Molecular interactions: metal ions and protein chaperones in the urease system from Helicobacter pylori

Although nickel is a toxic metal for living organisms in its soluble form, its importance in many biological processes recently emerged. In this view, the investigation of the nickel-dependent enzymes urease and [NiFe]-hydrogenase, especially the mechanism of nickel insertion into their active sites, represent two intriguing case studies to understand other analogous systems and therefore to lead to a comprehension of the nickel trafficking inside the cell. Moreover, these two enzymes have been demonstrated to ensure survival and colonization of the human pathogen H. pylori, the only known microorganism able to proliferate in the gastric niche. The right nickel delivering into the urease active site requires the presence of at least four accessory proteins, UreD, UreE, UreF and UreG. Similarly, analogous process is principally mediated by HypA and HypB proteins in the [NiFe]-hydrogenase system. Indeed, HpHypA and HpHypB also have been proposed to act in the activation of the urease enzyme from H. pylori, probably mobilizing nickel ions from HpHypA to the HpUreE-HpUreG complex. A complete comprehension of the interaction mechanism between the accessory proteins and the crosstalk between urease and hydrogenase accessory systems requires the determination of the role of each protein chaperone that strictly depends on their structural and biochemical properties. The availability of HpUreE, HpUreG and HpHypA proteins in a pure form is a pre-requisite to perform all the subsequent protein characterizations, thus their purification was the first aim of this work. Subsequently, the structural and biochemical properties of HpUreE were investigated using multi-angle and quasi-elastic light scattering, as well as NMR and circular dichroism spectroscopy. The thermodynamic parameters of Ni2+ and Zn2+ binding to HpUreE were principally established using isothermal titration calorimetry and the importance of key histidine residues in the process of binding metal ions was studied using site-directed mutagenesis. The molecular details of the HpUreE-HpUreG and HpUreE-HpHypA protein-protein assemblies were also elucidated. The interaction between HpUreE and HpUreG was investigated using ITC and NMR spectroscopy, and the influence of Ni2+ and Zn2+ metal ions on the stabilization of this association was established using native gel electrophoresis, light scattering and thermal denaturation scanning followed by CD spectroscopy. Preliminary HpUreE-HpHypA interaction studies were conducted using ITC. Finally, the possible structural architectures of the two protein-protein assemblies were rationalized using homology modeling and docking computational approaches. All the obtained data were interpreted in order to achieve a more exhaustive picture of the urease activation process, and the correlation with the accessory system of the hydrogenase enzyme, considering the specific role and activity of the involved protein players. A possible function for Zn2+ in the chaperone network involved in Ni2+ trafficking and urease activation is also envisaged.

Structural and biochemical studies of Sporosarcina pasteurii UreE: a nickel-chaperone involved in the urease activation process

Urease is a nickel-dependent enzyme that catalyzes hydrolysis of urea in the last step of organic nitrogen mineralization. Its active site contains a dinuclear center for Ni(II) ions that must be inserted into the apo-enzyme through the action of four accessory proteins (UreD, UreE, UreF, UreG) leading to activation of urease. UreE, acting as a metallo-chaperone, delivers Ni(II) to the preformed complex of apo-urease-UreDFG and has the capability to enhance the GTPase activity of UreG. This study, focused on characterization of UreE from Sporosarcina pasteurii (SpUreE), represents a piece of information on the structure/mobility-function relationships that control nickel binding by SpUreE and its interaction with SpUreG. A calorimetric analysis revealed the occurrence of a binding event between these proteins with positive cooperativity and a stoichiometry consistent with the formation of the (UreE)2-(UreG)2 hetero-oligomer complex. Chemical Shift Perturbations induced by the protein-protein interaction were analyzed using high-resolution NMR spectroscopy, which allowed to characterize the molecular details of the protein surface of SpUreE involved in the complex formation with SpUreG. Moreover, backbone dynamic properties of SpUreE, determined using 15N relaxation analysis, revealed a general mobility in the nanoseconds time-scale, with the fastest motions observed at the C-termini. The latter analysis made it possible for the first time to characterize of the C-terminal portions, known to contain key residues for metal ion binding, that were not observed in the crystal structure of UreE because of disorder. The residues belonging to this portion of SpUreE feature large CSPs upon addition of SpUreG, showing that their chemical environment is directly affected by protein-protein interaction. Metal ion selectivity and affinity of SpUreE for cognate Ni(II) and non cognate Zn(II) metal ions were determined, and the ability of the protein to select Ni(II) over Zn(II), in consistency with the proposed role in Ni(II) cations transport, was established.

Solvation-Driven Charge Transfer and Localization in Metal Complexes

In any physicochemical process in liquids, the dynamical response of the solvent to the solutes out of equilibrium plays a crucial role in the rates and products: the solvent molecules react to the changes in volume and electron density of the solutes to minimize the free energy of the solution, thus modulating the activation barriers and stabilizing (or destabilizing) intermediate states. In charge transfer (CT) processes in polar solvents, the response of the solvent always assists the formation of charge separation states by stabilizing the energy of the localized charges. A deep understanding of the solvation mechanisms and time scales is therefore essential for a correct description of any photochemical process in dense phase and for designing molecular devices based on photosensitizers with CT excited states. In the last two decades, with the advent of ultrafast time-resolved spectroscopies, microscopic models describing the relevant case of polar solvation (where both the solvent and the solute molecules have a permanent electric dipole and the mutual interaction is mainly dipole−dipole) have dramatically progressed. Regardless of the details of each model, they all assume that the effect of the electrostatic fields of the solvent molecules on the internal electronic dynamics of the solute are perturbative and that the solvent−solute coupling is mainly an electrostatic interaction between the constant permanent dipoles of the solute and the solvent molecules. This well-established picture has proven to quantitatively rationalize spectroscopic effects of environmental and electric dynamics (time-resolved Stokes shifts, inhomogeneous broadening, etc.). However, recent computational and experimental studies, including ours, have shown that further improvement is required. Indeed, in the last years we investigated several molecular complexes exhibiting photoexcited CT states, and we found that the current description of the formation and stabilization of CT states in an important group of molecules such as transition metal complexes is inaccurate. In particular, we proved that the solvent molecules are not just spectators of intramolecular electron density redistribution but significantly modulate it. Our results solicit further development of quantum mechanics computational methods to treat the solute and (at least) the closest solvent molecules including the nonperturbative treatment of the effects of local electrostatics and direct solvent−solute interactions to describe the dynamical changes of the solute excited states during the solvent response.

The Effect of Heavy Metal Stress on Avian Proximal Tubule Urate Secretion

In both humans and birds, urate is an important antioxidant when maintained at normal plasma concentrations. Though human kidneys primarily reabsorb filtered urate, while those of birds perform mostly secretion, both maintain urate levels at ~300microM. The importance of maintaining urate levels within the homeostatic range was observed when the study of several prominent diseases revealed an association with hyperuricemia. This study examined the effect of elevated zinc concentration on avian urate secretion. Here, acute exposure of chicken proximal tubule epithelial cells (cPTCs) to zinc stress had no effect on urate secretion, but prolonged zinc-induced cellular stress inhibited active transepithelial urate secretion with no change in Mrp4 expression, glucose transport, or transepithelial resistance. Moreover, zinc had no effect on urate transport by isolated brush border membrane vesicles, suggesting involvement of a more complex cellular stress adaptation. Previous work has demonstrated that AMP-activated protein kinase (AMPK), a critical metabolic regulator, conserves energy during cellular stress by shutting down ATP-utilizing processes and activating ATP-generating processes. Pharmacological activation of AMPK by AICAR produced decreased urate secretion by cPTCs similar to the effect seen with prolonged exposure to zinc, while the AMPK inhibitor Compound C prevented both AICAR and zinc inhibition of urate secretion, suggesting a stress induced mechanism of regulation. Supported by NSF. IACUC #A08-046.

Metallic Fluence Monitors and J-value standards

Utilizing the neutron-irradiation parameter J is one of the major uncertainties in 40Ar/39Ar dating. The associated error of the individual J-value for a sample of unknown age depends on the accuracy of the age of the geological standards, the fast-neutron fluence distribution in the reactor and the distances between standards and samples during irradiation. While it is generally assumed that rotating irradiation evens out radial neutron fluence gradients, we observed axial and radial variations of the J-values in sample irradiations in the rotating channels of two reactors. To quantify them, we included three-dimensionally distributed metallic fast- (Ni) and thermal- (Co) neutron fluence monitors in three irradiations and geological age standards in three more. Two irradiations were carried out under Cd-shielding in the FRG1 reactor in Geesthacht, Germany, and four without Cd-shielding in the LVR-15 reactor in Rez, Czech Republic. The 58Ni(nf,p)58Co activation reaction and ?-spectrometry of the 811 keV peak associated with the subsequent decay of 58Co to 58Fe allow to calculate the fast-neutron fluence. The fast-neutron fluences at known positions in the irradiation container correlate with the J-values determined by mass-spectrometric 40Ar/39Ar measurements of the geological age standards. Ra-dial neutron fluence gradients are up to 1.8 %/cm in FRG1 and up to 2.2 %/cm in LVR-15; the corre-sponding axial gradients are up to 5.9 and 2.1 %/cm. We conclude that sample rotation might not al-ways suffice to meet the needs of high-precision dating and gradient monitoring can be crucial.

Heavy metal concentrations in fauna from hydrothermal fields of the Mid-Atlantic Ridge

Distributions of Fe, Mn, Zn, Cu, Ni, Co, Cr, Pb, As, Ag, Cd, Se, Sb, and Hg in 128 samples of tissues of organisms that inhabit hydrothermal vent fields of the Mid-Atlantic Ridge (Menez Gwen, Snake Pit, and Rainbow) depending on the abiotic environmental parameters were studied. The majority of the elements studied showed direct correlations between their concentrations in fluids released and in tissues of hydrothermal organisms. A higher degree of bioaccumulation of metals was revealed in Bathymodiolus mussels and Rimicaris shrimps from the Rainbow hydrothermal vent field as compared to their analogues from the Menez Gwen and Snake Pit fields. This corresponds to maximal concentrations of the majority of the metals studied in the Rainbow high-temperature hydrothermal fluids. The highest degree of bioaccumulation of heavy metals was found in gills of symbiotrophic mussels Bathymodiolus and in maxillipeds of ectosymbiotic shrimps Rimicaris, i.e., in organs that function in dependence on chemosynthetic bacteria. Within the Rainbow vent field, the shrimps, which inhabit in biotopes with more high-temperature conditions and therefore are more strongly subjected to influence of fluids, were found to contain higher metal contents than mollusks. Fe-Mn hydroxide films that cover mussel shells might serve as important reservoirs of other metals related to Fe and Mn.

A comprehensive diagram to grow metal-polarity InGaN alloys by molecular beam epitaxy

he composition, strain and surface morphology of (0001)InGaN layers are investigated as a function of growth temperature (460–645 °C) and impinging In flux. Three different growth regimes: nitrogen-rich, metal-rich and intermediate metal-rich, are clearly identified and found to be in correlation with surface morphology and strain relaxation. Best epilayers’ quality is obtained when growing under intermediate metal-rich conditions, with 1–2 monolayers thick In ad-coverage. For a given In flux, the In incorporation decreases with increasing growth temperature due to InN thermal decomposition that follows an Arrhenius behavior with 1.84±0.12 eV activation energy.

Impact of metal-organic vapor phase epitaxy environment on silicon bulk lifetime for III–V-on-Si multijunction solar cells

With the final goal of integrating III-V materials on silicon substrates for tandem solar cells, the influence of the Metal-Organic Vapor Phase Epitaxy (MOVPE) environment on the minority carrier properties of silicon wafers has been evaluated. These properties will essentially determine the photovoltaic performance of the bottom cell in a III-V-on-Si tandem solar cell. A comparison of the base minority carrier lifetimes obtained for different thermal processes carried out in a MOVPE reactor on Czochralski silicon wafers has been carried out. An important degradation of minority carrier lifetime during the surface preparation (i.e. H2 anneal) has been observed. Three different mechanisms have been proposed for explaining this behavior: 1) the introduction of extrinsic impurities coming from the reactor; 2) the activation of intrinsic lifetime killing impurities coming from the wafer itself; and finally, 3) the formation of crystal defects, which eventually become recombination centers. The effect of the emitter formation by phosphorus diffusion has also been evaluated. In this sense, it has been reported that lifetime can be recovered during the emitter formation either by the effect of the P on extracting impurities, or by the role of the atomic hydrogen on passivating the defects.

Target gene search for the metal-responsive transcription factor MTF-1

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding α-fetoprotein, the liver-enriched transcription factor C/EBPα and tear lipocalin/von Ebner’s gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns γ-glutamylcysteine synthetasehc (γ-GCShc), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. γ-GCShc mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Photoconductivity and Electro-Optical Properties of Wide Band Gap Metal Oxides

The PhD activity described in this Thesis was focused on the study of metal-oxide wide-bandgap materials, aiming at fabricating new optoelectronic devices such as solar-blind UV photodetectors, high power electronics, and gas sensors. Photocurrent spectroscopy and DC photocurrent time evolution were used to investigate the performance of prototypes under different atmospheres, temperatures and excitation wavelengths (or dark conditions). Cathodoluminescence, absorption spectroscopy, XRD and SEM were used to assess structural, morphologic, electrical and optical properties of materials. This thesis is divided into two main sections, each describing the work done on a different metal-oxide semiconductor. 1) MOVPE-grown Ga2O3 thin films for UV solar-blind photodetectors and high power devices The semiconducting oxides, among them Ga2O3, have been employed for several decades as transparent conducting oxide (TCO) electrodes for fabrication of solar cells, displays, electronic, and opto-electronic devices. The interest was mainly confined to such applications, as these materials tend to grow intrinsically n-type, and attempts to get an effective p-type doping has consistently failed. The key requirements of TCO electrodes are indeed high electrical conductivity and good transparency, while crystallographic perfection is a minor issue. Furthermore, for a long period no high-quality substrates and epi-layers were available, which in turn impeded the development of a truly full-oxide electronics. Recently, Ga2O3 has attracted renewed interest, as large single crystals and high-quality homo- and hetero-epitaxial layers became available, which paved the way to novel application areas. Our research group spent the last two years in developing a low temperature (500-700°C) MOVPE growth procedure to obtain thin films of Ga2O3 on different substrates (Dept. of Physics and IMEM-CNR at UNIPR). We obtained a significant result growing on oriented sapphire epitaxial films of high crystalline, undoped, pure phase -Ga2O3 (hexagonal). The crystallographic properties of this phase were investigated by XRD, in order to clarify the lattice parameters of the hexagonal cell. First design and development of solar blind UV photodetectors based on -phase was carried out and the optoelectronic performance is evaluated by means of photocurrent spectroscopy. The UV-response is adequately fast and reliable to render this unusual phase a subject of great interest for future applications. The availability of a hexagonal phase of Ga2O3 stable up to 700°C, belonging to the same space group of gallium nitride, with high crystallinity and tunable electrical properties, is intriguing in view of the development of nitride-based devices, by taking advantage of the more favorable symmetry and epitaxial relationships with respect to the monoclinic β-phase. In addition, annealing at temperatures higher than 700°C demonstrate that the hexagonal phase converts totally in the monoclinic one. 2) ZnO nano-tetrapods: charge transport mechanisms and time-response in optoelectronic devices and sensors Size and morphology of ZnO at the nanometer scale play a key role in tailoring its physical and chemical properties. Thanks to the possibility of growing zinc oxide in a variety of different nanostructures, there is a great variety of applications, among which gas sensors, light emitting diodes, transparent conducting oxides, solar cells. Even if the operation of ZnO nanostructure-based devices has been recently demonstrated, the mechanisms of charge transport in these assembly is still under debate. The candidate performed an accurate investigation by photocurrent spectroscopy and DC-photocurrent time evolution of electrical response of both single-tetrapod and tetrapod-assembly devices. During the research done for this thesis, a thermal activation energy enables the performance of samples at high temperatures (above about 300°C). The energy barrier is related to the leg-to-leg interconnection in the assembly of nanotetrapods. Percolation mechanisms are responsible for both the very slow photo-response (minutes to hours or days) and the significant persistent photocurrent. Below the bandgap energy, electronic states were investigated but their contribution to the photocurrent are two-three order of magnitude lower than the band edge. Such devices are suitable for employ in photodetectors as well as in gas sensors, provided that the mechanism by which the photo-current is generated and gas adsorption on the surface modify the conductivity of the material are known.

Preparation of high metal content nanoporous carbon

Activated carbons with high metal content have been prepared by the pyrolysis of ethylene tar with dissolved metal acetylacetonates (Ti, V, Fe, Co, Ni and Cu) and subsequent activation with KOH of the pitch obtained in pyrolysis. These metal compounds decompose during the pyrolysis of ethylene tar yielding metal nanoparticles formed by metal and/or oxide which are homogeneously distributed in the pitch and remain in the activated carbon, so that the concentration of metal is, in most cases, 4–5 times higher than in the pristine ethylene tar. Since KOH is an effective activating agent, all activated carbons combine a high porosity development with a high metal content. In some of the carbons, such as P2FeA (3.3% Fe, pore volume 1.84 cm3/g, BET surface area 3270 m2/g), there is even an increase in the pore volume when compared to the activated carbon prepared in the same way without metal, in spite of the fact that the metal increases the weight of carbon without contributing to the adsorptive capacity. It seems that iron, on the one hand modifies the pyrolysis to give a pitch with larger mesophase content and on the other hand it locally catalyzes carbon gasification with the CO2 produced along the synthesis of the carbon. In addition to its influence on activation, iron promotes the formation of graphitic carbon fibers.