Titration Of Metal Ions Research Articles

Investigations of the Molecular Mechanism of Metal-Induced Aβ (1−40) Amyloidogenesis

Transition-metal ions (Cu2+ and Zn2+) play critical roles in the Abeta plaque formation. However, precise roles of the metal ions in the Abeta amyloidogenesis have been controversial. In this study, the molecular mechanism of the metal-induced Abeta oligomerization was investigated with extensive metal ion titration NMR experiments. Upon additions of the metal ions, the N-terminal region (1-16) of the Abeta (1-40) peptide was selectively perturbed. In particular, polar residues 4-8 and 13-15 were more strongly affected by the metal ions, suggesting that those regions may be the major binding sites of the metal ions. The NMR signal changes of the N-terminal region were dependent on the peptide concentrations (higher peptide concentrations resulted in stronger signal changes), suggesting that the metal ions facilitate the intermolecular contact between the Abeta peptides. The Abeta (1-40) peptides (>30 microM) were eventually oligomerized even at low temperature (3 degrees C), where the Abeta peptides are stable as monomeric forms without the metal ions. The real-time oligomerization process was monitored by 1H/15N HSQC NMR experiments, which provided the first residue-specific structural transition information. Hydrophobic residues 12-21 initially underwent conformational changes due to the intermolecular interactions. After the initial structural rearrangements, the C-terminal residues (32-40) readjusted their conformations presumably for effective oligomerization. Similar structural changes of the metal-free Abeta (1-40) peptides were also observed in the presence of the preformed oligomers, suggesting that the conformational transitions may be the general molecular mechanism of the Abeta (1-40) amyloidogenesis.

Solution Structure and Backbone Dynamics of an Endopeptidase HycI from scherichia coli: IMPLICATIONS FOR MECHANISM OF THE [NiFe] HYDROGENASE MATURATION

[NiFe] hydrogenases are metalloenzymes involved in many biological processes concerning the metabolism of hydrogen. The maturation of the large subunit of these hydrogenases requires the cleavage of a peptide at the C terminus by an endopeptidase before the final formation of the [NiFe] metallocenter. HycI is an endopeptidase of the M52 family and responsible for the C-terminal cleavage of the large subunit of hydrogenase 3 in Escherichia coli. Although extensive studies were performed, the molecular mechanism of recognition and cleavage of hydrogenase 3 remains elusive. Herein, we report the solution structure of E. coli HycI determined by high resolution nuclear magnetic resonance spectroscopy. This is the first solution structure of the apo form of endopeptidase of the M52 family reported thus far. The overall structure is similar to the crystal structure of holo-HybD in the same family. However, significant diversity was observed between the two structures. Especially, HycI shows an open conformation at the putative nickel-binding site, whereas HybD adopts a closed conformation. In addition, we performed backbone dynamic studies to probe the motional properties of the apo form of HycI. Furthermore, the metal ion titration experiments provide insightful information on the substrate recognition and cleavage processes. Taken together, our current structural, biochemical, and dynamic studies extend the knowledge of the M52 family proteins and provide novel insights into the biological function of HycI.

Truncated Semenogelin I Binds Zinc and Is Cleaved by Prostate‐Specific Antigen

Semenogelins I and II are major coagulum-forming proteins in semen, and they are secreted mainly by the seminal vesicles. These proteins bind Zn(2+) and act as substrates for prostate-specific antigen and transglutaminase. A variant semenogelin I lacking 60 amino acids has been described that occurs in different populations with an allele frequency of 1%-3%. To better understand the function of the semenogelins in vivo, our aim was to characterize the properties of the variant form and compare with the wild type. Recombinant proteins were synthesized in insect cells. Binding of Zn(2+) was studied by titration of metal ions in the presence of a zinc (II) fluorophore chelator. SDS-PAGE was used to visualize the results of cleavage by prostate-specific antigen and cross-linking with transglutaminase. We found that the truncated and wild-type semenogelin molecules had similar Zn(2+)-binding properties (ie, a stoichiometry of at least 9-10 mol per mol of protein and an average dissociation constant of 5 mumol/L per site), and they showed also similar susceptibility for degradation by prostate-specific antigen. Furthermore, like the wild-type form, the truncated semenogelin I was able to serve as a substrate for transglutaminase. These findings imply that the studied characteristics do not depend on a well-defined tertiary structure, or that the deletion has no major effect on the structure responsible for these features.

Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH3-d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH3-3TCTP because of a 160-fold acceleration of the catalytic constant kpol. Mechanistic insights into the rate enhancement were obtained using various alpha-boranophosphate nucleotides. The presence of the BH3 group renders kpol independent of amino acid substitutions present in RT. Indeed, the approximately 100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH3-dTTP. Metal ion titration studies show that alpha-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg2+ ions to the active site of the RT.DNA.dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

Binding of Cu(II) or Zn(II) in a de novo designed triple-stranded alpha-helical coiled-coil toward a prototype for a metalloenzyme.

We previously reported the IZ-3adH peptide, which formed a triple-stranded coiled-coil after binding Ni(II), Cu(II), or Zn(II). In this paper, we report the peptide, IZ-3aH, having a new metal binding specificity. The IZ-3aH peptide was found to bind Cu(II) and Zn(II) and form a triple-stranded coiled-coil. However, it did not bind Ni(II). Metal ion titrations monitored by circular dichroism revealed that the dissociation constants, K(d) were 9 microm for Zn(II) and 10 microm for Cu(II). The bound Cu(II) ion has a planar tetragonal geometry, where the coordination positions are three nitrogens of the His residues and one H(2)O.

Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif.

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.

Single-molecule transition-state analysis of RNA folding.

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.

Solvent protection of the hammerhead ribozyme in the ground state: evidence for a cation-assisted conformational change leading to catalysis.

Tertiary folding of the hammerhead ribozyme has been analyzed by hydroxyl radical footprinting. Three hammerhead constructs with distinct noncore sequences, connectivities, and catalytic properties show identical protection patterns, in which conserved core residues (G5, A6, U7, G8, and A9) and the cleavage site (C17, G1.1, and U1.2) become reproducibly protected from nucleolytic attack by radicals. Metal ion titrations show that all protections appear together, suggesting a single folding event to a common tertiary structure, rather than an ensemble of different folds. The apparent binding constants for folding and catalysis by Mg(2+) are lower than those for Li(+) by 3 orders of magnitude, but in each case the protected sites are identical. For both Mg(2+) and Li(+), the ribozyme folds into the protected tertiary structure at significantly lower cation concentrations than those required for cleavage. The sites of protection include all of the sites of reduced solvent accessibility calculated from two different crystal structures, including both core and noncore nucleotides. In addition, experimentally observed protected sites include additional sequences adjacent to those predicted by the crystal structures, suggesting that the solution structure may be folded into a more compact shape. A 2'-deoxy substitution at G5 abolishes all protection, indicating that the 2'-OH is essential for folding. Together, these results support a model in which low concentrations of metal ions fold the ribozyme into a stable ground state tertiary structure that is similar to the crystallographic structures, and higher concentrations of metal ions support a transient conformational change into the transition state for catalysis. These data do not themselves address the issue as to whether a large- or small-scale conformational change is required for catalysis.

The bulge region of HIV-1 TAR RNA binds metal ions in solution.

Binding of Mg2+, Ca2+ and Co(NH3)6(3+) ions to the HIV-1 TAR RNA in solution was analysed by 19F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for 19F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg2+ and Ca2+ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K(d)) of 0.9 +/- 0.6 and 2.7 +/- 1.7 mM, respectively. Argininamide, inducing largest (19)F chemical shifts changes at FU-23, was used as a reference ligand (K(d) = 0.3 +/- 0.1 mM). In the Pb2+-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg2+ and Ca2+ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb2+-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH3)6(3+) > Mg2+ > Ca2+] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.

Development of Novel Redox Systems by Use of Ligand Effect and Its Application to Potentiometry

The redox potential of a system involving metal ions can be modified by the complexation of metal ions with a suitable ligand. By using this phenomenon, novel redox systems which are applicable to quantitative analyses of metal ions have been developed. Some potentiometric titrations of metal ions and new types of spectrophotometric determinations of metal ions and complexing agents based on the ligand effect are presented in full detail. Estimation of complexing capacity in natural water and a new kinetic-catalytic method for copper(II) are also discussed. In addition, novel potentiometric flow titration has been proposed based on the relationship of the flow rates between titrant and sample solutions.

Estimation of the role of side reactions in the simulation of complexometric titration

A mathematical model was selected to describe the titration of metal ions with an arbitrary reagent in the presence of masking agents. The model takes into account the stepwise character of the complexation reaction and side reactions of protonation, hydrolysis and masking. The computed titration curves were in good agreement with the data of potentiometric titration. The corresponding program was used to optimize the titration conditions, to select promising titrants (other than complexones), and to predict systematic errors due to the shift of the titration jump in the presence of masking agents. Because of the last-named effect, the results of analysis may be underestimated several times

Sulphonated azoligand for metal ion determination in ion interaction chromatography

An azosulphonated ligand, namely cyclo-tris-7-(1-azo-8-hydroxynaphthalene-3,6-disulphonic acid), Calcion, which is generally used as a metallochromic indicator for metal ion titrations, has been studied in order to verify its suitability as a new ligand for metal ion separation in a liquid chromatography technique based on the ion interaction mechanism. Before chromatographic method development, studies on the complexation capabilities of Calcion with Ni 2+, Cu 2+, Fe 3+ and Al 3+ metal ions were performed, determining the stability constants of the complexes between such metal ions and the ligand. The procedure is based on ion-exchange chromatography and involves the retention of the negatively charged complexes on an anion-exchange resin and the determination of free and complexed metal by inductively coupled plasma atomic emission spectrometry. Chromatographic separation of Ni 2+–Cu 2+and Fe 3+–Al 3+ was reached, using a silica-based octyl stationary phase, through pre-complexation with Calcion, and UV–visible detection of the resulting anionic complexes. The effect of the main mobile phase parameters (concentration of the ion interaction reagent tetrabutylammonium chloride, organic modifier methanol and pH) on k′ of analytes was studied and optimization of the metal ion separation was achieved. In order to obtain the simultaneous determination of metal ions, the concentration of CH 3OH and pH gradient elution were also considered. The method developed has been applied to tap water analysis.

Amperometric end-point detection of complexometric titrations with platinum microelectrodes

An amperometric method is proposed for end-point detection of complexometric titrations of metal ions in unbuffered solutions with disodium ethylenediaminetetraacetate (Na2H2Y). It is based on the use of a platinum disc microelectrode as a sensor for monitoring the hydrogen ions delivered during the titration. Synthetic solutions containing Ca2+ and Mg2+ ions, each alone or in mixture, are examined. The method is also applied to the determination of the total concentration of Ca2+ and Mg2+, i.e., the total hardness, in mineral and tap waters. The precision of the method is satisfactory, the relative standard deviation being not greater than 2% for at least three replicates. Comparative measurements performed by the classical procedures using a metallochromic indicator give data agreement within 3.5%.

Metal Ion Induced Self-Assembly of a Designed Peptide into a Triple-Stranded α-Helical Bundle: A Novel Metal Binding Site in the Hydrophobic Core

Coiled coils, which mediate the associations and regulate the functions of various proteins, have a representative amino acid sequence of (defgabc)n heptad repeats and usually have hydrophobic residues at the a and d positions. We have designed a triple-stranded parallel α-helical coiled coil, in which the amino acid sequence is YGG(IEKKIEA)4. To construct a peptide that undergoes metal ion induced self-assembly into a triple-stranded coiled coil, we engineered a metal binding site in the hydrophobic core of the coiled coil. We replaced two Ile residues of the third heptad with His residues. The peptide had a random structure in aqueous solution. In contrast, in the presence of a transition metal ion, the peptide exhibited an α-helical conformation. The metal-complexed peptide was triple stranded and had a parallel orientation, as determined by sedimentation equilibrium and fluorescence quenching analyses. Metal ion titrations monitored by circular dichroism revealed that the dissociation constants, Kd, w...

Metal binding properties of three Cys 2X 2 (X = His, Asp) metallothionein-related peptides

The metal binding properties of two chemically synthesized peptides, Ac-Gly-Asp-His-Gly-Cys-Ser-Gly-Ala-Ser-Ser-Cys-Thr-His-Ala-Ser-NH 2 and Ac-Pro-Asp-His-Ser-Cys-Ser-Thr-Gly-Gly-Ser-Cys-Thr-His-Thr-Ser-NH 2, with coordination sites Cys 2His 2 were characterized. The peptides are based on a metallothionein consensus sequence in which two cysteine residues have been replaced by histidine. Spectrophotometric titrations using Co 2+ as a probe indicated that both peptides form 1:1 peptide-Co(II) complexes, and allowed determination of the corresponding affinity constants. The absorption spectrum of the peptide-Co(II) complexes is highly suggestive of tetrahedral coordination involving two cysteine and two histidine residues. Assuming that Zn(II) and Cd(II) have the same coordination environments as Co(II), the affinity constants for the peptide complexes with Zn 2+ and Cd 2+ were determined via a series of competitive metal ion titrations. For both peptides the trend in relative affinities for the metals studied follows the order Cd(II) ≈ Zn(II) > Co(II). Analogous spectroscopic studies with a third metallothionein-related peptide of sequence Ac-Gly-Asp-Cys-Gly-Asp-Ser-Gly-Ala-Ser-Ser-Cys-Thr-Asp-Ala-Ser-NH 2, with a putative coordination site Cys 2Asp 2, showed that this peptide does not bind Co 2+ with tetrahedral geometry.

Design and Synthesis of a Transition Metal Responsive Semisynthetic Myoglobin-Bearing Iminodiacetic Acid Moiety

Iminodiacetic acid appended myoglobins (IDAn-Mb, n = 1, 2) were synthesized by conventional reconstitution of chemically modified hemes with apomyoglobin. The metal responsive property of the obtained IDAn-Mb was studied by metal ion titration, pH titration, circular dichroism (CD) and 1H NMR spectroscopies, and reduction with ascorbate. IDAn-Mb quantitatively bound various transition metal cations (Co2+, Ni2+, Zn2+, Cd2+, and Cu2+) but not Mg2+. The binding stoichiometry of IDA2-Mb was 1:1 for Co2+, Ni2+, Zn2+, and Cd2+ and 1:2 for Cu2+, whereas the stoichiometry of 1:1 was shown for IDA1-Mb to all transition metals (Co2+, Ni2+, Zn2+, Cd2+, and Cu2+). The acidic pKa shift of the H2O coordinated to the heme iron(III) was clearly observed upon the binding of transition metals, suggesting the microenvironmental change of the heme crevice. This was supported by the CD and 1H NMR spectra of IDAn-Mb. The transition metal induced structural changes of IDAn-Mb were reflected in their redox behavior, i.e., the re...

Modification of the N-terminus of human factor IX by defective propeptide cleavage or acetylation results in a destabilized calcium-induced conformation: effects on phospholipid binding and activation by factor XIa.

The propeptide of human coagulation factor IX (FIX) directs the gamma-carboxylation of the first 12 glutamic acid residues of the mature protein into gamma-carboxyglutamic acid (Gla) residues. The propeptide is normally removed before secretion of FIX into the blood. However, mutation of Arg-4 in the propeptide abolishes propeptide cleavage and results in circulating profactor IX in the blood. We studied three such genetic variants, factor IX Boxtel (Arg-4-->Trp), factor IX Bendorf (Arg-4-->Leu) and factor IX Seattle C (Arg-4-->Gln). These variant profactor IX molecules bind normally to anti-FIX:Mg(II) antibodies, which indicates that the mutations do not seriously affect gamma-carboxylation. Metal ion titration of the binding of variant profactor IX to conformation-specific antibodies demonstrates that the calcium-induced conformation is destabilized in the variant molecules. Also the binding of FIX Boxtel to phospholipids and its activation by factor XIa requires a high (>5 mM) calcium concentration. The three-dimensional structure of the Gla domain of FIX in the presence of calcium indicates that the acylation of the amino-terminus, rather than the presence of the propeptide, was responsible for the destabilization of the calcium-induced conformation. In order to confirm this, the alpha-amino group of Tyr1 of FIX was acetylated. This chemically modified FIX showed a similar destabilization of the calcium-induced conformation to variant profactor IX. Our data imply that the amino-terminus of FIX plays an important role in stabilizing the calcium-induced conformation of the Gla domain of FIX. This conformation is important for the binding to phospholipids as well as for the activation by factor XIa. Our results indicate that mutations in FIX that interfere with propeptide cleavage affect the function of the protein mainly by destabilizing the calcium-induced conformation.

Paraformaldehyde as an end-point indicator in hydrolytic thermometric titration of metal ions and iodine

The use of paraformaldehyde (PF) as an end-point indicator in hydrolytic titrations involving precipitation, complexation and redox reactions was investigated. The decrease in temperature caused by depolymerization of PF, catalysed by hydroxyl ions produced by hydrolysis of excess of titrant, was employed to locate the end-point of the titration. The results of titrations of alkaline earth metals with carbonate, several metal ions with EDTA and iodine with arsenite are presented.

Voltammetry of Pb(II), Cd(II) and Zn(II) ions in the presence of the sulphated polysaccharide λ-carrageenan

A systematic study of the voltammetric redox processes of Pb(II), Cd(II) and Zn(II) in the presence of λ-carrageenan has been made by normal pulse, reverse pulse, and differential pulse polarography, as well as by differential pulse anodic stripping voltammetry. Titrations of metal ions with λ-carrageenan allow the determination of the global formation constant of each system and their dependences on KNO 3 (supporting electrolyte) concentration and pH values. Reversibility of the redox couples M(II) M(Hg) is not strongly affected by the presence of λ-carrageenan, which gives small non-Faradaic signals. In the time scale of used techniques M( II)/ λ-carrageenan systems seem to be of labile nature.

Potentiometric ion‐pair titrations of metal ions forming complexes with 1,10‐phenanthroline and its derivatives

AbstractSimple coated‐wire electrodes, prepared using an ordinary aluminum wire conductor covered with the plasticized poly(vinyl chloride) membrane, were used in potentiometric titrations of metal ions forming complexes with 1,10‐phenanthroline and its derivatives. The method precipitation of ion pairs formed by metal phenanthroline complexes and tetraphenylborate. Titractions were accompanied with titration curves yielding very sharp potential breaks, so that very reproducible results could be taken even in titrations of quite diluted solutions (down to 2.5 × 10−7 mol of the metal ion in the 25 cm3 of the sample titrated). Using the procedure described the following cations were titratable in forms of their 1.10‐phenanthroline complexes: Zn2+, Cd2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Vo2+, Ga3−, In3−, and Tl3−. Some of the metal ion solutions, prepared from the salts of analytical‐reagent‐grade purity, can be recommended for use in standardization procedures of the sodium tetraphenylborate titrant. 2.9‐Dimethyl‐1,10‐phenanthroline complexes of Zn2+, Cd2+, and Cu+ were also titrable, but the results were significantly erroneous. Metal 4,7‐diphenyl‐1,10‐phenathroline complexes were insoluble in water as well as in water/ethanol mixtures.