mg/l culture.

Arsenite Transporting ATPases↗

PubMed2003

Cloning, preliminary characterization and crystallization of nucleoside hydrolases from Caenorhabditis elegans and Campylobacter jejuni.

The nucleoside hydrolases (NHs) are a family of nucleoside-modifying enzymes. They play an important role in the purine-salvage pathway of many pathogenic organisms which are unable to synthesize purines de novo. Although well characterized in protozoan parasites, their precise function and mechanism remain unclear in other species. For the first time, NHs from Caenorhabditis elegans and Campylobacter jejuni, which are representatives of mesozoa and bacteria, respectively, have been cloned and purified. Steady-state kinetics indicate a different substrate-specificity profile to previously described hydrolases. Native diffraction data sets were collected from crystals of NH from each organism. The hexagonal crystals (space group P6(2)22 or P6(4)22) of NH from C. elegans diffracted to a resolution of 2.8 A, while the data set from the orthorhombic crystals (space group I222 or I2(1)2(1)2(1)) of NH from C. jejuni could be processed to 1.7 A resolution. The unit-cell parameters were a = b = 102.23, c = 117.27 A in the former case and a = 101.13, b = 100.13, c = 81.37 A in the latter.

Animals↗

PubMed2003

Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.

Arsenite Transporting ATPases↗

PubMed2002

All intermediates of the arsenate reductase mechanism, including an intramolecular dynamic disulfide cascade.

The mechanism of pI258 arsenate reductase (ArsC) catalyzed arsenate reduction, involving its P-loop structural motif and three redox active cysteines, has been unraveled. All essential intermediates are visualized with x-ray crystallography, and NMR is used to map dynamic regions in a key disulfide intermediate. Steady-state kinetics of ArsC mutants gives a view of the crucial residues for catalysis. ArsC combines a phosphatase-like nucleophilic displacement reaction with a unique intramolecular disulfide bond cascade. Within this cascade, the formation of a disulfide bond triggers a reversible "conformational switch" that transfers the oxidative equivalents to the surface of the protein, while releasing the reduced substrate.

Arsenates↗