Mercury-binding Protein Research Articles

NeMeHg, genetically encoded indicator for mercury ions based on mNeonGreen green fluorescent protein and merP protein from Shigella flexneri.

The detection of mercury ions is an important task in both environmental monitoring and cell biology research. However, existing genetically encoded sensors for mercury ions have certain limitations, such as negative fluorescence response, narrow dynamic range, or the need for cofactor supplementation. To address these limitations, we have developed novel sensors by fusing a circularly permutated version of the mNeonGreen green fluorescent protein with the merP mercury-binding protein from Gram-negative bacteria Shigella flexneri. The developed NeMeHg and iNeMeHg sensors responded to mercury ions with positive and negative fluorescence changes, respectively. We characterized their properties in vitro. Using the developed biosensors, we were able to successfully visualize changes in mercury ion concentration in mammalian cultured cells.

Cadmium transport activity of four mercury transporters (MerC, MerE, MerF and MerT) and effects of the periplasmic mercury-binding protein MerP on Mer-dependent cadmium uptake.

Mercury superfamily proteins, i.e. inner membrane-spanning proteins (MerC, MerE, MerF and MerT) and a periplasmic mercury-binding protein (MerP), transport mercury into the cytoplasm. A previous study demonstrated that a Mer transporter homolog exhibits cadmium transport activity; based on this, the present study aimed to evaluate the cadmium transport activity of MerC, MerE, MerF and MerT and the effects of MerP co-expression in Escherichia coli. Bacteria expressing MerC, MerE, MerF or MerT without MerP were more sensitive to cadmium and significantly absorbed more cadmium than did the control strain. Expression of MerP in combination with MerC, MerE, MerF or MerT increased the bacterial sensitivity to cadmium and cadmium accumulation compared to a single expression of MerC, MerE, MerF or MerT. Cadmium uptake mediated by MerC, MerE, MerF or MerT was inhibited under cold or acidic conditions. These findings suggest that MerC, MerE, MerF and MerT are broad-spectrum heavy metal transporters that mediate both mercury and cadmium transport into cells and that MerP accelerates the cadmium transport ability of MerC, MerE, MerF and MerT.

Genetically encoded FRET-based optical sensor for Hg2+ detection and intracellular imaging in living cells.

Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10-7 M, thus providing a detection range for Hg2+ quantification between 0.210µM and 1.196µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.

Characterization of mercury-binding proteins in rat blood plasma.

Mercury-binding protein profiles in rat plasma at different levels of mercury exposure in vitro and in vivo were systematically investigated using column gel electrophoresis coupled with inductively coupled plasma-mass spectrometry. The current finding provided various protein candidates that may play critical roles in mercury binding and transporting in plasma.

Mercury speciation and mercury-binding protein study by HPLC-ICP-MS on the estimation of mercury toxicity between maternal and infant rats

A comparative study of methylmercury (MeHg) metabolism and its conversion into inorganic mercury (I-Hg) in organic tissues (brain, liver and kidney) and the subcellular fractions (nucleus, mitochondrion, lysosome, microsome and cytosol) of maternal and infant rats after infant in utero and lactational exposure to methylmercury was achieved by extraction preconcentration with HPLC-ICP-MS determination. The quantification of Hg-containing proteins in brain cytosol was studied by HPLC hyphenated on-line isotope dilution ICP-MS and the proteins were analyzed by SDS-PAGE and MALDI-TOF-MS. The results demonstrated that the methods could be successfully used in the study of mercury species in sub-compartments of organ cells. The study showed that the distribution patterns of MeHg and I-Hg in the levels of organs, the subcellular fractions of organs and proteins are all significantly different between mothers and their offspring, indicating their significantly different metabolism, transportation and accumulation behavior of mercury species, which may be all involved in the toxic mechanism of mercury for adults and young children.

Crystal Structure of the Oxidized Form of the Periplasmic Mercury-binding Protein MerP from Ralstonia metallidurans CH34

In Ralstonia metallidurans CH34, the gene merP encodes for a periplasmic mercury-binding protein which is capable of binding one mercury atom. The metal-binding site of MerP consists of the highly conserved sequence GMTCXXC found in the family that includes metallochaperones and metal-transporting ATPases. We purified MerP from R. metallidurans CH34 and solved its crystal structure under the oxidized form at 2.0 Å resolution. Superposition with structures of other metal-binding proteins shows that the global structure of R. metallidurans CH34 oxidized MerP follows the general topology of the whole family. The largest differences are observed with the NMR structure of oxidized Shigella flexneri MerP. Detailed analysis of the metal-binding site suggests a direct role for Y66 in stabilizing the thiolate group of C17 during the mercury-binding reaction. The metal-binding site of oxidized MerP is also similar to the metal-binding sites of oxidized copper chaperone for superoxide dismutase and Atx1, two copper-binding proteins from Saccharomyces cerevisiae. Finally, the packing of the MerP crystals suggests that F38, a well-conserved residue in the MerP family may be important in mercury binding and transfer. We propose a possible mechanism of mercury transfer between two CXXC motifs based on a transient bi-coordinated mercury intermediate.

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from Ralstonia metallidurans CH34.

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. Deltaa has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTC XXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl-methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. (15)N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first beta-strand and the first alpha-helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.

Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay

Thirty mercury-resistant (Hg R) Bacillus strains were isolated from mercury-polluted sediment of Minamata Bay, Japan. Mercury resistance phenotypes were classified into broad-spectrum (resistant to inorganic Hg 2+ and organomercurials) and narrow-spectrum (resistant to inorganic Hg 2+ and sensitive to organomercurials) groups. Polymerase chain reaction (PCR) product sizes and the restriction nuclease site maps of mer operon regions from all broad-spectrum Hg R Bacillus were identical to that of Bacillus megaterium MB1. On the other hand, the PCR products of the targeted merP (extracellular mercury-binding protein gene) and merA (intracellular mercury reductase protein gene) regions from the narrow-spectrum Hg R Bacillus were generally smaller than those of the B. megaterium MB1 mer determinant. Diversity of gene structure configurations was also observed by restriction fragment length polymorphism (RFLP) profiles of the merA PCR products from the narrow-spectrum Hg R Bacillus. The genetic diversity of narrow-spectrum mer operons was greater than that of broad-spectrum ones.

Selectivity in heavy metal- binding to peptides and proteins.

The metal-binding affinities and three-dimensional structures of three synthetic 18-residue peptides with sequences derived from that of the highly conserved metal-binding motif MXCXXC found in many heavy metal-binding proteins were determined. A change in register of the cysteines and alanines of the sequence from the periplasmic mercury-binding protein, MerP, i.e., CAAC, CACA, and CCAA, affects the specificity of metal binding, in particular, the peptide with vicinal cysteines binds only mercury. The three-dimensional structures of the mercury-bound forms of the three peptides determined in solution by NMR spectroscopy peptides differ considerably, even though they are all linear bicoordinate complexes. The three-dimensional structure of the peptide with CAAC bound to Cd(II) demonstrates that the metal-binding loop is malleable enough to accommodate modes of coordination other than linear bicoordinate.

Mercury-binding proteins from the marine mussel, Mytilus edulis.

The marine mussel, Mytilus edulis, possesses low molecular weight, metal-binding proteins which can be induced by and, in turn, bind mercury when individuals are exposed to low, but elevated concentrations of mercury as HgCl2. Induction of the proteins by exposure of mussels to copper, cadmium, or mercury is associated with enhanced tolerance to mercury toxicity. Mercury-binding proteins isolated from gills of mussels occur as two molecular weight variants of about 20-25 and 10-12 kdaltons, respectively, on Sephadex G-75. These have been designated as HgBP20 and HgBP10 following the nomenclature used for cadmium-binding proteins. HgBP20 represents the primary mercury-binding species. These exist as dimers which can be dissociated into subunits by treatment with 1% 2-mercaptoethanol. Further purification of HgBP20 by DEAE-cellulose ion-exchange chromatography resulted in the resolution of three major mercury-binding protein peaks; analysis of two of these showed that both had similar amino acid compositions with 26% half-cystine, 16% glycine, and very low levels of the aromatic amino acids phenylalanine and tyrosine (0.3-0.5%), histidine (0.4%), methionine (about 0.5%), and leucine (about 1%). These are similar to the compositions of proteins reported as mussel thioneins by others. Separation of HgBP20 by anion-exchange high-performance liquid chromatography resulted in the resolution of six peaks, indicating a more complex situation than was evident from DEAE-cellulose separations. Although not completely purified, these also contain cysteine- and glycine-rich proteins.

Characterization of mercury-binding proteins from the gills of marine mussels exposed to mercury

1. A low molecular weight, mercury-binding protein was isolated from the gills of Mytilus edulis and partially characterized. 2. This protein had a minimum mol. wt of 14,400 daltons and 115 amino acid residues per protein molecule. 3. The relatively low amounts of half-cystine (7.8 mol%) in this protein and the presence of aromatic amino acids and histidine were not characteristic of metallothionein. 4. The amino acid composition was similar to a cadmium-binding protein of mussels reported by other researchers.

Mechanism of fetal poisoning by mercury. Distribution of mercury-binding protein in the placenta and fetus of pregnant mice

Mechanism of fetal poisoning by mercury. Distribution of mercury-binding protein in the placenta and fetus of pregnant mice