Effect Of Metal Ion Binding Research Articles

Aggregation of poly(N-isopropylacrylamide) homopolymer by Cu2+ and Zn2+: Significance for ratiometric metal ion indicators

Aqueous transition metal dications Cu2+ and Zn2+ bind to the amide groups of the responsive polymer poly(N-isopropylacrylamide) (PNIPAm). Ratiometric detection by the response of PNIPAm is based on the effect of metal ion binding to ligands that are copolymerized into PNIPAm. Metal ions bound to copolymerized ligands cause a polarity change that shifts the ratio of coil and globule polymer phases, whereby the coil:globule state is monitored by the FRET process of a pair of fluorophores distributed throughout the polymer. This work shows that aqueous Cu2+ or Zn2+ ([M2+] = 2.5 × 10−2 M to 2.5 × 10−4 M) cause aggregation of the PNIPAm homopolymer (Mn = 8400 and 10,300) in the globule phase above 32 °C, as determined by second-order and dynamic light-scattering. Conditional formation constants (pH 6.3) for Cu2+ binding PNIPAm were measured as log Kc = 2.64 ± 0.07 and 4.02 ± 0.05 for PNIPAm concentrations of 1 mg/mL and 0.05 mg/mL, respectively. The interaction of Cu2+ and Zn2+ with PNIPAm is therefore in competition with the copolymerized ligands and when detecting millimolar to high micromolar concentrations, this interaction poses a potential interference to metal ion detection. Several aggregation processes are implicated based on Cu2+ or Zn2+ from 8 × 10−5 to 2.5 × 10−2 M. These processes are fully reversible by reducing the sample temperature.

Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins.

The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of "in solution" techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity.

Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438-452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.

Structural studies of the tethered N-terminus of the Alzheimer's disease amyloid-β peptide

Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid-β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal-binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1-16 fused to the N-terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti-Aβ N-terminal antibody WO2. The structure demonstrates that Aβ residues 10-16, which are not in complex with the antibody, adopt a mixture of local polyproline II-helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10-16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13-metal-His14 coordination in the Aβ1-16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N-terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal-binding-induced neurotoxicity.

Bipyridyl-modified phosphonium polyelectrolytes: synthesis, photophysics, metal ion coordination and layer-by-layer assembly with anionic conjugated polymers

A novel class of chromophoric polyelectrolytes bearing phosphonium moieties in their backbones has been prepared to incorporate metal-binding 2,2′-bipyridyl (bipy) units. The effect of metal ion binding on the photophysical properties of the polyelectrolyte was probed. By binding metal ions to the polycation, the relative charge loading could be progressively increased as a strategy to control its supramolecular assembly with polyanions. The metallated and metal-free polyelectrolytes were utilized in layer-by-layer self-assembly with poly(acrylic acid) or anionic conjugated polymers including a poly(p-phenylene vinylene) and polythiophene derivative. The photophysical properties, nanomorphology, and substrate dependence of the resultant multilayer films were examined, and indicate that layer density, composition, and morphology can be tuned by metallation and ion pairing effects.

Role of metal ions in aggregation of intrinsically disordered proteins in neurodegenerative diseases

Neurodegenerative diseases constitute a set of pathological conditions originating from the slow, irreversible, and systematic cell loss within the various regions of the brain and/or the spinal cord. Depending on the affected region, the outcomes of the neurodegeneration are very broad and diverse, ranging from the problems with movements to dementia. Some neurodegenerative diseases are associated with protein misfolding and aggregation. Many proteins that misfold in human neurodegenerative diseases are intrinsically disordered; i.e., they lack a stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) functionally complement ordered proteins, being typically involved in regulation and signaling. There is accumulating evidence that altered metal homeostasis may be related to the progression of neurodegenerative diseases. This review examines the effects of metal ion binding on the aggregation pathways of IDPs found in neurodegenerative diseases.

Remarkable Metal Counterion Effect on the Internucleotide J-Couplings and Chemical Shifts of the N−H···N Hydrogen Bonds in the W−C Base Pairs

The effects of metal ion binding on the (2h) J(NN)-coupling and delta( (1)H)/Deltadelta( (15)N) chemical shifts of N-H...N H-bond units in internucleotide base pairs were explored by a combination of density functional theory calculations and molecular dynamics (MD) simulations. Results indicate that the NMR parameters vary considerably upon cation binding to the natural GC or AT base pairs, and thus can be used to identify the status of the base pairs, if cation-perturbed. The basic trend is that cation perturbation causes (2h) J(NN) to increase, Deltadelta( (15)N) to decrease, and delta( (1)H) to shift upfield for GC, and in the opposite directions for AT. The magnitudes of variation are closely related to the Lewis acidity of the metal ions. For both base pair series (M(z+)GC and M(z+)AT), these NMR parameters are linearly correlated among themselves. Their values depend strongly on the energy gaps (Delta(ELP-->sigma*)) and the second-order interaction energies ( E(2)) between the donor N lone pair (LP(N)) and the acceptor sigma* N-H localized NBO orbitals. In addition, the (2h) J NN changes are also sensitive to the amount of sigma charge transfer from LP(N) to sigma*(N-H) NBOs or from the purine to the pyrimidine moieties. The different trends are a consequence of the different H-bond patterns combined with the polarization effect of the metal ions in the cationized M(z+)AT series, M(z+) <-- A --> T, and the cationized GC series, M(z+) <-- G <-- C. The predicted cation-induced systematic trends of (2h) J(NN) and delta( (15)N, (1)H) in N-H...N H-bond units may provide a new approach to the determination of H-bond structure and strength in Watson-Crick base pairs, and provide an alternative probe of the heterogeneity of DNA sequences.

Hydrophobic Environment of Cysteine Residues in Pig Brain S-100 Proteins

Abstract The effects of metal ion binding to pig brain S-100 proteins on the hydrophobicity of their cysteine (Cys) residues were examined using the fluorescent thiol reagent, N-(1-anilinonaphthyl-4)maleimide (ANM). The hydrophobicity order was Ca2+/S-100 &amp;lt; K+/S-100 &amp;lt; Mg2+/S-100 &amp;lt; S-100 &amp;lt; Zn2+/S-100.

Effects of Metal Ion Binding on Structural Dynamics of Human Hemopexin

Hemopexin (Hx) functions as a major heme scavenging protein in blood plasma and as such circulates without heme bound. In recent work, we have demonstrated that Hx binds metal ions in vitro in a manner that varies from one metal ion to another and that changes with heme binding. The structural consequences of metal ion binding to the form of Hx that dominates in plasma have now been evaluated by monitoring metal ion-linked changes in tertiary structure of the protein as reflected by changes in the near-UV CD spectrum and the ultraviolet absorption spectrum as a function of temperature. As part of this analysis we have developed thermally induced difference absorption maps (TIDAMs) to afford efficient visualization of temperature-dependent changes in the UV spectrum of Hx that are induced by binding of metal ions. The results are interpreted in terms of recent models proposed for metal ion binding sites on Hx and have implications for the possible modulation of heme binding to Hx by metal ions in vivo.

Monovalent cation-promoted ordering of a glycine-rich cyclic peptide

This report describes an accelerated self-assembly of a synthetic cyclic hexapeptide in the presence of alkali metal ions. Time-dependent aggregation of hexapeptide was considerably influenced upon co-incubation with monovalent metal ions, of which K + afforded the most significant effect both on the time-scale required for self-assembly and on the morphology of aged structures. Metal ion adducts formation ability of the hexapeptide was confirmed by electrospray ionization mass spectrometry measurements and 13C NMR spectrometry. The effect of metal ion binding on peptide structure was also probed by circular dichroism, optical microscopy, and scanning electron microscopy. K + ions not only interacted more efficiently with the hexapeptide enabling it to reach conformational state(s) conducive for self-assembly, but also altered the morphologies of the aged peptide fibers, when compared to the unmetalated peptide.

Computational studies on siblings en-route to fullerenes: Study of curved polycyclic aromatic hydrocarbons

Computational studies at ab initio, DFT and semiempirical levels were carried out on a large number of buckybowl molecules. Corannulene 2 and Sumanene 3 constitute the primary buckybowls as they are readily identifiable C 60 fragments along the C 5 and C 3 axes, respectively. These studies are directed at understanding the most elegant strategy to modulate the curvature of buckybowl molecules. The importance of choosing the right precursor for the successful synthesis of the target buckybowl molecules were highlighted. The effect of sequential ring annelation to the rim of buckybowl on their structures, curvature and inversion dynamics were studied. The energy profiles of the substituted buckybowls were fit to a double-well potential, which results in an empirical correlation between the bowl-to-bowl inversion and the bowl depths. The effect of metal ion binding to the concave and convex surfaces of the bowl structure and the inversion dynamics was also studied.

The effect of surface attachment on ligand binding: studying the association of Mg2+, Ca2+and Sr2+by 1-thioglycerol and 1,4-dithiothreitol monolayers

The difference in the heterogeneous binding of Mg(2+), Ca(2+) and Sr(2+) ions by 1-thioglycerol (TG) and 1,4-dithiothreitol (DTT) spontaneously adsorbed monolayers on Au has been studied following the changes in the double layer capacity. A mathematical treatment, based on calculating the electrochemical potential difference at the monolayer-electrolyte interface, has followed our recent work which dealt with the acid-base equilibrium at the interface as a means of calculating the pK of ionizable SAMs and their binding with Cd(2+). Experimentally, spontaneously adsorbed monolayers of TG and DTT were assembled on Au surfaces and studied by impedance spectroscopy and alternating current voltammetry (ACV). The capacity was measured for each of the modified surfaces at increasing concentrations of the divalent metal ions separately. The goal of this study has been to examine the effect of metal ion binding by similar ligands that are differently attached onto the surface. TG and DTT monolayers differ in their flexibility, which is a result of their attachment to the surface through one and two arms, respectively. The general trend of the apparent heterogeneous association constants of the divalent metal ions, which were calculated from the capacity measurements, was substantially different from the classical Irving-Williams series that is applicable to homogeneous systems. This difference could be nicely explained by the reduction of the degree of freedom and flexibility of the attached ligands.

Effect of metal ion binding of chitosan on the printability of pretreated wool fabric

In this study, chitosan was used as a pretreatment agent for wool fabric to increase the metal binding during the mordanting process with copper sulphate. The copper(II) binding was measured by measuring volumetrically the concentration of metal ions that remained in the solution after mordanting. The pretreated mordanted wool samples were then subjected to printing with a natural dye, 2-hydroxy-1,4-naphthoquinone (lawsone). The results indicate that color strength and wash fastness of the pretreated printed wool samples were enhanced compared to the untreated samples. This enhancement depends mainly on the molecular weight and concentration of chitosan.

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp(220), Asp(318), and Asp(319) for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzyme-bound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.

Multicopy Crystallographic Refinement of a Relaxed Glutamine Synthetase from Mycobacterium tuberculosis Highlights Flexible Loops in the Enzymatic Mechanism and Its Regulation

The crystal structure of glutamine synthetase (GS) from Mycobacterium tuberculosis determined at 2.4 A resolution reveals citrate and AMP bound in the active site. The structure was refined with strict 24-fold noncrystallographic symmetry (NCS) constraints and has an R-factor of 22.7% and an R-free of 25.5%. Multicopy refinement using 10 atomic models and strict 24-fold NCS constraints further reduced the R-factor to 20.4% and the R-free to 23.2%. The multicopy model demonstrates the range of atomic displacements of catalytic and regulatory loops in glutamine synthesis, simulating loop motions. A comparison with loop positions in substrate complexes of GS from Salmonella typhimurium shows that the Asp50 and Glu327 loops close over the active site during catalysis. These loop closures are preceded by a conformational change of the Glu209 beta-strand upon metal ion or ATP binding that converts the enzyme from a relaxed to a taut state. We propose a model of the GS regulatory mechanism based on the loop motions in which adenylylation of the Tyr397 loop reverses the effect of metal ion binding, and regulates intermediate formation by preventing closure of the Glu327 loop.

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.

Effects of metal-ion binding on nucleobase pairing: stabilization, prevention and mismatch formation †

Depending on the site of metal binding to a nucleic acid constituent and the geometry of the metal entity, either a stabilization of a nucleobase ensemble, complementary or not (‘metal-modified’ base pair, triplet or quartet), or a prevention of nucleobase pairing may result.

Effects of metal ion binding on an oncomodulin mutant containing a novel calcium-binding loop

The Ca(2+)-binding protein oncomodulin was altered by cassette mutagenesis of the CD site (CDOM33) with a sequence that was derived by a consensus method using over 250 known Ca(2+)-binding loop sequences. This mutant was studied using time-resolved and steady-state fluorescence from the Trp residue included at position 7 of the loop (position 57 of the protein sequence). The fluorescence characteristics of this species in the absence and presence of metal ions were compared to those of a tetradecapeptide containing the loop and the single Trp mutant of oncomodulin, Y57W. The fluorescence properties of CDOM33 were quite different from the peptide, both in the apo form and in response to metal binding. The consensus CD loop in CDOM33 exhibited the characteristics of a Ca(2+)/Mg(2+) site in contrast to the Ca(2+) specificity of the wild-type CD loop. The Trp analogue, 5-hydroxytryptophan (5HW), was incorporated into both oncomodulin mutants to produce Y75(5HW) and 5HW-CDOM33. Results showed that this intrinsic probe was relatively insensitive to structural changes in the mutants upon metal binding compared to Trp itself.

Effect of metal ion binding on the secondary structure of bovine .alpha.-lactalbumin as examined by infrared spectroscopy

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

D-Glucose adducts with zinc-group metal ions. Synthesis, and spectroscopic and structural characterization of Zn(II), Cd(II), and Hg(II) complexes with d-glucose, and the effects of metal-ion binding on the sugar anomeric structures

The interaction of the zinc-group metal halide salts in solution with d-glucose has been investigated. Solid adducts of the type M( d-glucose)X 2· nH 2O, where M = Zn(II), Cd(II), or Hg(II); X = Cl − or Br −; and n = 4 or 2, were isolated and characterized by f.t.-i.r. and n.m.r. spectroscopy, and by molar conductivity measurements. The Zn(II)- and Cd(II)-glucose adducts appear structurally similar, binding to one d-glucose molecule, possibly through the 1-OH and 2-OH groups as well as to four H 2O molecules, forming a six-coordinate metal cation. The Hg(II) ion may be bonded to two sugar molecules (unidentately) and to two H 2O molecules, resulting in a four-coordinate structure around mercury. The binding of Zn(II) and Cd(II) is via the α anomer of d-glucose. The possibility cannot be excluded that the Hg(II) ion binds to both the α and β anomers.