Absence Of Magnesium Ions Research Articles

Phage display identifies Affimer proteins that direct calcium carbonate polymorph formation.

A key factor in biomineralization is the use of organic molecules to direct the formation of inorganic materials. However, identification of molecules that can selectively produce the calcium carbonate polymorphs calcite or aragonite has proven extremely challenging. Here, we use a phage display approach to identify proteins - rather than the short peptides typically identified using this method - that can direct calcium carbonate formation. A 1.3 × 1010 library of Affimer proteins was displayed on modified M13 phage, where an Affimer is a ≈13 kDa protein scaffold that displays two variable regions of 9-13 residues. The phage displaying the Affimer library were then screened in binding assays against calcite and aragonite at pH 7.4, and four different strongly-binding proteins were identified. The two aragonite-binding proteins generated aragonite when calcium and magnesium ions were present at a 1 : 1 ratio, while the calcite-binding proteins produce magnesium-calcite under the same conditions. Calcite alone formed in the presence of all four proteins in the absence of magnesium ions. In combination with molecular dynamics simulations to evaluate the conformations of the proteins in solution, this work demonstrates the importance of conformation in polymorph control, and highlights the importance of magnesium ions, which are abundant in seawater, to reduce the energetic barriers associated with aragonite formation.

Regioselective formation of RNA strands in the absence of magnesium ions.

The oligomerization of ribonucleotides can produce short RNA strands in the absence of enzymes. This reaction gives one of two regioisomeric phosphodiester linkages, a 2′,5′- or a 3′,5′-diester. The former, non-natural linkage is detrimental for duplex stability, and is known to form preferentially in oligomerizations occurring in homogeneous solution with preactivated nucleotides in the presence of magnesium cations. We have studied ribonucleotide oligomerization with in situ activation, using NMR as monitoring technique. Unexpectedly, the known preference for 2′,5′-linkages in the oligomerization of AMP was reversed in the absence of magnesium ions at slightly basic pH. Further, oligomerization was surprisingly efficient in the absence of Mg2+ salts, producing oligomers long enough for duplex formation. A quantitative systems chemistry analysis then revealed that the absence of magnesium ions favors the activation of nucleotides, and that the high concentration of active species can compensate for slower coupling. Further, organocatalytic intermediates can help to overcome the unfavorable regioselectivity of the magnesium-catalyzed reactions. Our findings allay concerns that RNA may have been difficult to form in the absence of enzymes. They also show that there is an efficient path to genetic material that does not require mineral surfaces or cations known to catalyze RNA hydrolysis.

The third structural switch in the archaeal translation initiation factor 2 (aIF2) molecule and its possible role in the initiation of GTP hydrolysis and the removal of aIF2 from the ribosome.

The structure of the γ subunit of archaeal translation initiation factor 2 (aIF2) from Sulfolobus solfataricus (SsoIF2γ) was determined in complex with GDPCP (a GTP analog). Crystals were obtained in the absence of magnesium ions in the crystallization solution. They belonged to space group P1, with five molecules in the unit cell. Four of these molecules are related in pairs by a common noncrystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 γ-subunit structures in the GTP-bound state show that (i) the magnesium ion is necessary for the formation and the maintenance of the active form of SsoIF2γ and (ii) in addition to the two previously known structural switches 1 and 2, eukaryotic translation initiation factor 2 (eIF2) and aIF2 molecules have another flexible region (switch 3), the function of which may consist of initiation of the hydrolysis of GTP and the removal of e/aIF2 from the ribosome after codon-anticodon recognition.

Investigating the effect of copper and magnesium ions on nitrogen removal capacity of pure cultures by modified non-competitive inhibition model

Copper, a common heavy metal, may be beneficial for or poisonous to microbial activity. The objective of this study was to determine the effect of different copper ion concentrations on the nitrogen removal performance of Arthrobacter arilaitensis strain Y-10 and Pseudomonas taiwanensis strain J488. The non-competitive inhibition model was employed to evaluate the 50% inhibition concentrations (IC50 values) of copper ions toward the pure strains. In the absence of magnesium ions, a low concentration of copper (0.1 mg/L) significantly enhanced the ammonium removal ability of strain Y-10 and its removal efficiency increased by 10.88% compared with the control treatment. Copper ranging from 0 to 0.1 mg/L had no significant effect on the ammonium removal capacity of strain J488. After adding 9.90 mg/L of magnesium to the basal medium, the effects of copper on nitrification of ammonium or denitrification of nitrate or nitrite were also assessed. In these conditions, 0.25 mg/L copper ions could strongly inhibit the ammonium, nitrate and nitrite removal activities for strain Y-10. For strain J488, no clear deterioration in ammonium removal efficiency was observed at copper ion concentrations below 0.5 mg/L, but 0.25 mg/L copper ions significantly inhibited nitrate and nitrite removal efficiencies, which were only 45.88% and 6.35%, respectively. The IC50 values of copper ions for nitrate and nitrite removal by strain Y-10 were 0.195 and 0.090 mg/L respectively; for strain J488, the IC50 values were 0.175 and 0.196 mg/L. The magnesium ions could improve the cell growth, nitrogen removal efficiency and copper ion resistance of bacteria.

Binding of Potassium Ions Inside the Access Channel at the Cytoplasmic Side of Na+,K+-ATPase

Binding of potassium ions through an access channel from the cytoplasmic side of Na+,K+-ATPase and the effect of pH and magnesium ions on this process have been studied. The studies were carried out by a previously developed method of measuring small increments of the admittance (capacitance and conductivity) of a compound membrane consisting of a bilayer lipid membrane with adsorbed membranes fragments containing Na+,K+-ATPase. The capacitance change of the membrane with the Na+,K+-ATPase was induced abruptly by release of protons from a bound form (caged H+) upon a UV-light flash in the absence of magnesium ions. The change of admittance consisted of an initial fast jump and a slow relaxation to a stationary value within a time of about 1–2 s. The kinetics of the capacitance relaxation depended on pH and the concentration of magnesium and potassium ions. The dependence of the rapid capacitance jump on the potassium concentration corresponded to the predictions of the model developed earlier that describes binding of sodium or potassium ions in competition with protons. The effect of magnesium ions can be explained by the assuming that they bind to the Na+,K+-ATPase and affect binding of potassium ions because of either changes in protein conformation or the creation of an electrostatic field in the access channel on the cytoplasmic side.

Biomineralization in Mediterranean Corals: The Role of the Intraskeletal Organic Matrix

The precipitation of calcium carbonate was carried out in the presence of the intraskeletal organic matrix (OM) extracted from Mediterranean corals. They were diverse in growth form and trophic strategy, Balanophyllia europaea and Leptopsammia pruvoti—solitary corals, only the first zooxanthellate coral—and Cladocora caespitosa and Astroides calycularis—colonial corals, only the first zooxanthellate coral. The results showed that, although the OM marked differences among species, the diverse influence over the calcium carbonate precipitation was evident only for B. europaea. This OM was the most prone to favor the precipitation of aragonite in the absence of magnesium ions, according to overgrowth and solution precipitation experiments. In artificial seawater, where magnesium ions were present, this OM, as well the one from A. calycularis, precipitated mainly a form of amorphous calcium carbonate different from that obtained with SOM from L. pruvoti or C. caespitosa. The amorphous calcium carbonate from B...

Single-Step Purification and Characterization of Recombinant Aspartase of Aeromonas media NFB-5

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

Formation of Caries-Like Lesions in vitro on the Root Surfaces of Human Teeth in Solutions Simulating Plaque Fluid

Lesion formation on root surfaces of human posterior teeth was studied in acetate/lactate buffers with a background electrolyte composition based on plaque fluid analyses. Lesion depth after 28 days at 37°C was measured in relation to: the presence or absence of cementum; the concentration of undissociated buffer; the presence or absence of magnesium ions at plaque fluid concentration. Each factor was evaluated at several values of –log(ion activity product for hydroxyapatite): pI_HA. Solutions were formulated to minimize variation in pH, which varied by ≤0.03 for a given comparison (individual pI_HA) and by 0.42–0.82 over the range of pI_HA within experiments. Lesions on surfaces from which cementum had been ground were significantly deeper than on intact surfaces, but this is considered to be due to subsurface mechanical damage and not to a solubility difference. Neither the concentration of undissociated buffer nor the presence of magnesium ions significantly affected lesion depth. Lesion depth was strongly influenced by the correlated variations in pI_HA and pH. At pI_HA 54 and 55, only extremely shallow lesions formed. From pI_HA 56, lesion depth increased with increasing pI_HA. The results confirm that the solubility of the mineral of root tissues is higher than that of hydroxyapatite, but indicate that it is probably lower than suggested by Hoppenbrouwers et al. [Arch Oral Biol 1987;32:319–322]. For calcium concentrations of 3–12 mM, the critical pH for root tissue mineral was calculated as 5.22–5.66 assuming solubility equivalent to pI_HA 54 and 5.08–5.51 assuming pI_HA 55.

The mechanism of acidic hydrolysis of esters explains the HDV ribozyme activity

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme that catalyzes the site-specific trans-esterification reaction. Using high hydrostatic pressure (HHP) technique we showed that HDV ribozyme catalyzes the reaction of RNA cleavage in the absence of magnesium ions according to mechanism of acidic hydrolysis of esters. HHP induces changes of water structure, lowering pH and effect ribozyme catalytic site structure formation without magnesium. HHP, similarly to magnesium ion at ambient pressure stabilizes the higher order RNA structure of HDV, but Mg(2+) is not involved in the catalysis. Our results clearly support the new mechanism of HDV hydrolysis and show advantages of using HHP in analysis of macromolecules interaction.

DNA Bubble Formation in Transcription Initiation

The properties of the DNA bubble in the transcription open complex have been characterized by topological analysis of DNA circles containing the lac UV5 promoter or the PR promoter from bacteriophage lambda. Topological analysis is particularly well suited to this purpose since it quantifies the changes in DNA duplex geometry caused by bubble formation as well as by superhelical DNA wrapping. The duplex unwinding that results from bubble formation is detected as a reduction in topological linking number of the DNA circle, and the precision of this measurement has been enhanced in the current study through the use of 8 or 10 promoter copies per circle. Several lines of evidence indicate that the linking number change induced by open complex formation is essentially all due to bubble generation, with very little derived from superhelical wrapping. Accordingly, the linking number change of -1.17 measured for the lac UV5 promoter indicates that the size of the lac UV5 bubble is about 12.3 base pairs, while the change of -0.98 measured for the lambda PR promoter indicates that the lambda PR bubble is 10.3 base pairs. It was also found that the presence or absence of magnesium ion had little effect on the value of the linking number change, a result that resolves the uncertainty associated with use of chemical probes to study the effect of magnesium on bubble size. Finally, the magnitude of linking number change increases progressively when the 3' end of a transcript is extended to +2 and +3 in an abortive initiation complex. This indicates that the transcription bubble expands at its leading edge in the abortive complex, results that confirm and extend the proposal of a DNA "scrunching" mechanism at the onset of transcription. These results are relevant to several models for the structure of DNA in the functional open complex in solution, and provide an important complement to the structural information available from recent crystal structures.

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.

Sister chromatid junctions in the hyperthermophilic archaeon Sulfolobus solfataricus

Although the Archaea exhibit an intriguing combination of bacterial- and eukaryotic-like features, it is not known how these prokaryotic cells segregate their chromosomes before the process of cell division. In the course of our analysis of the third replication origin in the archaeon Sulfolobus solfataricus, we identify and characterise sister chromatid junctions in this prokaryote. This pairing appears to be mediated by hemicatenane-like structures, and we provide evidence that these junctions persist in both replicating and postreplicative cells. These data, in conjunction with fluorescent in situ hybridisation analyses, suggest that Sulfolobus chromosomes have a significant period of postreplicative sister chromatid synapsis, a situation that is more reminiscent of eukaryotic than bacterial chromosome segregation mechanisms.

Oriented Aggregation and Novel Phase Transformation of Vaterite Controlled by the Synergistic Effect of Calcium Dodecyl Sulfate andn-Pentanol

Calcium dodecyl sulfate (CDS) was used for the first time both as an anionic surfactant and as the source of mineral ions in the precipitation process of calcium carbonate (CaCO3). The simple reaction of the enriched Ca2+ ions at the so-called organic-inorganic interfaces with the slowly bubbled CO2 gas resulted in the metastable vaterite polymorph with various structures. The single-crystalline vaterite of the hexagonal platelets, the lens-shaped structures with hexagonal symmetry, the olive-shaped superstructures and these with a concave at each top of olives, and another metastable polymorph of aragonite were obtained, respectively, depending upon the concentration ratio between CDS and n-pentanol. The synergistic effect of CDS and n-pentanol is believed to play a crucial role in driving the oriented aggregation of metastable nanoparticles. Simultaneously, the novel phase transformation of vaterite to aragonite was observed, implying the possible formation mechanism of aragonite at room temperature and in the absence of magnesium ions.

Internal Bulge and Tetraloop of the Catalytic Domain 5 of a Group II Intron Ribozyme Are Flexible: Implications for Catalysis

RNA molecules have an inherent flexibility that enables recognition of other interacting partners through potential disorder–order transitions, yet studies to quantify such motional dynamics remain few. With an increasing database of three-dimensional structures of biologically important RNA molecules, quantifying such motions becomes important to link structural deformations with function. One such system studied intensely is domain 5 (D5) from the self-splicing group II introns, which is at the heart of its catalytic machinery. We report the dynamics of a 36 nucleotide D5 from the Pylaiella littoralis group II intron in the presence and absence of magnesium ions, and at a range of temperatures (298K–318 K). Using high-resolution NMR experiments of heteronuclear nuclear Overhauser enhancement (NOE), spin-lattice ( R 1), and spin–spin ( R 2) 13C relaxation rates, we determined the rotational diffusion tensor of D5 using the ROTDIF program modified for RNA dynamic analysis (ROTDIF_RNA). The D5 rotational diffusion tensor has an axial symmetric ratio ( D ||/ D ⊥) of 1.7 ± 0.3, consistent with an estimated overall rotational correlation time of τ m = (2 D || + 4 D ⊥) −1 of 6.1(±0.3) ns at 298 K and 4.1(±0.2) ns at 318 K. The measured relaxation data were analyzed with the reduced spectral density mapping formalism using assumed values of the chemical shift anisotropy of the 13C spins. Both the relaxation data and the values of the spectral density function reveal that the functional groups in D5 implicated in magnesium ion binding and catalysis (catalytic triad, internal bulge, and tetraloop regions) exhibit thermally induced motion on a wide variety of timescales. Because these motions parallel those observed in the intramolecular stem–loop of the U6 element within the spliceosome, we hypothesize that such extensive dynamic disorder likely facilitates D5 engaging both binding and catalytic regions of the ribozyme, and these may be a conserved feature of the catalytic machinery essential for catalysis.

Highly Stable DNA Triplexes Formed with Cationic Phosphoramidate Pyrimidine α‐Oligonucleotides

The ability of cationic phosphoramidate pyrimidine alpha-oligonucleotides (ONs) to form triplexes with DNA duplexes was investigated by UV melting experiments, circular dichroism spectroscopy and gel mobility shift experiments. Replacement of the phosphodiester linkages in alpha-ONs with positively charged phosphoramidate linkages results in more efficient triplex formation, the triplex stability increasing with the number of positive charges. At a neutral pH and in the absence of magnesium ions, it was found that a fully cationic phosphoramidate alpha-TFO (triplex-forming oligonucleotide) forms a highly stable triplex that melts at a higher temperature than the duplex target. No hysteresis between the annealing and melting curves was noticed; this indicates fast association. Moreover, the recognition of a DNA duplex with a cationic alpha-TFO through Hoogsteen base pairing is highly sequence-specific. To the best of our knowledge, this is the first report of stable triplexes in the pyrimidine motif formed by cationic alpha-oligonucleotides and duplex targets.

Dynamic Profiles of DNA: Analysis of CAP- and LexA Protein-Binding Regions with Endonucleases

We examined ssDNA and dsDNA containing a CAP-binding region or a lexA protein-binding region in the recA gene of Escherichia coli with endonucleases (E. coli endonuclease I and bovine DNase I) to deduce the structural conformation of dsDNA as well as ssDNA. Each nuclease produced its own cleavage pattern. Some cleavages occurred at common sites in ssDNA and dsDNA. The common cleavage sites for endonuclease I included ones that have been inferred to be functionally important. Efficient cleavage occurred at bent sites in the CAP-binding region, and at "SOS box" sites in the recA gene. NTP (dNTP) greatly increased the cleavage at these sites in the ssDNA and dsDNA. Next, we investigated whether mutations which affect function can be ascribed to variation in the conformation of DNA. Polynucleotides containing a single base substitution derived from the mutants in the CAP-binding region were cleaved by endonuclease I. The cleavage pattern varied predominantly around the substituted nucleotide in dsDNA. Thus, we confirmed that DNA structure is closely related to function. Complexes of endonuclease I and synthetic polynucleotides that had one cleavage site were confirmed to exist in the absence of magnesium ions by gel-shift assay.

The Saccharomyces cerevisiae High Mobility Group Box Protein HMO1 Contains Two Functional DNA Binding Domains

High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K(d) = 39.6 +/- 5.0 nm and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 +/- 2.2 nm, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 +/- 1.1 nm). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure.

NMR studies of the alternative conformation of hammerhead ribozyme in the solution

The conformation of the hammerhead ribozyme was studied by NMR. In the absence of magnesium ion, the U turn does not exist at the core region. Consequently, stem II extends to stem I. G5 in the core region forms a base pair with C17 at the cleavage site. On the mutation of G16.2C15.2 in stem III by C16.2G15.2, G5 and U4 at the core region interact with C16.2 and U16.1, respectively, where stem III becomes shorter, while stem I does longer. On the addition of magnesium ions, U4 turn occurs in wild type, but does not in the mutation.

Holliday Junction Binding and Resolution by the Rap Structure-specific Endonuclease of Phage λ

Rap endonuclease targets recombinant joint molecules arising from phage λ Red-mediated genetic exchange. Previous studies revealed that Rap nicks DNA at the branch point of synthetic Holliday junctions and other DNA structures with a branched component. However, on X junctions incorporating a three base-pair core of homology or with a fixed crossover, Rap failed to make the bilateral strand cleavages characteristic of a Holliday junction resolvase. Here, we demonstrate that Rap can mediate symmetrical resolution of 50 bp and χ Holliday structures containing larger homologous cores. On two different mobile 50 bp junctions Rap displays a weak preference for cleaving the phosphodiester backbone between 5′-GC dinucleotides. The products of resolution on both large and small DNA substrates can be sealed by T4 DNA ligase, confirming the formation of nicked duplexes. Rap protein was also assessed for its capacity to influence the global conformation of junctions in the presence or absence of magnesium ions. Unlike the known Holliday junction binding proteins, Rap does not affect the angle of duplex arms, implying an unorthodox mode of junction binding. The results demonstrate that Rap can function as a Holliday junction resolvase in addition to eliminating other branched structures that may arise during phage recombination.

How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.