Ferric Ion Binding Research Articles

Microbial oil, alone or paired with β-glucans, can control hypercholesterolemia in a zebrafish model

Dyslipidemia is often associated with unhealthy dietary habits, and many mammalian studies have explored the mode of action of certain bioactive compounds such as β-glucans and n-3 PUFAs to understand their potential to normalize the lipid metabolism. There are only a few investigations that adopted omic approaches to unveil their combined effect on hypercholesterolemia. Zebrafish (Danio rerio) was used as a model organism to reveal the efficacy of Schizochytrium oil and β-glucans (from Euglena gracilis and Phaeodactylum tricornutum) against cholesterol-rich diet induced dyslipidemia. One of the folowing four diets was fed to a particular group of fish: a control high-cholesterol diet, a Schizochytrium oil diet or one of the two diets containing the oil and β-glucan. The plasma HDL, expression of hepatic genes linked to, among others, ferric ion binding and plasma phosphatidylcholines were higher and plasma cholesterol esters and triacylglycerols were lower in the microbial oil-fed fish compared to the fish fed high cholesterol diet. While the fish fed a mix of microbial oil and Euglena β-glucan had lower plasma triacylglycerols and expression of hepatic genes linked to PPAR signaling pathway and enriched biosynthesis of plasma unsaturated fatty acids, the fish fed microbial oil-Phaeodactylum β-glucan combination had lower abundance of triacylglycerols rich in saturated and mono-unsaturated fatty acids and cholesterol esters in the plasma.

Glycoproteomics meets thermodynamics: A calorimetric study of the effect of sialylation and synergistic anion on the binding of iron to human serum transferrin

The thermodynamic parameters for the binding of ferric ions to human serum transferrin (hTf) as the major mediator of iron transport in blood plasma were determined by isothermal titration calorimetry in the presence of carbonate and oxalate as synergistic anions at pH 7.4. The results indicate that the binding of ferric ions to the two binding sites of hTf is driven both enthalpically and entropically in a lobe-dependent manner: binding to the C-site is mainly enthalpically driven, whereas binding to the N-site is mainly entropically driven. Lower sialic acid content of hTf leads to more exothermic apparent binding enthalpies for both lobes, while the increased apparent binding constants for both sites were found in the presence of carbonate. Sialylation also unequally affected the heat change rates for both sites only in the presence of carbonate, but not in the presence of oxalate. Overall, the results suggest that the desialylated hTf has a higher iron sequestering ability, which may have implications for iron metabolism.

Enabling single-molecule localization microscopy in turbid food emulsions.

Turbidity poses a major challenge for the microscopic characterization of food systems. Local mismatches in refractive indices, for example, lead to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the accessible optical resolution in food microscopy, we added adaptive optics (AO) and flat-field illumination to our previously published open microscopy framework, the miCube. In the detection path, we implemented AO via a deformable mirror to compensate aberrations and to modulate the emission wavefront enabling the engineering of point spread functions (PSFs) for single-molecule localization microscopy (SMLM) in three dimensions. As a model system for a non-transparent food colloid such as mayonnaise, we designed an oil-in-water emulsion containing the ferric ion binding protein phosvitin commonly present in egg yolk. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain two- and three-dimensional images of phosvitin covered oil droplets with sub 100 nm resolution. Our data indicated that phosvitin is homogeneously distributed at the interface. With the possibility to obtain super-resolved images in depth, our work paves the way for localizing biomacromolecules at heterogeneous colloidal interfaces in food emulsions. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Deferoxamine-Modified Hybrid Materials for Direct Chelation of Fe(III) Ions from Aqueous Solutions and Indication of the Competitiveness of In Vitro Complexing toward a Biological System.

Deferoxamine (DFO) is one of the most potent iron ion complexing agent belonging to a class of trihydroxamic acids. The extremely high stability constant of the DFO–Fe complex (log β = 30.6) prompts the use of deferoxamine as a targeted receptor for scavenging Fe(III) ions. The following study aimed at deferoxamine immobilization on three different supports: poly(methyl vinyl ether-alt-maleic anhydride), silica particles, and magnetite nanoparticles, leading to a class of hybrid materials exhibiting effectiveness in ferric ion adsorption. The formed deferoxamine-loaded hybrid materials were characterized with several analytical techniques. Their adsorptive properties toward Fe(III) ions in aqueous samples, including pH-dependence, isothermal, kinetic, and thermodynamic experiments, were investigated. The materials were described with high values of maximal adsorption capacity qm, which varied between 87.41 and 140.65 mg g–1, indicating the high adsorptive potential of the DFO-functionalized materials. The adsorption processes were also described as intense, endothermic, and spontaneous. Moreover, an exemplary magnetically active deferoxamine-modified material has been proven for competitive in vitro binding of ferric ions from the biological complex protoporphyrin IX–Fe(III), which may lead to a further examination of the materials’ biological or medical applicability.

Fabrication of iron loaded whey protein isolate/gum Arabic nanoparticles and its adsorption activity on oil-water interface

Design and fabrication of iron fortifier is very essential in the prevention or improvement of iron-deficiency anemia in food industry. In this study whey protein isolate (WPI) and gum arabic (GA) loaded with ferric ions was expected to be self-assembled to form stable nanoparticles by heating at 85 °C for 15 min at pH 4.0. It is illustrated that WPI/GAFe3+ nanoparticles were more uniformly dispersed in comparison with the easily aggregative GAFe3+ complex. The tightly binding of ferric ions was confirmed by FTIR and XPS. During formation of WPI/GAFe3+ aggregates induced by heating, the disulfide bond was formed and the surface hydrophobicity was significantly enhanced. The ferric ions release rate of WPI/GAFe3+ nanoparticles was significantly slower than that of GAFe3+ complex in simulated gastric juice due to the fact that the assembled structure exhibited a delayed effect on the hydrolysis of peptides catalyzed by pepsin during its internal diffusion into nanoparticles. WPI/GAFe3+ nanoparticles contributed to a good adsorption activity on oil-water interface between WPI and GAFe3+ complex because of its high steric hindrance. The decrease of interfacial tension with sequential adsorption demonstrated that WPI/GAFe3+ nanoparticles could be electrostatically adsorbed onto positively charged sodium caseinate interface, or co-adsorbed onto non-ionic Tween 80 interface, forming a more stable interface with higher steric hindrance and lower interfacial energy. Thus, the self-assembled WPI/GAFe3+ nanoparticles would have great potential in the application for iron fortification especially in food emulsion system.

Investigating the ferric ion binding site of magnetite biomineralisation protein Mms6

The biomineralization protein Mms6 has been shown to be a major player in the formation of magnetic nanoparticles both within the magnetosomes of magnetotactic bacteria and as an additive in synthetic magnetite precipitation assays. Previous studies have highlighted the ferric iron binding capability of the protein and this activity is thought to be crucial to its mineralizing properties. To understand how this protein binds ferric ions we have prepared a series of single amino acid substitutions within the C-terminal binding region of Mms6 and have used a ferric binding assay to probe the binding site at the level of individual residues which has pinpointed the key residues of E44, E50 and R55 involved in Mms6 ferric binding. No aspartic residues bound ferric ions. A nanoplasmonic sensing experiment was used to investigate the unstable EER44, 50,55AAA triple mutant in comparison to native Mms6. This suggests a difference in interaction with iron ions between the two and potential changes to the surface precipitation of iron oxide when the pH is increased. All-atom simulations suggest that disruptive mutations do not fundamentally alter the conformational preferences of the ferric binding region. Instead, disruption of these residues appears to impede a sequence-specific motif in the C-terminus critical to ferric ion binding.

Functional characterisation of two ferric-ion coordination modes of TtFbpA, the periplasmic subunit of an ABC-type iron transporter from Thermus thermophilus HB8.

The ferric ion binding protein A of Thermus thermophilus HB8 (TtFbpA) is the periplasmic subunit of an ABC-type iron transporter. Two Fe3+-bound crystal structures at pH 5.5 and pH 7.5 and one apo structure have been reported for TtFbpA. In addition to three Tyr residues, TtFbpA coordinates with Fe3+ using two monodentate HCO3- and one H2O to form a six-coordinated mode at pH 5.5 or one bidentate CO32- to form a five-coordinated mode at pH 7.5. We investigated the biological significance of these Fe3+-bound forms of TtFbpA and the synergistic anions (HCO3- and CO32-). Quantum mechanical calculations in silico indicated that only these coordination modes were plausible out of six possibilities. Comparison of the crystal structures revealed a key motif, RZX1X2L(I/V), that would couple the Fe3+ coordination mode and the TtFbpA protein conformation. Both gel filtration chromatography and isothermal titration calorimetry showed that TtFbpA could bind Fe3+ at pH 7.5 but not at pH 5.5. Isothermal titration calorimetry also revealed that the binding at pH 7.5 was a three-step endothermic reaction that required NaHCO3. These results indicate that the holo structure at pH 5.5 is unstable in solution and may correspond to a transition state of TtFbpA-Fe3+ binding at pH 7.5 because HCO3- is much more abundant than CO32- at both pH values. Reorganisation of the synergistic ions and coupled protein conformational change will occur to form the stable TtFbpA-Fe3+ complex at pH 7.5, but not at pH 5.5. Identification of such a transition state will contribute to molecular design of novel FbpA inhibitors.

Luminescent properties of 4-aminobenzo-15-crown-5 after preferential binding of ferric ions in aqueous solutions.

We report a combined approach that introduces the use of 4-aminobenzo-15-crown-5 (4AB15C5) for the detection of ferric(III) ions by colorimetric, ultraviolet (UV)-visible light absorption, fluorescence, and live-cell imaging techniques along with density functional theory (DFT) calculations. We have found that 4AB15C5 is sensitive and selective for binding ferric(III) ions in aqueous solutions. DFT calculations using the polarizable continuum model have been used to explain the strong binding of the ferric ion by 4AB15C5 in aqueous solutions. The detection limit in the fluorescence quenching measurements was found to be as low as 50μM for the ferric ion with a determined Stern-Volmer constant of 1.52×104 M-1 . Fluorescence intensity did not change for other ions tested, Fe2+ , Co2+ , Mn2+ , Mg2+ , Zn2+ , Ca2+ , NH4+ , Na+ , and K+ ions. Live-cell fluorescence imaging was also used to check the intracellular variations in ferric ion levels. Our spectroscopic data indicated that 4AB15C5 can bind ferric ions selectively in aqueous solutions.

Tyrosine modification increases the affinity of gastrin for ferric ions.

The peptide hormone gastrin17, which occurs naturally in both tyrosine sulphated and unsulphated forms, binds two ferric ions with pM affinities. The aim of this study was to investigate the hypothesis that sulphation or phosphorylation of gastrin17 altered ferric ion binding, and/or affinity for the CCK1 or CCK2 receptor. To investigate the effect of tyrosine modification on ferric ion binding, the changes in absorbance of gastrin17, gastrin17SO4 and gastrin17PO4 on addition of Fe3+ ions were monitored. Binding of gastrin17, gastrin17SO4 and gastrin17PO4 to the human CCK1 and CCK2 receptors was assessed by competition with [125I]-Bolton and Hunter-labelled cholecystokinin8 in transiently transfected COS cells. Tyrosine sulphation or phosphorylation increased the affinity of gastrin17 for the first ferric ion bound from 267 to 83 pM and 14 pM, respectively, but had no effect on the stoichiometry of ferric ion binding. In contrast the affinity of gastrin17 for the second ferric ion bound was reduced from 94 pM to 7.32 µM and 671 nM, respectively. While sulphation of gastrin17 increased its affinity for the CCK2 receptor approximately 50 fold, phosphorylation had no effect on receptor binding. These results demonstrate that tyrosine modification may have profound effects on the interaction of gastrins with ferric ions and with the CCK2 receptor.

Retro-inverso forms of gastrin5–12 are as biologically active as glycine-extended gastrin in vitro but not in vivo

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived ‘super agonists’ using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function.

A novel six-coordinated ferric ion binding mode of TtFbpA

Iron is an essential element for the growth and survival of nearly all living organisms. However, it is difficult for most organisms to get enough iron from the environment, because of the extremely low solubility of ferric ion. One of the strategies for iron acquisition is to use the ATP-binding cassette (ABC) transport system. In Gram-negative bacteria, a typical iron uptake ABC transporter consists of a ferric ion-binding protein (Fbp) located in periplasm (FbpA), two transmembrane proteins that form a pathway for ferric ions (FbpB), and two peripheral ATP-binding proteins located at the cytoplasm side of the inner membrane (FbpC). TtFbpA is a ferric ion-binding protein of a putative iron uptake ABC transporter from Thermus thermophilus HB8. Here we report the crystal structures of the apo-form and ferric ion-bound form of TtFbpA at 1.8-Å and 1.7-Å resolutions, respectively [1]. The crystal structure of the ferric ion-bound TtFbpA shows that a ferric ion binds to a specific site of TtFbpA to form a six-coordinated complex by three tyrosine residues, two bicarbonates and a water molecule, revealing a novel mode of coordination to a ferric ion. Another crystal structure of ferric ion-bound TtFbpA reported earlier showed the bound ferric ion is five-coordinated by three tyrosine residues and a carbonate bound in the bidentate manner [2]. The different modes of the coordination would probably result from the different pHs used for crystallization: pH 5.5 (six-coordinated) vs. pH 7.5 (five-coordinated). The Gram negative bacterium T. thermophilus HB8 can live in a wide pH range of 3.4–9.6. We propose that TtFbpA, a periplasmic protein of T. thermophilus HB8, can act as a ferric ion-binding protein over the wide pH range by taking at least two different coordination manners to a ferric ion depending on pH. This is the first example of a periplasmic ferric iron-binding protein that can coordinate a ferric ion via multiple types of coordination complex formation.

Direct thermodynamic and kinetic measurements of Fe2 + and Zn2 + binding to human serum transferrin

Human serum transferrin (hTf) is a single-chain bilobal glycoprotein that efficiently delivers iron to mammalian cells by endocytosis via the transferrin/transferrin receptor system. While extensive studies have been directed towards the study of ferric ion binding to hTf, ferrous ion interactions with the protein have never been firmly investigated owing to the rapid oxidation of FeII to FeIII and the difficulty in maintaining a fully anaerobic environment. Here, the binding of Fe2+ and Zn2+ ions to hTf has been studied under anaerobic and aerobic conditions, respectively, in the presence and absence of bicarbonate by means of isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The ITC data indicate the presence of one class of strong binding sites with dissociation constants of 25.2nM for Fe2+ and 6.7nM for Zn2+ and maximum binding stoichiometries of 1 Zn2+ (or 1 Fe2+) per hTf molecule. With either metal, the binding interaction was achieved by both favorable enthalpy and entropy changes (ΔH0~−12kJ/mol and ΔS0~106J/mol·K for Fe2+ and ΔH0~−18kJ/mol and ΔS0~97J/mol·K for Zn2+). The large and positive entropy values are most likely due to the change in the hydration of the protein and the metal ions upon interaction. Rapid kinetics stopped-flow fluorescence spectroscopy revealed two different complexation mechanisms with different degrees of conformational changes upon metal ion binding. Our results are discussed in terms of a plausible scenario for iron dissociation from transferrin by which the highly stable Fe3+–hTf complex might be reduced to the more labile Fe2+ ion before iron is released to the cytosol.

1P010 TtFbpA による新規鉄結合様式の解明(蛋白質 : 構造,ポスター,第52回日本生物物理学会年会(2014年度))

1P010 TtFbpA による新規鉄結合様式の解明(蛋白質 : 構造,ポスター,第52回日本生物物理学会年会(2014年度))

Electronic Structure Study of Certain Rhizoferrin Analogs and Its Ferric-Ion Complexes

We have used first-principles electronic structure methods to examine the ferric-ion binding specificity of rhizoferrin analogs: (a) the homo-rhizoferrin and nor-rhizoferrin that fall in the category of variable length diamine bridge; (b) monodesoxy-rhizoferrin and didesoxy-rhizoferrin having different functional groups in the citric-acid moieties of rhizoferrin, and (c) cyclized dehydration product of rhizoferrin, named imido-rhizoferrin. A particular focus is the effect of deprotonation of hydroxyl and carboxyl groups on binding energy of ferric-ion complexes of rhizoferrin-analogs has been investigated. In addition, we have calculated Nuclear Magnetic Resonance properties, such as chemical shifts for13C and 1H, and indirect spin-spin couplings for methylene protons, and compared our results with available experimental data. The nuclear quadrupole interaction parameters will be discussed within the context of charge distributions and electric field gradients. Our study provides a structural basis for ferric-ion chelation by rhizoferrin analogs for these biomolecules, for which there exist no experimentally determined structures. Argonne National Laboratory is a US DOE Science Laboratory operated under contract no. DE-AC02-06CH11357 by UChicago Argonne, LLC. The research was performed, in part, at Argonne National Laboratory as a research participant in the Visiting Faculty Program. The program is administered by Argonne's Division of Communication, Education, and Public Affairs (CEPA) with funding provided by the U.S. Department of Energy.

A novel mode of ferric ion coordination by the periplasmic ferric ion-binding subunit FbpA of an ABC-type iron transporter fromThermus thermophilusHB8

Crystal structures of FbpA, the periplasmic ferric ion-binding protein of an iron-uptake ABC transporter, from Thermus thermophilus HB8 (TtFbpA) have been solved in apo and ferric ion-bound forms at 1.8 and 1.7 Å resolution, respectively. The latter crystal structure shows that the bound ferric ion forms a novel six-coordinated complex with three tyrosine side chains, two bicarbonates and a water molecule in the metal-binding site. The results of gel-filtration chromatography and dynamic light scattering show that TtFbpA exists as a monomer in solution regardless of ferric ion binding and that TtFbpA adopts a more compact conformation in the ferric ion-bound state than in the apo state in solution.

Structural insights into the coordination of iron by Thermus thermophilus HB8 ferric binding protein A

The ferric binding protein belongs to the substrate-binding protein super-family and transports ferric ions across the periplasmic space in gram negative bacteria. This process involves the binding and release of ferric ions through conformational changes of the ferric binding protein, and the assistance of a synergistic anion. Here we report the crystal structure of Thermus thermophilus HB8’s (TtFbpA) ferric binding protein A in four different forms, which represent the apo state (apo-TtFbpA), the carbonate-bound state (TtFbpACO3),and the iron- and carbonate-bound state (TtFbpAFeCO3). The ferric ion in TtFbpAFeCO3 is bound by three tyrosine residues from TtFbpA and one synergistic carbonate ion. Structural comparisons among the three different states reveal the molecular mechanisms of iron-binding by TtFbpA. Our results, together with previous studies on other bacterial periplasmic ferric binding proteins, provide a complete understanding of the structural basis for iron binding and release in the periplasm of gram-negative bacteria.

Ferric ion (hydr)oxo clusters in the “Venus flytrap” cleft of FbpA: Mössbauer, calorimetric and mass spectrometric studies

Isothermal calorimetric studies of the binding of iron(III) citrate to ferric ion binding protein from Neisseria gonorrhoeae suggested the complexation of a tetranuclear iron(III) cluster as a single step binding event (apparent binding constant K(app) (ITC) = 6.0(5) × 10(5) M(-1)). High-resolution Fourier transform ion cyclotron resonance mass spectrometric data supported the binding of a tetranuclear oxo(hydroxo) iron(III) cluster of formula [Fe(4)O(2)(OH)(4)(H(2)O)(cit)](+) in the interdomain binding cleft of FbpA. The mutant H9Y-nFbpA showed a twofold increase in the apparent binding constant [K(app) (ITC) = 1.1(7) × 10(6) M(-1)] for the tetranuclear iron(III) cluster compared to the wild-type protein. Mössbauer spectra of Escherichia coli cells overexpressing FbpA and cultured in the presence of added (57)Fe citrate were indicative of the presence of dinuclear and polynuclear clusters. FbpA therefore appears to have a strong affinity for iron clusters in iron-rich environments, a property which might endow the protein with new biological functions.

Interaction between ciprofloxacin and ferric-ion binding protein from pathogenic bacteria

Objective:To investigate the interaction between ciprofloxacin (CIP) and ferric-ion binding protein (FBP) from pathogenic bacteria,so as to determine whether FBP is the target of CIP.Methods: Reactions between ciprofloxacin and FBP from N.gonorrhoeae,which was expressed in E.coli,were monitored by UV-visible and NMR spectroscopy.Results: Fe3+ was removed from holo-FBP by CIP in 10 mmol/L Tris-Cl buffer,pH 7.40,at 298 K,and half of the loaded Fe3+ was taken off when 50 molar equivalence of CIP was present.Conclusion: CIP can remove Fe3+ from holo-FBP,indicating that CIP can interfere with the Fe3+ capture of the bacteria and influence the growth and virulence of the bacteria.

Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

Ferric ion binding proteins (Fbps) transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III) is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues) together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed.

Folding process of silk fibroin induced by ferric and ferrous ions

Bombyx mori silk fiber has useful mechanical properties largely due to a high content of ordered β-sheet crystallites separated by non-crystalline spacers. Metallic ions present in the silk dope in nature could affect the β-sheet content. In this work, we used solid-state 13C NMR, EPR and Raman spectroscopy to investigate how the ferric/ferrous ions affect the folding process of the silk fibroin. NMR and Raman results indicate that ferric and ferrous ions have different effects on the secondary structure of silk fibroin. Ferric ions can induce a conformation change from helix to β-sheet form in silk fibroin when their concentration exceeds a critical value, while ferrous ions cannot. EPR results indicate that the ferric ions bound with silk fibroin have a high-spin state ( S = 5/2) with g-value of g 1 = 1.950, g 2 = 1.990 and g 3 = 1.995, zero-field splitting interaction D of 1.2–2 cm −1, and symmetric character of E/ D = 1/3, resulting in an effective g-value of g′ = 4.25. The hydrophilic spacer GTGSSGFGPYVAN(H)GGYSGYEYAWSSESDFGT in the heavy chain of silk fibroin is likely to be involved in the binding of ferric ions, and His, Asn and Tyr residues are considered as the potential binding sites.