Isolation and thermal stabilization of mouse ferroportin.

Ferroportin (Fpn) is an essential mammalian iron transporter that is negatively regulated by the hormone hepcidin. Our current molecular understanding of Fpn-mediated iron efflux and regulation is limited due to a lack of biochemical, biophysical and high-resolution structural studies. A critical step towards understanding the transport mechanism of Fpn is to obtain sufficient quantities of pure and stable protein for downstream studies. As such, we detail here an expression and purification protocol for mouse Fpn yielding milligram quantities of pure protein. We have generated deletion constructs exhibiting enhanced thermal stability and which retained iron-transport activity and hepcidin responsiveness, providing a platform for further biophysical studies of Fpn.

Membrane Protein Solubilization and Quality Control: An Example of a Primary Active Transporter.

When purifying a membrane protein, finding a detergent for solubilization is one of the first steps to master. Ideally, only little time is invested to identify the best-suited detergent, which on the one hand would solubilize large amounts of the target protein but on the other hand would sustain the protein's activity. Here we describe the solubilization screen and subsequent activity assay we have optimized for the bacterial P-type ATPase KdpFABC. In just 2 days, more than 70 detergents were tested for their solubilization potential. Afterwards, a smaller selection of the successful detergents was assayed for their ability to retain the activity of the membrane protein complex.

Developing Colorimetric and Luminescence-Based High-Throughput Screening Platforms for Monitoring the GTPase Activity of Ferrous Iron Transport Protein B (FeoB).

Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.

Overexpression of TtNRAMP6 enhances the accumulation of Cd in Arabidopsis.

The uptake and translocation of non-essential heavy metals in plant are always through metal transporters for essential micronutrient transport, such as NRAMP (Natural Resistance-Associated Macrophage Protein). NRAMPs from different species exhibit different biological functions, although their sequences are highly identical. In the present study, a NRAMP6 was isolated from Ailanmai (Triticum turgidum L. ssp. turgidum). TtNRAMP6, localized on chromosome 3B, was mainly expressed in roots, followed by other tissues varied with different growth stages. At the seedling stage, TtNRAMP6 was significantly regulated by Cd stress in roots, but not by the deficiency of Zn, Fe or Mg. Subcellular localization analysis indicated that TtNRAMP6 encoded a plasma membrane protein. Expressing-TtNRAMP6 significantly enhanced the Cd concentration in yeast, and increased the Cd sensitivity. Meanwhile, overexpression of TtNRAMP6 also increased the Cd concentration in roots, stems, leaves and the whole plant of Arabidopsis, which indicated that overexpression of TtNRAMP6 enhanced the Cd accumulation. Thus, genetic manipulation of TtNRAMP6 may reduce the uptake of Cd from external solution to wheat, finally protecting the safety of wheat food.

Expression and purification of functionally active ferrous iron transporter FeoB from Klebsiella pneumoniae.

The acquisition of ferrous iron (Fe2+) is an important virulence factor utilized by several hospital-acquired (nosocomial) pathogens such as Klebsiella pneumoniae to establish infection within human hosts. Virtually all bacteria use the ferrous iron transport system (Feo) to acquire ferrous iron from their environments, which are often biological niches that stabilize Fe2+ relative to Fe3+. However, the details of this process remain poorly understood, likely owing to the few expression and purification systems capable of supplying sufficient quantities of the chief component of the Feo system, the integral membrane GTPase FeoB. This bottleneck has undoubtedly hampered efforts to understand this system in order to target it for therapeutic intervention. In this study, we describe the expression, solubilization, and purification of the Fe2+ transporter from K. pneumoniae, KpFeoB. We show that this protein may be heterologously overexpressed in Escherichia coli as the host organism. After testing several different commercially-available detergents, we have developed a solubilization and purification protocol that produces milligram quantities of KpFeoB with sufficient purity for enzymatic and biophysical analyses. Importantly, we demonstrate that KpFeoB displays robust GTP hydrolysis activity (kcatGTP of ∼10-1 s-1) in the absence of any additional stimulatory factors. Our findings suggest that K. pneumoniae may be capable of using its Feo system to drive Fe2+ import in an active manner.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.

Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the Atox1 Metallochaperone.

Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle.

[From the discovery of microbial Mep-Amt ammonium transporters to human Rhesus factors]. / De la découverte des transporteurs d'ammonium Mep-Amt microbiens aux facteurs Rhésus humains.

Ammonium, ubiquitous on Earth, plays major and distinct roles in most organisms. While it can be a nitrogen source for many microorganisms and plants, it is a cytotoxic metabolic product actively detoxified by the liver in animals. Furthermore, in the latter, ammonium synthesis in the kidney is involved in acid/base homeostasis. Ammonium transport is ensured by a family of proteins, called Mep-Amt-Rh. This family is conserved in all domains of life and comprises the human Rh factors, notably known in transfusional medicine. While the study of bacterial, fungal and vegetal Mep-Amt transporters reveals a fine-tuned and rapid regulation of these proteins in function of environmental changes, the regulation of animal Rh proteins has been poorly addressed. This review notably highlights the importance of the yeast model in the study of the regulation of these proteins as well as in the functional characterization of Mep-Amt-Rh members of diverse origins.

A streptococcal NRAMP homologue is crucial for the survival of Streptococcus agalactiae under low pH conditions.

Streptococcus agalactiae or Group B Streptococcus (GBS) is a commensal bacterium of the human gastrointestinal and urogenital tracts as well as a leading cause of neonatal sepsis, pneumonia and meningitis. Maternal vaginal carriage is the main source for GBS transmission and thus the most important risk factor for neonatal disease. Several studies in eukaryotes identified a group of proteins natural resistance-associated macrophage protein (NRAMP) that function as divalent cation transporters for Fe(2+) and Mn(2+) and confer on macrophages the ability to control replication of bacterial pathogens. Genome sequencing predicted potential NRAMP homologues in several prokaryotes. Here we describe for the first time, a pH-regulated NRAMP Mn(2+) /Fe(2+) transporter in GBS, designated MntH, which confers resistance to reactive oxygen species (ROS) and is crucial for bacterial growth and survival under low pH conditions. Our investigation implicates MntH as an important colonization determinant for GBS in the maternal vagina as it helps bacteria to adapt to the harsh acidic environment, facilitates bacterial adherence, contributes to the coexistence with the vaginal microbiota and plays a role in GBS intracellular survival inside macrophages.

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.

Expression, purification and spin labelling of the ferrous iron transporter FeoB from Escherichia coli BL21 for EPR studies.

The ferrous iron transporter FeoB is an important factor in the iron metabolism of various bacteria. As a membrane bound GTPase it also represents an interesting evolutionary link between prokaryotic and eukaryotic membrane signalling pathways. To date, structural information for FeoB is limited to the cytosolic GTPase domain and structural features such as the oligomeric state of the transporter in the membrane, and thereby the nature of the transport pore are a matter of constant debate. Recently, EPR distance measurements have become an important tool to investigate such questions in frozen solution. As a prerequisite for these experiments, we designed protocols to express and purify both the cytosolic domain of FeoB (NFeoB) and full-length FeoB from Escherichia coli BL21 in purity, quantity and quality needed for EPR studies. Since FeoB from E. coli contains 12 native cysteines, we incorporated the unnatural amino acid para-acetylphenylalanine (pAcF) into the protein. We spin labelled the mutant protein using the HO4120 spin label and performed preliminary EPR experiments using cw-X-band EPR spectroscopy. Our results provide new insights concerning the oligomeric state of full-length FeoB.

Identification and characterization of calcium transporter gene family in finger millet in relation to grain calcium content.

Calcium (Ca) is an essential mineral for proper growth and development of plants as well as animals. In plants including cereals, calcium is deposited in seed during its development which is mediated by specialized Ca transporters. Common cereal seeds contain very low amounts of Ca while the finger millet (Eleusine coracana) contains exceptionally high amounts of Ca in seed. In order to understand the role of Ca transporters in grain Ca accumulation, developing seed transcriptome of two finger millet genotypes (GP-1, low Ca and GP-45 high Ca) differing in seed Ca content was sequenced using Illumina paired-end sequencing technology and members of Ca transporter gene family were identified. Out of 109,218 and 120,130 contigs, 86 and 81 contigs encoding Ca transporters were identified in GP-1 and GP-45, respectively. After removal of redundant sequences, a total of 19 sequences were confirmed as Ca transporter genes, which includes 11 Ca(2+) ATPases, 07 Ca(2+)/cation exchangers and 01 Ca(2+) channel. The differential expressions of all genes were analyzed from transcriptome data and it was observed that 9 and 3 genes were highly expressed in GP-45 and GP-1 genotypes respectively. Validation of transcriptome expression data of selected Ca transporter genes was performed on different stages of developing spikes of both genotypes grown under different concentrations of exogenous Ca. In both genotypes, significant correlation was observed between the expression of these genes, especially EcCaX3, and on the amount of Ca accumulated in seed. The positive correlation of seed mass with the amount of Ca concentration was also observed. The efficient Ca transport property and responsiveness of EcCAX3 towards exogenous Ca could be utilized in future biofortification program.

Overexpression, purification and biophysical characterisation of E. coli MerT.

Mercury resistance is the most widespread of all anti-microbial resistance occurring in a wide variety of Gram-negative and Gram-positive bacterial genera. The systems that are most studied and best understood are those encoded in mercury resistance (Mer) operons in Gram-negative bacteria. The mercury detoxification functions by the importation of highly toxic Hg(2+) into cytoplasm and enzymic reduction to volatile Hg(0). MerT is a small (13kDa) inner membrane protein involved in mercuric ion transport system. We have overexpressed recombinant 6His-tagged MerT from Escherichia coli in a native folded form and purified it to homogeneity in n-dodecyl-ß-d-maltopyranoside (DDM) by immobilized metal affinity chromatography (IMAC). Circular dichroism showed that the protein is largely α-helical. Size-exclusion chromatography (SEC) in a variety of detergents showed that the protein exists in a multiple of oligomeric states as also confirmed by SEC coupled with multiple-angle light scattering.

Transport of magnesium by a bacterial Nramp-related gene.

Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5-2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.

Identification of major zinc-binding proteins from a marine cyanobacterium: insight into metal uptake in oligotrophic environments.

Marine cyanobacteria make a significant contribution to primary production whilst occupying some of the most nutrient poor regions of the world's oceans. The low bioavailability of trace metals can limit the growth of phytoplankton in ocean waters, but only scarce data are available on the requirements of marine microbes for zinc. Recent genome mining studies suggest that marine cyanobacteria have both uptake systems for zinc and proteins that utilize zinc as a cofactor. In this study, the oligotrophic strain Synechococcus sp. WH8102 was grown at different zinc concentrations. Using metalloproteomics approaches, we demonstrate that even though this organism's growth was not affected by extremely low zinc levels, cells accumulated significant quantities of zinc, which was shown to be protein-associated by 2D liquid chromatography and ICP-MS. This indicates that the mechanisms for zinc uptake in Synechococcus sp. WH8102 are extremely efficient. Significantly, expression of SYNW2224, a putative porin, was up-regulated during growth in zinc-depleted conditions. Furthermore, along with 30 other proteins, SYNW2224 was captured by immobilised zinc affinity chromatography, indicating the presence of surface-exposed site(s) with metal-binding capacity. It is proposed that this porin plays a role in high-affinity zinc uptake in this and other cyanobacteria.

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.

In vitro thermodynamic dissection of human copper transfer from chaperone to target protein.

Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

The transcriptome and proteome are altered in marine polychaetes (Annelida) exposed to elevated metal levels.

Polychaetes are often used in toxicological studies to understand mechanisms of resistance and for biomarker detection, however, we know of only a few genetic pathways involved in resistance. We found the marine polychaete Ophelina sp.1 (Opheliidae) in sediment containing high copper levels and investigated this phenomenon by measuring metal accumulation in the worms and changes in gene and protein expression. We sequenced the transcriptome of Ophelina sp.1 from both the impacted and reference sediments using 454-sequencing and analysed their proteomes using differential in gel electrophoresis (DIGE). We used the sequenced transcriptome to guide protein identification. Transcripts coding for the copper chaperone, Atox1, were up-regulated in the worms inhabiting the high copper sediment. In addition, genes coding for respiratory proteins, detoxification proteins and cytoskeletal proteins were significantly altered in metal-exposed worms; many of these changes were also detected in the proteome. This dual approach has provided a better understanding of heavy metal resistance in polychaetes and we now have a wider range of suitable indicator genes and proteins for future biomarker development.

Wheat grain yield on saline soils is improved by an ancestral Na⁺ transporter gene.

The ability of wheat to maintain a low sodium concentration ([Na(+)]) in leaves correlates with improved growth under saline conditions. This trait, termed Na(+) exclusion, contributes to the greater salt tolerance of bread wheat relative to durum wheat. To improve the salt tolerance of durum wheat, we explored natural diversity in shoot Na(+) exclusion within ancestral wheat germplasm. Previously, we showed that crossing of Nax2, a gene locus in the wheat relative Triticum monococcum into a commercial durum wheat (Triticum turgidum ssp. durum var. Tamaroi) reduced its leaf [Na(+)] (ref. 5). Here we show that a gene in the Nax2 locus, TmHKT1;5-A, encodes a Na(+)-selective transporter located on the plasma membrane of root cells surrounding xylem vessels, which is therefore ideally localized to withdraw Na(+) from the xylem and reduce transport of Na(+) to leaves. Field trials on saline soils demonstrate that the presence of TmHKT1;5-A significantly reduces leaf [Na(+)] and increases durum wheat grain yield by 25% compared to near-isogenic lines without the Nax2 locus.

New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Mesostigma viride.

Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name "channelrhodopsins." Channelrhodopsins have become useful molecular tools for light control of cellular activity. Only four channelrhodopsins, identified in Chlamydomonas reinhardtii and Volvox carteri, have been reported so far. By screening light-induced currents among algal species, we identified that the phylogenetically distant flagellate Mesostigma viride showed photoelectrical responses in vivo with properties suggesting a channelrhodopsin especially promising for optogenetic use. We cloned an M. viride channelrhodopsin, MChR1, and studied its channel activity upon heterologous expression. Action spectra in HEK293 cells match those of the photocurrents observed in M. viride cells. Comparison of the more divergent MChR1 sequence to the previously studied phylogenetically clustered homologs and study of several MChR1 mutants refine our understanding of the sequence determinants of channelrhodopsin function. We found that MChR1 has the most red-shifted and pH-independent spectral sensitivity so far reported, matches or surpasses known channelrhodopsins' channel kinetics features, and undergoes minimal inactivation upon sustained illumination. This combination of properties makes MChR1 a promising candidate for optogenetic applications.