Ferritin Shell Research Articles

Intra- and interparticle magnetism of cobalt-doped iron-oxide nanoparticles encapsulated in a synthetic ferritin cage

We present an in-depth examination of the composition and magnetism of cobalt $({\mathrm{Co}}^{2+})$-doped iron-oxide nanoparticles encapsulated in Pyrococcus furiosus ferritin shells. We show that the ${\mathrm{Co}}^{2+}$ dopant ions were incorporated into the $\ensuremath{\gamma}\text{\ensuremath{-}}{\mathrm{Fe}}_{2}{\mathrm{O}}_{3}/{\mathrm{Fe}}_{3}{\mathrm{O}}_{4}$ core, with small paramagnetic-like clusters likely residing on the surface of the nanoparticle that were observed for all cobalt-doped samples. In addition, element-specific characterization using M\ossbauer spectroscopy and polarized x-ray absorption indicated that ${\mathrm{Co}}^{2+}$ was incorporated exclusively into the octahedral B sites of the spinel-oxide nanoparticle. Comparable superparamagnetic blocking temperatures, coercivities, and effective anisotropies were obtained for 7%, 10%, and 12% cobalt-doped nanoparticles, and were only slightly reduced for 3% cobalt, indicating a strong effect of cobalt incorporation, with a lesser effect of cobalt content. Due to the regular particle size and separation that result from the use of the ferritin cage, a comparison of the effects of interparticle interactions on the disordered assembly of nanoparticles was also obtained that indicated significantly different behaviors between undoped and cobalt-doped nanoparticles.

Local packing modulates diversity of iron pathways and cooperative behavior in eukaryotic and prokaryotic ferritins

Ferritin-like molecules show a remarkable combination of the evolutionary conserved activity of iron uptake and release that engage different pores in the conserved ferritin shell. It was hypothesized that pore selection and iron traffic depend on dynamic allostery with no conformational changes in the backbone. In this study, we detect the allosteric networks in Pseudomonas aeruginosa bacterioferritin (BfrB), bacterial ferritin (FtnA), and bullfrog M and L ferritins (Ftns) by a network-weaving algorithm (NWA) that passes threads of an allosteric network through highly correlated residues using hierarchical clustering. The residue-residue correlations are calculated in the packing-on elastic network model that introduces atom packing into the common packing-off model. Applying NWA revealed that each of the molecules has an extended allosteric network mostly buried inside the ferritin shell. The structure of the networks is consistent with experimental observations of iron transport: The allosteric networks in BfrB and FtnA connect the ferroxidase center with the 4-fold pores and B-pores, leaving the 3-fold pores unengaged. In contrast, the allosteric network directly links the 3-fold pores with the 4-fold pores in M and L Ftns. The majority of the network residues are either on the inner surface or buried inside the subunit fold or at the subunit interfaces. We hypothesize that the ferritin structures evolved in a way to limit the influence of functionally unrelated events in the cytoplasm on the allosteric network to maintain stability of the translocation mechanisms. We showed that the residue-residue correlations and the resultant long-range cooperativity depend on the ferritin shell packing, which, in turn, depends on protein sequence composition. Switching from the packing-on to the packing-off model reduces correlations by 35%-38% so that no allosteric network can be found. The influence of the side-chain packing on the allosteric networks explains the diversity in mechanisms of iron traffic suggested by experimental approaches.

Prussian Blue Modified Ferritin as Peroxidase Mimetics and Its Applications in Biological Detection

Ferritins are natural nanoscale structures composed with 24 subunits endowed with similar three-dimensional structures. The iron is stored in the form of ferrihydrite phosphate in the hollow spherical ferritin shells. Prussian blue nanoparticles (PBNPs) have been certified a kind of mimetic enzyme with the advantages of stability, high catalytic activity and low prices. In this context, we designed a strategy to synthesize PBNPs of small size using ferritin as template and meanwhile retain the biological properties of ferritin. Our results show the resulting nanostructures (Prussian blue modified ferritin nanoparticles, PB-Ft NPs) got very small size and relatively high catalytic activity, furthermore, PB-Ft NPs successfully combined the intrinsic enzyme mimetic activity of PBNPs and the specificity of ferritin. Peroxidase-like activity which fits well the Michaelis-Menten kinetics was found strongly depending on pH, temperature and the concentration of PB-Ft NPs. Then a sensitive method for glucose detection was developed using glucose oxidase (GOx) and PB-Ft NPs. The consequence of Enzyme-linked immunosorbent assay (ELISA) shows PB-Ft NPs possess both specificity and peroxidase-like activity, which suggests that PB-Ft NPs can be served as a useful reagent in some biological detections.

The extension peptide of plant ferritin from sea lettuce contributes to shell stability and surface hydrophobicity

Plant ferritins have some unique structural and functional features. Most of these features can be related to the plant-specific "extension peptide" (EP), which exists in the N-terminus of the mature region of a plant ferritin. Recent crystallographic analysis of a plant ferritin revealed the structure of the EP, however, two points remain unclear: (i) whether the structures of well-conserved EP of plant ferritins are common in all plants, and (ii) whether the EP truly contributes to the shell stability of the plant ferritin oligomer. To clarify these matters, we have cloned a green-plant-type ferritin cDNA from a green alga, Ulva pertusa, and investigated its crystal structure. Ulva pertusa ferritin (UpFER) has a plant-ferritin-specific extension peptide composed of 28 amino acid residues. In the crystal structure of UpFER, the EP lay on and interacted with the neighboring threefold symmetry-related subunit. The amino acid residues involved in the interaction were very highly conserved among plant ferritins. The EPs masked the hydrophobic pockets on the ferritin shell surface by lying on them, and this made the ferritin oligomer more hydrophilic. Furthermore, differential scanning calorimetric analysis of the native and its EP-deletion mutant suggested that the EP contributed to the thermal stability of the plant ferritin shell. Thus, the shell stability and surface hydrophobicity of plant ferritin were controlled by the presence or absence of the plant-ferritin-specific EP. This regulation can account for those processes such as shell stability, degradation, and association of plant ferritin, which are significantly related to iron utilization in plants.

Cobalt driven enhancement of nanomagnetism in iron-oxide nanoparticles encapsulated in a synthetic ferritin shell

We have examined the effects of Co doping (0–12% relative to total metal) on the magnetic properties of iron-oxide nanoparticles incorporated in a synthetic ferritin shell. Mössbauer spectroscopy revealed that the nanoparticles consisted of a mixture of Fe3O4 and γ-Fe2O3, and that Co was integrated exclusively into the B-sites of the Fe3O4 phase. Cobalt doping resulted in the formation of CoxFe3-xO4/γ-Fe2O3 nanoparticles with x = 0, 0.25, 0.51, 0.56, and 0.63. Magnetometry and susceptometry experiments showed that substantial enhancements in the coercivities, blocking temperatures and anisotropy constants of the magnetoferritin nanoparticles occurred for all Co doping levels. Our results show that the Co altered significantly the local atomic Fe magnetism and enhanced the magnetic nanoparticle anisotropy considerably.

Characteristics and Kinetics of Iron Releasefrom the Ferritin under the EGCG reduction

The mechanism of iron release from ferritin in vivo is still unclear even though it represents a key step of the metabolism of iron in vivo. Here, both interaction intensity and binding stability between epigallocatechin gallate (EGCG) from tea and liver ferritin of Dasyatis akajei (DALF) were investigated using UV-visible, fluorescence and circular dichroism (CD) spectrometry, respectively. The results indicated that EGCG could reduce the iron within the ferritin shell directly in the absence of chemical reducers such as Na(2)S(2)O(4), but this process was strictly pH-dependent, and the rate of iron release is faster at low pH than at high pH. The kinetic study of iron release showed that this process fitted the law of zero order reaction, which differed from that of first order reaction by various chemical reducers such as Vitamin C. In addition, Both fluorescence and CD spectrometry were further used to study the reduction mechanism of iron release in vitro, showing that there was a slight conformation change of the ferritin shell during EGCG reduction because of a complex formation of DALF-EGCG. It appears that chemical reducers with large molecular sizes reduce the iron across the protein shell by the way of an electron transfer pathway (ETP). A novel pathway for iron release from DALF with EGCG reduction is suggested to explain for a reductive route of iron metabolism by biological reducers in vivo.

PH-Dependent Structures of Ferritin and Apoferritin in Solution: Disassembly and Reassembly

The pH-dependent structures of the ferritin shell (apoferritin, 24-mer) and the ferrihydrite core, under physiological conditions that permit enzymatic activity, were investigated by synchrotron small-angle X-ray scattering (SAXS). The solution structure of apoferritin was found to be nearly identical to the crystal structure. The shell thickness and hollow core volumes were estimated. The intact hollow spherical apoferritin was stable over a wide pH range, 3.40-10.0, and the ferrihydrite core was stable over the pH range 2.10-10.0. The apoferritin subunits underwent aggregation below pH 0.80, whereas the ferrihydrite cores aggregated below pH 2.10 as a result of the disassembly of the ferritin shell under the strongly acidic conditions. As the pH decreased from 3.40 to 0.80, apoferritin underwent stepwise disassembly by first forming a hollow sphere with two holes, then a headset-shaped structure, and, finally, rodlike oligomers. As the pH was increased from pH 1.96, the disassembled rodlike oligomers recovered only to the headset-shaped structure, and the disassembled headset-shaped intermediates recovered only to the hollow spherical structure with two hole defects. The apoferritin hole defects that formed during the disassembly process did not heal as the pH was increased to neutral or slightly basic conditions. The pH-induced apoferritin disassembly and reassembly processes were not fully reversible, although they were pseudoreversible over a limited pH range, between 10.0 and 2.66.

Role of iron in pathogenesis of Parkinson disease

Parkinson’s disease is one of the most frequent human neurodegenerations. Motor symptoms of Parkinson’s disease are the consequence of the destruction of nervous cells in the substantia nigra (SN), a small (about 500 mg) structure located deep in human brain. The concentration of iron in SN is comparable to that in liver and is equal to about 180 ± 60 ng/mg of wet tissue and the iron in SN is mostly bound to ferritin. For many years it has been believed that the degeneration of nervous cells in SN in Parkinson’s disease is related to an important increase in the concentration of iron. Our own studies based on Mossbauer spectroscopy and other studies conducted with the use of various techniques have not confirmed this finding. The ratio of the concentration of iron in PD vs. control SN evaluated by Mossbauer spectroscopy was found to be equal 1.00 ± 0.13. We also confirmed that most of iron in SN is located within ferritin. ELISA studies demonstrated a significant decrease in L ferritin in parkinsonian SN compared to the control group. As L-ferritin is related to safe keeping of iron within the ferritin shell, its decrease may lead to an efflux of iron and increase in the concentration of labile iron. Indeed our studies did show a difference in the concentration of labile iron between PD and control SN (135 ± 10 ng/g vs. 76 ± 5 ng/g). This labile iron, which may initiate Fenton reaction, may be the cause of the oxidative stress leading to the death of nervous cells in PD.

Oxygen catalyzed mobilization of iron from ferritin by iron(iii) chelate ligands

Tridentate chelate ligands of 2,6-bis[hydroxy(methyl)amino]-1,3,5-triazine family rapidly release iron from human recombinant ferritin in the presence of oxygen. The reaction is inhibited by superoxide dismutase, catalase, mannitol and urea. Suggested reaction mechanism involves reduction of the ferritin iron core by superoxide anion, diffusion of iron(II) cations outside the ferritin shell, and regeneration of superoxide anions through oxidation of iron(II) chelate complexes with molecular oxygen.

Crystal Structure of Plant Ferritin Reveals a Novel Metal Binding Site That Functions as a Transit Site for Metal Transfer in Ferritin

Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-A resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu(173) and carbonyl oxygen of Thr(168), which correspond, respectively, to Glu(140) and Thr(135) of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.

Magnetic–fluorescent Langmuir–Blodgett films of fluorophore-labeled ferritin nanoparticles

We have covalently coupled fluorophore 4-(2-hydroxyethoxy)-7-nitro-2,1,3-benzoxadiazole (NBD) to the external ferritin shell through lysine residues. An increase in the luminescence quantum yield of the fluorescent ferritin particles and a blue shift in its emission peak compared to individual fluorophore were observed. The study of the particles by transmission electron microscopy showed that the native iron core ferritin is intact and that no degradation occurs during chemical functionalization of the protein shell. The NBD-labeled ferritin particles are water soluble, which allowed their controlled deposition by the Langmuir–Blodgett (LB) technique. Superparamagnetic and fluorescent properties of the particles are preserved within the LB film.

Electrochemical properties of SWNT/ferritin composite for bioapplications

A functionalized single-wall carbon nanotube (SWNT), fabricated using an acid treatment, was used to prepare SWNT/ferritin composites. Different physical and chemical composites were synthesized on a glassy carbon electrode, and their structures and electrochemical properties were analyzed. The specific redox reaction due to ferritin appeared in both composites, but depended on the structure of the composite, which influenced the electrochemical properties. The redox reaction of ferritin was analyzed using fulfilled core, holoferritin, and coreless, apoferritin. From the electrochemical results, we confirmed that electron transfer through the ferritin shell is possible, and that the core of the ferritin facilitates electron transfer in the composites. The chemical composites showed a significant catalytic activity towards hydrogen peroxide, and the electrochemical results show that this type of composite has potential as biosensors and in bioapplications.

Fluorescence resonance energy transfer in ferritin labeled with multiple fluorescent dyes

We simultaneously labeled ferritin with two Alexa Fluor fluorophores (AF350 and AF430). When both fluorophores label the same ferritin subunit, fluorescence resonance energy transfer (FRET) takes place from the excited AF350 to the acceptor AF430. By varying the number and the ratio of labeling fluorophores, we can modulate FRET such that the ferritin particles can exhibit multiple colors under UV illumination. Labeling of the ferritin shell does not affect the properties of the metallic core.

ELISA reveals a difference in the structure of substantia nigra ferritin in Parkinson's disease and incidental Lewy body compared to control

Iron released from ferritin may trigger oxidative stress leading to progressive neurodegeneration of substantia nigra resulting in Parkinson's disease (PD). Change in the structure of ferritin may allow an easier efflux of iron. We compared with the use of ELISA the structure of ferritin (concentrations of H and L ferritins) in substantia nigra (SN) in ten cases of PD, six of incidental Lewy body (ILB) cases and 20 controls. SN concentration of L ferritin in ILB (50.6±11.5 ng/mg) and in PD (52.5±26.0) was lower than in control (97.9±54.9). H ferritin in PD (534.2±223.1) was higher than in ILB (336.9±87.7) and control (374.8±169.3). The decrease of L ferritin in SN in PD and ILB may suggest that the whole process of neurodegeneration starts with a higher availability of free iron, which is released from the ferritin shell.

Characterization of the l-ferritin variant 460InsA responsible of a hereditary ferritinopathy disorder

Hereditary ferritinopathies are dominant inherited movement disorders associated with extensive alterations of the l-ferritin C-terminus peptide caused by nucleotide insertions in l-ferritin gene (FTL). We describe the characterization of the most common variant, produced by the 460InsA mutations and here named Ln1. The recombinant Ln1 assembled into 24-mer ferritin shells with low efficiency, however, it was able to form heteropolymers that showed a reduced capacity to incorporate iron in vitro. The Ln1 expressed in HeLa cells formed hybrid ferritins, with the endogenous H and L chains, and caused an iron excess phenotype. Ferritin inactivation and faster degradation in Ln1 transfectants concurred in increasing iron availability, which was probably responsible for the higher sensitivity to H(2)O(2) toxicity and higher level of oxidized proteins. The findings suggest that the pathogenic effects of Ln1 expression are more likely due to deregulation of cellular iron homeostasis rather than to protein conformational problems.

Mutations of Ferritin H Chain C-Terminus Produced by Nucleotide Insertions Have Altered Stability and Functional Properties

Ferritin is an iron storage protein made of 24 subunits. Previous mutational analyses showed that ferritin C-terminal region has a major role in protein stability and assembly but is only marginally involved in the mechanism of iron incorporation. However, it has recently been shown that patients who carry alterations of ferritin C-terminal sequence caused by nucleotide insertions show neurological disorders possibly related to altered protein functionality and cellular iron deregulation. To re-evaluate the role of this region, five mutants of mouse H-ferritin were produced by 2-nucleotide insertions that modified the last 6-29 residues and extended the sequence of 14 amino acids. The mutants were expressed in Escherichia coli and analysed for solubility, stability and capacity to incorporate iron. The alteration of the last 6-residue non-helical extension had no evident effect on the properties of ferritin, while solubility and capacity to assemble in ferritin shells decreased progressively with the extension of the modified region. The results also showed that the modification of even a part of the terminal E-helix abolished the capacity of ferritin to incorporate iron during expression in the cells, probably caused by conformational modification of the hydrophobic channels. The data support the hypothesis that the pathogenic mutations alter cellular iron homeostasis.

Electron Exchange between Fe(II)-Horse Spleen Ferritin and Co(III)/Mn(III) Reconstituted Horse Spleen andAzotobacter vinelandiiFerritins

Azotobacter vinelandii bacterioferritin (AvBF) containing 800-1500 Co or Mn atoms as Co(III) and Mn(III) oxyhydroxide cores (Co-AvBF, Mn-AvBF) was synthesized by the same procedure used previously for horse spleen ferritin (HoSF). The kinetics of reduction of Co-AvBF and Mn-AvBF by ascorbic acid are first-order in each reactant. The rate constant for the reduction of Mn-AvBF (8.52 M(-1) min(-1)) is approximately 12 times larger than that for Co-AvBF (0.72 M(-1) min(-1)), which is consistent with a previous observation that Mn-HoSF is reduced approximately 10-fold faster than Co-HoSF [Zhang, B. et al. (2005) Inorg. Chem. 44, 3738-3745]. The rates of reduction of M-AvBF (M = Co and Mn) are more than twice that for the reduction of the corresponding M-HoSF. HoSF containing reduced Fe(II) cores (Fe(II)-HoSF), prepared by methyl viologen and CO, also reduces M-HoSF and M-AvBF species, with both cores remaining within ferritin, suggesting that electrons transfer through the ferritin shell. Electron transfer from Fe(II)-HoSF to Co-AvBF occurs at a rate approximately 3 times faster than that to Co-HoSF, indicating that the Co cores in AvBF are more accessible to reduction than the Co cores in HoSF. The presence of nonconductive (SiO2) or conductive (gold) surfaces known to bind ferritins enhances the rate of electron transfer. A more than approximately 4-fold increase in the apparent reaction rate is observed in the presence of gold. Although both surfaces (SiO2 and gold) enhance reaction by providing binding sites for molecular interaction, results show that ferritins with different mineral cores bound to a gold surface transfer electrons through the gold substrate so that direct contact of the reacting molecules is not required.

Size control for two-dimensional iron oxide nanodots derived from biological molecules

We demonstrated the fabrication of size-controlled two-dimensional iron oxide nanodots derived from the heat treatment of ferritin molecules self-immobilized on modified silicon surfaces. Ferritin molecules were immobilized onto 3-aminopropyltrimethoxysilane (3-APMS)-modified silicon surfaces by electrostatic interactions between negatively charged amino acids of ferritin molecules and amino terminal functional groups of 3-APMS. Heat treatments were performed at 400 °C for 60 min to fabricate two-dimensional nanodots based on ferritin cores. XPS and FT-IR results clearly indicate that ferritin shells were composed of amino acids and 3-APMS modifiers on silicon surfaces were eliminated by heat treatment. Nanodots on substrate surfaces corresponded to iron oxides. The size of nanodots was tunable in the range of 0–5 (±0.75) nm by in situ reactions of iron ion chelators with ferritin molecules immobilized on substrates before heat treatment.

Rate of iron transfer through the horse spleen ferritin shell determined by the rate of formation of Prussian Blue and Fe-desferrioxamine within the ferritin cavity

Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN) 6] 3−, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible. We report the rate of Fe 2+ penetrating into the ferritin interior by adding external Fe 2+ to [Fe(CN) 6] 3− encapsulated in the HoSF interior and measuring the rate of formation of the resulting encapsulated PB. The rate at which Fe 2+ reacts with [Fe(CN) 6] 3− in the HoSF interior is much slower than the formation of free PB in solution and is proceeded by a lag period. We assume this lag period and the difference in rate represent the transfer of Fe 2+ through the HoSF protein shell. The calculated diffusion coefficient, D ∼5.8 × 10 − 20 m 2/s corresponds to the measured lag time of 10–20 s before PB forms within the HoSF interior. The activation energy for Fe 2+ transfer from the outside solution through the protein shell was determined to be 52.9 kJ/mol by conducting the reactions at 10∼40 °C. The reaction of Fe 3+ with encapsulated [Fe(CN) 6] 4− also readily forms PB in the HoSF interior, but the rate is faster than the corresponding Fe 2+ reaction. The rate for Fe 3+ transfer through the ferritin shell was confirmed by measuring the rate of the formation of Fe-DES inside HoSF and an activation energy of 58.4 kJ/mol was determined. An attempt was made to determine the rate of iron (2+ and 3+) transit out from the ferritin interior by adding excess bipyridine or DES to PB trapped within the HoSF interior. However, the reactions are slow and occur at almost identical rates for free and HoSF-encapsulated PB, indicating that the transfer of iron from the interior through the protein shell is faster than the rate-limiting step of PB dissociation. The method described in this work presents a novel way of determining the rate of transfer of iron and possibly other small molecules through the ferritin shell.

Release of Iron from Ferritin by Aceto- and Benzohydroxamic Acids

The release of iron from ferritin by aceto- and benzohydroxamic acids was studied at two different iron chelator concentrations (100 and 10 mM), at two pH values (7.4 and 5.2), and in the presence or absence of urea. Collectively, the results demonstrate that both aceto- and benzohydroxamic acids remove iron from ferritin. Aceto- and benzohydroxamic acids penetrate the ferritin shell and react directly with the iron core of the ferritin cavity probably forming mono(hydroxamate) iron(III) complexes which exit ferritin and react with the excess hydroxamate in the solution to produce bis(hydroxamate) iron(III) complexes. The sizes of both the benzohydroxamic acid and the mono(benzohydroxamate) iron(III) complex, 6 and 7 A, respectively, are larger than that of the ferritin channels which indicates the flexibility of the channels to allow the entry and exit of these molecules. The size of the hydroxamic acid influenced the effectiveness of the iron release from ferritin following the expected trend with smaller iron chelators showing greater effectiveness. Likewise, the percentage of iron removed from ferritin was pH-dependent; the percentage of iron removed at pH 5.2 was greater than that at pH 7.4. Finally, the presence of urea, capable of opening the ferritin channels, dramatically increased the effectiveness of the iron chelator in removing iron from ferritin, especially at pH 7.4.