Effects Of Zinc Binding Research Articles

Effects of zinc binding on the binding of epigallocatechin gallate (green tea) to bovine serum albumin and myoglobin

Serum albumin as a zinc carrier binds 80% plasma zinc to facilitate zinc absorption. Epigallocatechin gallate (EGCG, green tea) is reported to bind to serum albumin to perform its biological functions in vivo. However, the available information on how zinc binding affects the binding of EGCG to proteins or how EGCG binding affects the binding of zinc to proteins are very limited. In the present study, the effects of zinc binding on the binding of EGCG to bovine serum albumin and myoglobin were investigated using isothermal titration calorimetry, fluorescence quenching and molecular docking. The obtained results suggested that binding of zinc to bovine serum albumin and myoglobin could increase the EGCG binding affinity of proteins as indicated by the thermodynamic parameters. In addition, the formation of protein/zinc complex shifted the EGCG binding site of proteins. For myoglobin, the electron transfer from EGCG to myoglobin was facilitated by zinc binding induced stronger EGCG binding to myoglobin. Such study provides very fundamental and useful knowledge for zinc and EGCG nutrition.

Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

Effects of zinc binding on the structure and thermal stability of camel alpha-lactalbumin

The interaction between zinc ion and camel alpha-lactalbumin (α-La) has been studied using different techniques of fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry, and differential scanning calorimetry (DSC). There are two sets of independent binding sites for Zn2+, two ions bind with the binding constant of 4.53 × 104 M−1 and four ions with the binding constant of 963 M−1. The Protein-Zn2+complexation is an entropy-driven process. Circular dichroism studies do not show any significant change in the secondary structure of the protein after the binding of zinc ion on the α-La. The interaction leads to a conformational change of the protein and exposure of hydrophobic patches on α-Lato the solvent as observed in ANS fluorescence spectroscopy. DSC was applied to elucidate the effect of Zn2+ binding on the protein stability. Binding of Zinc ion to α-La induces a partially folded structure of protein. At the Zn2+/α-La molar ratio of 5 and 10, a mixture of native and partially folded structures is present. When the ratio reaches 20, all molecules adopt partially folded structure. Chemometric analysis confirmed the experimental results and provided insight into the process of denaturation of α-La via characterizing the native, intermediate, and denatured conformations in the presence of different concentrations of Zn2+ and different thermal conditions. Thermal stability of the partially folded structure of α-La in the presence of Zinc ion is lower than in the native protein.

Effects of Zinc Binding and Chelation on Structures and Dynamics in Amyloid-Beta Dimers

Amyloid-β (Aβ) is a protein of 39-43 amino acids that self-associates into a diverse array of neurotoxic aggregates linked to Alzheimer's disease (AD). Recently, Aβ dimers and trimers have been shown to cause memory deficits when they are recruited to the synapse, perhaps due to zinc release during neurotransmission. We have employed single-molecule fluorescence methods to investigate dimer structures, to understand zinc-induced structural change and determine whether it may be reversed by metal chelation. Single-pair FRET measurements were performed using Aβ40 peptides labeled at the N-termini with donor and acceptor dyes; the donor peptide was additionally labeled at the C-terminus with a Lys-biotin moiety to permit tethering to a functionalized cover slip. Time-dependent FRET efficiencies (EFRET) were determined by measuring fluorescence from individual surface-tethered dimers, affording insight into dimer structures and structural dynamics. Characteristic EFRET values were determined for dimers in metal-free samples and in the presence of Zn2+ (1 equiv.), with and without excess amounts of the zinc chelator clioquinol (CQ, 10 equiv.). Under all sample conditions, dimers appear to exhibit at least two characteristic structures, as evidenced by at least two broad peaks in each of the EFRET ensemble histograms. Zinc binding causes a slight structural change that is not reversed by chelation. Strikingly, zinc also severely limits dimer structural dynamics: while 29% of dimers in metal-free samples visit two EFRET values over time, only 12% of dimers in the presence of zinc exhibit structural change. Chelation does not reverse this effect, as only 9% of dimers in the presence of CQ show structural dynamics. These results lend new insights into the role(s) of zinc in Aβ association and the use of metal chelators in AD prevention and treatment.

Structures and Dynamics in Amyloid-Beta Dimers: Effects of Zinc Binding and Chelation

Alzheimer's disease (AD) is causally linked to the self-association of amyloid-β peptide (Aβ), a small protein of 39-43 amino acids. Historically, Aβ fibrils found in extracellular senile plaques were thought to be the pathogenic agents; however, recent evidence suggests that Aβ oligomers as small as dimers are more closely linked to Alzheimer's symptoms and progression. We have employed Förster resonance energy transfer (FRET) measurements to probe structures and dynamics in dimers of Aβ40. Results for single, surface-tethered dimers reveal two characteristic FRET efficiencies, perhaps indicative of two preferred dimer structures; these values are confirmed by fluorescence lifetime measurements in bulk solution.

Effects of Zinc Binding on the Structure and Stability of Glycylglycine Dipeptide: A Computational Study

Energies and structures of six different isomers resulted of complex between glycylglycine dipeptide and zinc cation were calculated at the BLYP and B3LYP levels of theory with 6-311G** and 6-311++G** basis sets. The energies of the glycylglycine-Zn2+different complexes at the BLYP/6-311++G** obtained more stable than another methods. In this method, the most stable complex was zinc cation bound to the two oxygen sites of the glycylglycine zwitterion and acted as a bidentate ligand. The vibrational frequencies, thermodynamic and electronic properties, HOMO, LUMO, and energy gap for the most stable isomer in different solvents were calculated at the BLYP/6-311++G** level of theory.

Effect of Zinc Binding on β-Amyloid Structure and Dynamics: Implications for A β Aggregation

Assembly of β-amyloid (A β) peptide into toxic oligomers is widely believed to initiate Alzheimer's disease pathogenesis. Under in vitro physiological conditions, zinc (Zn(II)) can bind to A β and redirect its assembly from amyloid fibrillar toward less toxic amorphous aggregation. Propensity of A β to go toward a specific form of aggregate state is determined by structural and dynamical properties of the initial monomeric as well as the aggregate state. Here we probe the structural and dynamical impact of binding of Zn(II) to monomeric A β40 using NMR spectroscopy. To obtain further support for the importance of intrinsic dynamics in the aggregation precursor, 15N relaxation measurements were also performed for A β42, the more fibrillar aggregation-prone variant of A β. The combined data suggest that, upon Zn(II)-binding to the N-terminus of A β40, a relatively rigid turnlike structure is induced at residues Val 24-Lys 28 whereas the residues flanking this region become more mobile on the picosecond-to-nanosecond timescale. This is in contrast to the increased rigidity of A β42 at the C-terminus, and proposed to be linked to the higher propensity of Zn(II)-bound peptide to form amorphous aggregates with less entropic penalties than their fibrillar counterparts.

Inhibition of proton pumping in membrane reconstituted bovine heart cytochrome c oxidase by zinc binding at the inner matrix side

A study is presented on the effect of zinc binding at the matrix side, on the proton pump of purified liposome reconstituted bovine heart cytochrome c oxidase (COV). Internally trapped Zn 2+ resulted in 50% decoupling of the proton pump at level flow. Analysis of the pH dependence of inhibition by internal Zn 2+ of proton release in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase indicates that Zn 2+ suppresses two of the four proton pumping steps in the cycle, those taking place when the 2 OH − produced in the reduction of O 2 at the binuclear center are protonated to 2 H 2O. This decoupling effect could be associated with Zn 2+ induced conformational alteration of an acid/base cluster linked to heme a 3 .

Effects of Zinc Binding on the Conformational Distribution of the Amyloid-β Peptide Based on Molecular Dynamics Simulations

A number of experiments suggested that metal binding can promote the aggregation of the amyloid-beta peptide. In this work, the effects of the zinc binding on the conformational distributions of the full length amyloid-beta peptide are investigated on the basis of extensive molecular dynamics simulations. By comparing the conformational distributions of the apo-peptide and the holo-peptide, we show that the zinc binding can affect the conformational distribution of the amyloid-beta monomer dramatically. Compared with the apo-peptide, the holo-peptide samples more beta-strand conformation for the central hydrophobic cluster 17-21. Meanwhile, the formation probabilities of the salt bridge Asp23-Lys28 and the turn comprising 23-28 are also increased significantly at room temperature. Since these local structures are essential for the amyloid-beta aggregation, the observed effects of the zinc binding indicate that the metal induced conformational change of the monomer is one of the possible mechanisms for the metal promoted aggregation of the amyloid-beta peptide.

Effects of Zinc Binding on the Structure and Dynamics of the Intrinsically Disordered Protein Prothymosin α: Evidence for Metalation as an Entropic Switch

Prothymosin alpha (ProTalpha) is a small acidic protein that is highly conserved among mammals. The human form has 110 amino acid residues (M.W. 12.1 kDa; pI approximately 3.5) and is found to be expressed in a wide variety of tissues. ProTalpha plays an essential role in cell proliferation and apoptosis, and it is involved in transcriptional regulation of oxidative stress-protecting genes. Despite the multiple biological functions ProTalpha has, the protein does not adopt a well-defined three-dimensional structure under physiological conditions. Previous studies have shown that the interaction between ProTalpha and some of its protein targets is significantly enhanced in the presence of zinc ions, suggesting that zinc binding plays a crucial role in the protein's function. In this work, we use nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry to characterize the structure and dynamics of ProTalpha and its complexation with Zn2+. We found that zinc binding causes partial folding of the C-terminal half of ProTalpha, especially the Glu-rich region, while the N-terminal portion of the protein remains largely unstructured. The metalated protein also exhibits a significantly reduced flexibility. ProTalpha shows a high specificity for Zn2+, and the interactions with other divalent cations (Ca2+, Mg2+) are much weaker. On the basis of the site-specific information obtained here, as well as the results from previous studies, we propose that the conformational and dynamic changes upon zinc binding may act as an entropic switch that greatly facilitates the binding to other proteins.

Desiccation and Zinc Binding Induce Transition of Tomato Abscisic Acid Stress Ripening 1, a Water Stress- and Salt Stress-Regulated Plant-Specific Protein, from Unfolded to Folded State

Abscisic acid stress ripening 1 (ASR1) is a low molecular weight plant-specific protein encoded by an abiotic stress-regulated gene. Overexpression of ASR1 in transgenic plants increases their salt tolerance. The ASR1 protein possesses a zinc-dependent DNA-binding activity. The DNA-binding site was mapped to the central part of the polypeptide using truncated forms of the protein. Two additional zinc-binding sites were shown to be localized at the amino terminus of the polypeptide. ASR1 protein is presumed to be an intrinsically unstructured protein using a number of prediction algorithms. The degree of order of ASR1 was determined experimentally using nontagged recombinant protein expressed in Escherichia coli and purified to homogeneity. Purified ASR1 was shown to be unfolded using dynamic light scattering, gel filtration, microcalorimetry, circular dichroism, and Fourier transform infrared spectrometry. The protein was shown to be monomeric by analytical ultracentrifugation. Addition of zinc ions resulted in a global change in ASR1 structure from monomer to homodimer. Upon binding of zinc ions, the protein becomes ordered as shown by Fourier transform infrared spectrometry and microcalorimetry, concomitant with dimerization. Tomato (Solanum lycopersicum) leaf soluble ASR1 is unstructured in the absence of added zinc and gains structure upon binding of the metal ion. The effect of zinc binding on ASR1 folding and dimerization is discussed.

Mechanism and Kinetics of Metalloenzyme Phosphomannose Isomerase: Measurement of Dissociation Constants and Effect of Zinc Binding Using ESI-FTICR Mass Spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was used to study the noncovalent complexation of a metalloenzyme, phosphomannose isomerase (PMI), which catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The zinc cofactor binding effect and the noncovalent interactions of the holoenzyme with its two natural substrates and two inhibitors, erythrose 4-phosphate and mannitol 1-phosphate, were investigated. Under nondenaturing conditions, the intact zinc-containing monomeric protein ions were reproducibly observed with no dissociation. Molecular ions corresponding to apo-PMI monomer were obtained by dialyzing the holoenzyme against EDTA. The binding/release of the metal ion did not alter the charge-state distributions of the protein to any significant extent, but changed the binding affinity of the substrates by at least 5-fold. Using ESI-FTICR mass spectrometry, the binding stoichiometry and specificity of the enzyme-substrate and enzyme-inhibitor complexes were directly determined. The first time report of the apparent dissociation constant for the isomeric substrates of PMI was measured to be 88.8 microM. The relative dissociation constant of the two inhibitors derived from gas-phase noncovalent complexation was very similar to the relative inhibition constant derived from solution phase kinetics.

Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor

Metal‐induced precipitation of protein therapeutics is being used and further developed as a processing step in protein formulation and may have utility in protein purification and bulk storage. In such processes, it is imperative that native protein structure is maintained and the metal complexation is reversible. In the current study, we investigated the effects of zinc‐induced precipitation on recombinant human growth hormone (rhGH) and recombinant human nerve growth factor (rhNGF). On the addition of ethylenediaminetetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific metal binding sites and require specific molar ratios of zinc to protein to initiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc complexes and zinc‐induced precipitates, as measured by infrared and circular dichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiary structural alterations, whereas zinc binding did not alter the tertiary structure of rhNGF. These studies indicated that metal‐induced precipitation provides a method to maintain proteins in their native state in precipitates, which may be useful for purification, storage, and formulation. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 1480–1485, 2000

Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom.

Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an alpha-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an alpha-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.