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Martina Huber

Publications and source records attributed to Martina Huber.

14 recordsLinked to original sources
PubMed2006

Spin-density distribution in the copper site of azurin.

A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand. Approximately 45 % of the spin density in the paramagnetic site can be attributed to copper and 30 % to sulfur.

Anisotropy↗
PubMed2006

High-field (275 GHz) spin-label EPR for high-resolution polarity determination in proteins.

The polarity of protein surfaces is one of the factors driving protein-protein interactions. High-field, spin-label EPR at 95 GHz, i.e., 10 times higher than conventional EPR, is an upcoming technique to determine polarity parameters of the inside of proteins. Here we show that by 275 GHz EPR even the small polarity differences of sites at the protein surface can be discriminated. To do so, four single cysteine mutations were introduced at surface sites (positions 12, 27, 42, and 118) of azurin and spin labeled. By 275 GHz EPR in frozen solution, polarity/proticity differences between all four sites can be resolved, which is impossible by 95 GHz EPR. In addition, by 275 GHz EPR, two spectral components are observed for all mutants. The difference between them corresponds to one additional hydrogen bond.

Azurin↗
PubMed2006

Distance between a native cofactor and a spin label in the reaction centre of Rhodobacter sphaeroides by a two-frequency pulsed electron paramagnetic resonance method and molecular dynamics simulations.

The distance between the paramagnetic state of a native cofactor and a spin label is measured in the photosynthetic reaction centre from the bacterium Rhodobacter sphaeroides R26. A two-frequency pulsed electron paramagnetic resonance method [double-electron-electron spin resonance (DEER)] is used. A distance of 3.05 nm between the semiquinone anion state of the primary acceptor (Q(A)) and the spin label at the native cysteine at position 156 in the H-subunit is found. Molecular-dynamics (MD) simulations are performed to interpret the distance. A 6 ns run comprising the entire RC protein yields a distance distribution that is close to the experimental one. The average distance found by the MD simulation is smaller than the distance obtained by DEER by at least 0.2 nm. To better represent the experiments performed at low temperature (60K), a MD method to mimic the freezing-in of the room-temperature conformations is introduced. Both MD methods yield similar distances, but the second method has a trend towards a wider distance distribution.

Anions↗
PubMed2006

Identifying protein construct variants with increased crystallization propensity--a case study.

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.

Cloning, Molecular↗
PubMed2005

The ubiquitin-specific protease USP10 modulates androgen receptor function.

The role of the ubiquitin/proteasome system in degrading nuclear hormone receptors and regulating their transcriptional function has emerged in the last few years. We identified the ubiquitin-specific protease USP10 as part of DNA-bound androgen receptor (AR) complexes purified from nuclear extracts of PC-3 cells stably expressing the AR. The interaction between USP10 and the AR was confirmed by GST pull-down assays. Fluorescence microscopy documented that USP10 was localised in the nucleus and the cytoplasm. Cell-based transactivation assays in PC-3/AR cells revealed that overexpression of wild-type USP10, but not of an enzymatically inactive form, stimulated AR activity mediated by reporter constructs harbouring selective androgen response elements (AREs), non-selective steroid response elements (SREs) or the mouse mammary tumour virus (MMTV) promoter. Conversely, USP10 expression knock-down by siRNAs impaired the MMTV response to androgen. In summary, the data indicate that USP10 is a new cofactor that binds to the AR and stimulates the androgen response of target promoters. This finding underlines the role of the ubiquitin/proteasome system in modulating the AR function.

Androgens↗
PubMed2005

The substrate-bound type 2 copper site of nitrite reductase: the nitrogen hyperfine coupling of nitrite revealed by pulsed EPR.

A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replaced by alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. This mutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site of H145A and of its nitrite-bound form H145A (NO2(-)). The experiments yielded hyperfine and quadrupole parameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein and revealed the changes of these values induced by substrate binding (14NO2(-) and 15NO2(-)). The HYSCORE experiments displayed a signal of 15NO2(-) bound to H145A, from which hyperfine parameters of the nitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen (14N) suggests that the substrate binds in an axial position to the copper in the type 2 site and that the molecular orbital containing the unpaired electron extends onto the substrate. This and other changes in the EPR parameters occurring after nitrite binding suggest a change in electronic structure of the site, which most likely prepares the site for the catalytic reaction. We propose that this change is essential for the reaction to occur.

Alcaligenes faecalis↗
PubMed2005

Distances between the b-subunits in the tether domain of F(0)F(1)-ATP synthase from E. coli.

The arrangement of the b-subunits in the holo-enzyme F(0)F(1)-ATP synthase from E. coli is investigated by site-directed mutagenesis spin-label EPR. F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The hydrophilic F(1)-part and the hydrophobic membrane-integrated F(0)-part are connected by a central and a peripheral stalk. The peripheral stalk consists of two b-subunits. Cysteine mutations are introduced in the tether domain of the b-subunit at b-40, b-51, b-53, b-62 or b-64 and labeled with a nitroxide spin label. Conventional (9 GHz), high-field (95 GHz) and pulsed EPR spectroscopy reveal: All residues are in a relatively polar environment, with mobilities consistent with helix sites. The distance between the spin labels at each b-subunit is 2.9 nm in each mutant, revealing a parallel arrangement of the two helices. They can be in-register but separated by a large distance (1.9 nm), or at close contact and displaced along the helix axes by maximally 2.7 nm, which excludes an in-register coiled-coil model suggested previously for the b-subunit. Binding of the non-hydrolysable nucleotide AMPPNP to the spin-labeled enzyme had no significant influence on the distances compared to that in the absence of nucleotides.

Electron Spin Resonance Spectroscopy↗
PubMed2003

Kinetic stability of the peroxidase activity of unfolded cytochrome c: heme degradation and catalyst inactivation by hydrogen peroxide.

Unfolding converts Paracoccus versutus cytochrome c-550 into a potent peroxidase (Diederix, R. E. M.; Ubbink, M.; Canters, G. W. ChemBioChem 2002, 3, 110-112). The catalytic activity is accompanied by peroxide-driven inactivation that is prevented, in part, by reducing substrate. Here, the kinetics of inactivation are described, and evidence is presented for the occurrence of a labile intermediate on the catalytic peroxidase pathway of unfolded cytochrome c-550. This intermediate represents a branching point, whereby the protein proceeds along either the productive pathway or self-inactivates. Reducing substrate suppresses inactivation by decreasing the steady-state concentration of the labile intermediate. Inactivation is accompanied by heme degradation. Its chemical reactivity, UV-vis, and EPR properties identify the first intermediate as hydroxyheme-cytochrome c-550, i.e. with heme hydroxylated at one of the heme meso positions. The occurrence of this species argues for the peroxo-iron species in the peroxidase mechanism as the labile intermediate leading to inactivated cytochrome c-550.

Catalysis↗
PubMed2003

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r.

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.

Breast Neoplasms↗
PubMed2003

Reconstitution of the type-1 active site of the H145G/A variants of nitrite reductase by ligand insertion.

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.

Alcaligenes↗
PubMed2003

Single-crystal EPR study at 95 GHz of the type 2 copper site of the inhibitor-bound quercetin 2,3-dioxygenase.

An electron-spin-echo-detected, electron-paramagnetic-resonance study has been performed on the type 2 copper site of quercetin 2,3-dioxygenase from Aspergillus japonicus. In the protein, copper is coordinated by three histidine nitrogens and two sulfurs from the inhibitor diethyldithiocarbamate. A single crystal of the protein was studied at 95 GHz and the complete g-tensor determined. The electron-paramagnetic-resonance data are compatible with two orientations of the principal g-axes in the copper center, one of which is preferred on the basis of an analysis of the copper coordination and the d-orbitals that are involved in the unpaired-electron orbital. For this orientation, the principal z-axis of the g-tensor makes an angle of 19 degrees with the Cu-N(His112) bond and the N of His112 may be considered the axial ligand. The singly occupied molecular orbital contains a linear combination of copper dxy and dyz-orbitals, which are antibonding with atomic orbitals of histidine nitrogens and diethyldithiocarbamate sulfurs. The orientation of the g-tensor for the quercetin 2,3-dioxygenase is compared with that for type 1 copper sites.

Aspergillus↗
PubMed2002

EPR characterization of the mononuclear Cu-containing Aspergillus japonicus quercetin 2,3-dioxygenase reveals dramatic changes upon anaerobic binding of substrates.

Quercetin 2,3-dioxygenase (2,3QD) is a copper-containing dioxygenase that catalyses the oxidation of the flavonol quercetin to 2-protocatechuoylphloroglucinol carboxylic acid with concomitant production of carbon monoxide. In contrast to iron dioxygenases, very little is known about copper dioxygenases. We have characterized 2,3QD from the fungus Aspergillus japonicus by electron paramagnetic resonance spectroscopy (EPR). At pH 6.0, 2,3QD shows a mixture of two EPR species. The major form has parameters typical of type 2 Cu sites (g// = 2.330, A// = 13.7 mT), the minor one has a more distorted geometry (g// = 2.290, A// = 12.5 mT). Anaerobic addition of the substrate quercetin results in a different, single species EPR spectrum with g// = 2.336, A// = 11.4 mT, parameters, which are in-between those of the type 2 and type 1 Cu sites in the Peisach-Blumberg (g// vs. A//) plot. After turnover, a new EPR signal is observed, which is ascribed to the carboxylic acid ester product complex. This spectrum is similar to that of the native enzyme at pH 10.0 and has g-tensor parameters suggesting a trigonal bipyramidal site. Of a variety of flavonoids studied, only flavonols are able to bind to the copper centre of 2,3QD. Nine flavonols with different hydroxylation patterns at the A- and B-ring have been analysed. They cluster in two different regions of the Peisach-Blumberg plot and show that the presence of a 5-OH group has a large effect on the A// parameter. Several differences are noted between A. japonicus 2,3QD and the enzyme from A. niger German Collection of Microorganisms 821.

Aspergillus↗