Search

Transition metals such as copper and zinc are essential elements required for the survival of most organisms, from bacteria to humans. Yet, elevated levels of these elements are highly toxic. The Copper TRansporter protein family (CTRs) represents the only identified copper uptake proteins in eukaryotes and hence serves as key components for the maintenance of appropriate levels of the metal. Moreover, CTRs have been proposed to serve as an entry point into cells of certain cancer drugs and to constitute attractive drug-targets for novel antifungals. Nevertheless, the structure, function, and regulation of the CTRs remain elusive, limiting valuable information also for applied sciences. To this end, here we report procedures to isolate a range of CTR members using Saccharomyces cerevisiae as a production host, focusing on three homologs, human CTR1, human CTR2, and Candida albicans CTR. Using forms C-terminally-linked to a protease cleavage sequence, Green Fluorescent Protein (GFP), and a His-tag, assessment of the localization, quantification and purification was facilitated. Cellular accumulation of the proteins was investigated via live-cell imaging. Detergents compatible with acceptable solubilization yields were identified and fluorescence-detection size-exclusion-chromatography (F-SEC) revealed preferred membrane extraction conditions for the targets. For purification purposes, the solubilized CTR members were subjected to affinity chromatography and SEC, reaching near homogeneity. The quality and quantity of the CTRs studied will permit downstream efforts to uncover imperative biophysical aspects of these proteins, paving the way for subsequent drug-discovery studies.

Measurement of protein-facilitated copper flux across biological membranes is a considerable challenge. Here, we demonstrate a straightforward microfluidic-derived approach for visualization and measurement of membranous Cu flux. Giant unilamellar vesicles, reconstituted with the membrane protein of interest, are prepared, surface-immobilized, and assessed using a novel quencher-sensor reporter system for detection of copper. With the aid of a syringe pump, the external buffer is exchanged, enabling consistent and precise exchange of solutes, without causing vesicle rupture or uneven local metal concentrations brought about by rapid mixing. This approach bypasses common issues encountered when studying heavy metal-ion flux, thereby providing a new platform for in vitro studies of metal homeostasis aspects that are critical for all cells, health, and disease.

Work performed in the building industry contributes to the formation of many threats for employees. Microclimate factors, working at heights as well as dangerous substances used in the building space may cause a negative impact on the worker’s health.

Effect of high-temperature exposure on the microstructure and mechanical properties of the Al5Ti5Co35Ni35Fe20 High-Entropy Alloy was studied. High-entropy alloys belong to the group of multi-principal element alloys composed of at least five elements with a concentration between 5 and 35 atomic pct. Despite the multicomponent composition, the goal is to achieve a simple solid solution. The alloy was exposed to a temperature range of 650–900 °C for 168 h (7 days), after hot rolling and air-quenching. After hot rolling the investigated alloy was not fully supersaturated. The application of air cooling caused γ′ precipitation, which was uniformly distributed within the microstructure. High-temperature exposure caused the growth of γ′ particles. At 700 °C and higher, additional continuous and discontinuous precipitation of γ′ was observed. Finally, the microstructure of the investigated alloy consisted of γ matrix and three types of γ’ precipitates, i.e. spherical γ′ phases with bimodal distribution within the grains and elongated DP γ′ phases present at the grain boundaries. The highest mechanical properties were obtained after exposure to 650 °C, which was caused by γ′ precipitation. Further increase in temperature decreased the mechanical properties. Keywords: High entropy alloys, Aging, Microstructure, γ′, Discontinuous precipitation

The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex metalloclusters of nitrogenase. In addition, it can function as a reductase on its own and affect the ambient reduction of CO2 or CO to hydrocarbons. This review will provide an overview of the properties and functions of the Fe protein, highlighting the relevance of this unique FeS enzyme to areas related to the catalysis, biosynthesis, and applications of the fascinating nitrogenase system.

Copper-containing mixed metal oxides were studied as catalysts for selective catalytic oxidation of ammonia. The performed research shows a high correlation between Cu amount and catalyst efficiency, especially dinitrogen selectivity. Copper species, mainly in the form of highly dispersed CuO oxides, were found to be responsible for high N2 selectivity, which decreased with increasing Cu loading and the formation of CuO bulk-like species. The addition of cerium seemed to have an effect on material’s catalytic activity and N2 selectivity. The increase in selectivity towards N2 of Ce-impregnated materials was attributed to the formation of copper-cerium redox couples on the material’s surface. The catalytic tests performed under different NH3/O2 molar ratios revealed that the developed materials exhibit high catalytic activity in a wide range of ammonia concentrations and oxygen contents.

The majority of efforts on microbial and photosynthetic microbial fuel cells are both curiosity driven and made to possibly meet the future growing demand for sustainable energy. The most metabolically versatile purple bacteria Rhodobacter capsulatus is a potential candidate for this purpose. However, utilizing bacteria in such systems requires efficient electronic transfer communication between the microbial cells and the electrodes, which is one of the greatest challenges. Previous studies demonstrated that osmium redox polymers (ORPs) could be used for extracellular electron transfer between the cells and electrodes. Recently, heterotrophically grown R. capsulatus has been wired with ORP modified electrodes. Here in this communication, we report electron transfer from R. capsulatus grown under heterotrophic as well as under photoheterotrophic conditions to electrodes. The cells, immobilized on bare graphite and ORP modified graphite electrodes, were excited with visible light and subsequent photosynthetic electron transfer was recorded using cyclic voltammetric and chronoamperometric measurements. Photoheterotrophically grown R. capsulatus cells on bare graphite generate a significant photocurrent density of 3.46 mu A cm(-2), whereas on an ORP modified electrode the current density increases to 8.46 mu A cm(-2). Furthermore, when 1 mM p-benzoquinone is added to the electrolyte the photocurrent density reaches 12.25 mu A cm(-2). Our results could have significant implications in photosynthetic energy conversion and in development of photobioelectrochemical devices. (Less)

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na+/H+ antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity.

MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required.

It has long been known that the three largest subunits in the membrane domain (NuoL, NuoM and NuoN) of complex I are homologous to each other, as well as to two subunits (MrpA and MrpD) from a Na+/H+ antiporter, Mrp. MrpA and NuoL are more similar to each other and the same is true for MrpD and NuoN. This suggests a functional differentiation which was proven experimentally in a deletion strain model system, where NuoL could restore the loss of MrpA, but not that of MrpD and vice versa. The simplest explanation for these observations was that the MrpA and MrpD proteins are not antiporters, but rather single subunit ion channels that together form an antiporter. In this work our focus was on a set of amino acid residues in helix VIII, which are only conserved in NuoL and MrpA (but not in any of the other antiporter-like subunits.) and to compare their effect on the function of these two proteins. By combining complementation studies in B. subtilis and 23Na-NMR, response of mutants to high sodium levels were tested. All of the mutants were able to cope with high salt levels; however, all but one mutation (M258I/M225I) showed differences in the efficiency of cell growth and sodium efflux. Our findings showed that, although very similar in sequence, NuoL and MrpA seem to differ on the functional level. Nonetheless the studied mutations gave rise to interesting phenotypes which are of interest in complex I research.

Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c(550). Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c(550) domain in all the fusion proteins exhibited normal spectra and redox properties, with an E(m) of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.

Aims: To examine the effects of ammonium ion concentration and illumination intensity on the effectivness of the hydrogen generation process of Rhodobacter sphaeroides. Methods and Results: In all experiments the amount of evolved hydrogen, biomass growth, concentration of ammonium ions, pH and chemical oxygen demand were measured. A nonstructural kinetic model was applied in description of biomass growth, the amount of evolved hydrogen and consumption of organic compounds and ammonium ions. An increase of ammonium ions concentration resulted in a decrease of maximal specific hydrogen potential production. At higher ammonium ion concentrations, no hydrogen evolution was observed. The efficiency of malic acid conversion into hydrogen and the PHB content in the biomass were the highest with lower concentrations of nitrogen compounds. Conclusion: The presence of ammonium ions inhibits hydrogen photogeneration. A good agreement between the experimental data and model simulations were obtained. In all cases, hydrogen evolution started after an exhaustion of ammonium ions and the increase was proportional to the C/N ratio in the medium. The accumulation of PHB competes with the hydrogen evolution process while the conversion of acids into biomass was limited at higher levels of hydrogen generation. Significance and Impact of the Study: Confirmation of the suitability of the selected model for kinetic studies of hydrogen photoevolution.

(23)Na nuclear magnetic resonance (NMR) has previously been used to monitor Na(+) translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na NMR method was adapted for measurements of internal Na(+) concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na(+) concentration than an external one.

Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu(3), which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe's pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe's pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu(3) was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu(3) was found to be superior to the commercially available pH indicators.

Here, we present an unusual approach of modifying the growth pattern of electroactive bacteria for improving their communication with electrodes. Cells of a known exoelectrogen, Shewanella oneidensis MR-1, were treated with cisplatin, a drug that inhibits cell division but forces the cells to grow longer resulting in a characteristic elongated growth. Such elongated cells showed up to a 5-fold improvement in current densities compared to normal, untreated cells.

not Available.