RESUMO
Iron oxide biomineralization occurs in all living organisms and typically involves protein compartments ranging from 5 to 100nm in size. The smallest iron-oxo particles are formed inside dodecameric Dps protein cages, while the structurally related ferritin compartments consist of twice as many identical protein subunits. The largest known compartments are encapsulins, icosahedra made of up to 180 protein subunits that harbor additional ferritin-like proteins in their interior. The formation of iron-oxo particles in all these compartments requires a series of steps including recruitment of iron, translocation, oxidation, nucleation, and storage, that are mediated by ferroxidase centers. Thus, compartmentalized iron oxide biomineralization yields uniform nanoparticles strictly determined by the sizes of the compartments, allowing customization for highly diverse nanotechnological applications.
Assuntos
Ceruloplasmina/metabolismo , Compostos Férricos/metabolismo , Minerais/metabolismo , Compartimento Celular , Ferritinas/metabolismoRESUMO
Iron storage in biology is carried out by cage-shaped proteins of the ferritin superfamily, one of which is the dodecameric protein Dps. In Dps, four distinct steps lead to the formation of metal nanoparticles: attraction of ion-aquo complexes to the protein matrix, passage of these complexes through translocation pores, oxidation of these complexes at ferroxidase centers, and, ultimately, nanoparticle formation. In this study, we investigated Dps from Listeria innocua to structurally characterize these steps for Co2+, Zn2+, and La3+ ions. The structures reveal that differences in their ion coordination chemistry determine alternative metal ion-binding sites on the areas of the surface surrounding the translocation pore that captures nine La3+, three Co2+, or three Zn2+ ions as aquo clusters and passes them on for translocation. Inside these pores, ion-selective conformational changes at key residues occur before a gating residue to actively move ions through the constriction zone. Ions upstream of the Asp130 gate residue are typically hydrated, while ions downstream directly interact with the protein matrix. Inside the cavity, ions move along negatively charged residues to the ferroxidase center, where seven main residues adapt to the three different ions by dynamically changing their conformations. In total, we observed more than 20 metal-binding sites per Dps monomer, which clearly highlights the metal-binding capacity of this protein family. Collectively, our results provide a detailed structural description of the preparative steps for amino acid-assisted biomineralization in Dps proteins, demonstrating unexpected protein matrix plasticity.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Listeria/química , Metais Pesados/química , Proteínas de Bactérias/biossíntese , Proteínas de Ligação a DNA/biossíntese , Modelos Moleculares , Eletricidade EstáticaRESUMO
The first six peptides of multifunctional titanium binding peptide-1 bestowed recombinant L-ferritin, minT1-LF, was genetically engineered and used to fabricate multilayered nanoparticle architecture. The multifunctionality of minT1-LF enables specific binding of nanoparticle-accommodated minT1-LF to the silicon substrate surface and wet biochemical fabrication of gate oxide layer by its biomineralization activity. Three-dimensional (3D) nanoparticle architecture with multilayered structure was fabricated by the biological layer-by-layer method and embedded in a metal oxide-semiconductor device structure as a charge storage node of a flash memory device. The 3D-integrated multilayered nanoparticle architecture successfully worked as a charge storage node in flash memory devices that exhibited improved charge storage capacity compared with that of a conventional monolayer structure device.
Assuntos
Nanoestruturas/química , Microscopia Eletrônica de Transmissão , Nanoestruturas/ultraestrutura , Semicondutores , VolatilizaçãoRESUMO
The synthesis of magnetic, monodisperse nanoparticles has attracted great interest in nanoelectronics and nanomedicine. Here we report the fabrication of pure magnetite nanoparticles, less than ten nanometers in size, using the cage-shaped protein apoferritin (Fe(3)O(4)-ferritin). Crystallizable proteins were obtained through careful successive separation methods, including a magnetic chromatography that enabled the effective separation of proteins, including a Fe(3)O(4) nanoparticle (7.9 ± 0.8 nm), from empty ones. Macroscopic protein crystals allowed the fabrication of three-dimensional arrays of Fe(3)O(4) nanoparticles with interparticle gaps controlled by dehydration, decreasing their magnetic susceptibilities and increasing their blocking temperatures through enhanced dipole-dipole interactions.
Assuntos
Apoferritinas/química , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Animais , Cristalização , Cavalos , Nanotecnologia , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The adhesion process of osteoblast-like cells on hydroxyapatite (HAp) and oxidized polystyrene (PSox) was investigated using a quartz crystal microbalance with dissipation (QCM-D), confocal laser scanning microscope (CLSM), and atomic force microscope (AFM) techniques in order to clarify the interfacial phenomena between the surfaces and cells. The interfacial viscoelastic properties (shear viscosity (η(ad)), elastic shear modulus (µ(ad)), and tan δ) of the preadsorbed protein layer and the interface layer between the surfaces and cells were estimated using a Voigt-based viscoelastic model from the measured frequency (Δf) and dissipation shift (ΔD) curves. In the ΔD-Δf plots, the cell adhesion process on HAp was classified as (1) a mass increase only, (2) increases in both mass and ΔD, and (3) slight decreases in mass and ΔD. On PSox, only ΔD increases were observed, indicating that the adhesion behavior depended on the surface properties. The interfacial µ(ad) value between the material surfaces and cells increased with the number of adherent cells, whereas η(ad) and tanδ decreased slightly, irrespective of the surface. Thus, the interfacial layer changed the elasticity to viscosity with an increase in the number. The tan δ values on HAp were higher than those on PSox and exceeded 1.0. Furthermore, the pseudopod-like structures of the cells on HAp had periodic stripe patterns stained with a type I collagen antibody, whereas those on PSox had cell-membrane-like structures unstained with type I collagen. These results indicate that the interfacial layers on PSox and HAp exhibit elasticity and viscosity, respectively, indicating that the rearrangements of the extracellular matrix and cytoskeleton changes cause different cell-surface interactions. Therefore, the different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully monitored in situ and evaluated by the QCM-D technique combined with other techniques.
Assuntos
Adesão Celular , Durapatita/química , Osteoblastos/citologia , Poliestirenos/química , Quartzo , Células 3T3 , Animais , Camundongos , Microscopia de Força Atômica , OxirreduçãoRESUMO
The mineralized structure of aligned collagen fibrils in a tilapia fish scale was investigated using transmission electron microscopy (TEM) techniques after a thin sample was prepared using aqueous techniques. Electron diffraction and electron energy loss spectroscopy data indicated that a mineralized internal layer consisting of aligned collagen fibrils contains hydroxyapatite crystals. Bright-field imaging, dark-field imaging, and energy-filtered TEM showed that the hydroxyapatite was mainly distributed in the hole zones of the aligned collagen fibrils structure, while needle-like materials composed of calcium compounds including hydroxyapatite existed in the mineralized internal layer. Dark-field imaging and three-dimensional observation using electron tomography revealed that hydroxyapatite and needle-like materials were mainly found in the matrix between the collagen fibrils. It was observed that hydroxyapatite and needle-like materials were preferentially distributed on the surface of the hole zones in the aligned collagen fibrils structure and in the matrix between the collagen fibrils in the mineralized internal layer of the scale.
Assuntos
Colágeno/ultraestrutura , Tomografia com Microscopia Eletrônica , Tegumento Comum , Microscopia Eletrônica de Transmissão por Filtração de Energia , Minerais/análise , Tilápia , AnimaisRESUMO
Cage-shaped protein (CSP) complexes are frequently used in bionanotechnology, and they have a variety of different architectures and sizes. The smallest cage-shaped protein, Dps (DNA binding protein from starved cells), can naturally form iron oxide biominerals in a multistep process of ion attraction, translocation, oxidation, and nucleation. The structural basis of this biomineralization mechanism is still unclear. The aim of this paper is to further develop understanding of this topic. Time-resolved metal translocation of Yb3+ ions has been investigated on Dps surfaces using X-ray crystallography. The results reveal that the soak time of protein crystals with Yb3+ ions strongly affects metal positions during metal translocation, in particular, around and inside the ion translocation pore. We have trapped a dynamic state with ongoing translocation events and compared this to a static state, which is reached when the cavity of Dps is entirely filled by metal ions and translocation is therefore blocked. By comparison with La3+ and Co2+ datasets, the time-dependence together with the coordination sphere chemistry primarily determine metal-protein interactions. Our data can allow structure-based protein engineering to generate CSPs for the production of tailored nanoparticles.
RESUMO
Magnetic nanoparticle (MNP) composites with a magnetite (Fe(3)O(4)) core and a hydroxyapatite (HAp, Ca(10)(PO(4))(6)(OH)(2)) coating were prepared using a precipitation method and a subsequent hydrothermal treatment. The hydrothermal treatment diminished the lepidocrocite layer on the magnetite, enhanced the crystal growth of HAp and dissolved the MNPs. The divalent iron ions dissolved into solvent were not substituted for the HAp lattice. The three-dimensional (3D) nanostructure, the crystal morphology of HAp covered with the MNPs and the interfacial nanostructure of magnetite/HAp were analyzed using an energy-filter transmission electron microscopy (EF-TEM) and visualized by computer tomography in transmission electron microscopy (TEM). EF-TEM and 3D reconstruction images using a tilted series of high-angle annular dark-field images showed that the needlelike HAp nanocrystals covered with a magnetite core and the crystal growth of HAp attached to the magnetite surface was inhibited as a result of the lower density of the nucleation site of the lepidocrocite layer. The dissolution of iron ion from MNPs and the interfacial interaction of HAp and magnetite could cause the needlelike morphology of HAp nanocrystals.
RESUMO
Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.
Assuntos
Nanopartículas de Magnetita/química , Células-Tronco Mesenquimais/citologia , Coloração e Rotulagem/métodos , Animais , Apoferritinas/química , Cavalos , Humanos , Ferro/química , Células-Tronco Mesenquimais/metabolismoAssuntos
Ferritinas/química , Nanopartículas/química , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Nanotubos de Peptídeos/química , Adsorção , Animais , Cobalto/química , Escherichia coli/metabolismo , Cavalos , Micromanipulação , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Modelos Moleculares , Platina/química , Propriedades de Superfície , Titânio/químicaRESUMO
We report here, for the first time, a biotemplated synthesis of uniform CdSe nanoparticle (4.1 ± 0.5 nm) and a fabrication of two-dimensional CdSe nanoparticles (over one micrometre) with nanometric gaps by cage-like protein, Listeria-Dps.
Assuntos
Compostos de Cádmio/síntese química , Proteínas de Ligação a DNA/química , Nanopartículas/química , Compostos de Selênio/síntese química , Compostos de Cádmio/química , Listeria/química , Tamanho da Partícula , Compostos de Selênio/químicaRESUMO
Elemental distribution of calcium, phosphorus, oxygen, and carbon in a single collagen fibril obtained from tilapia fish scales was identified with an electron energy-loss spectroscopy and an energy-filtered transmission electron microscopy, for the first time. The carbon intensity profile of the single collagen fibril showed the specific D-periodic pattern at 67 nm of type I collagen fibrils. The calcium L(2,3)-edge and oxygen K-edge peak positions were detected at 347/350 eV and 137 eV, respectively, and these positions were identical to those of hydroxyapatite. Calcium, phosphorus, and oxygen were present in the hole zones as the amorphous phase, while carbon was present in the overlap zone. Our results indicated that the hole zones preferentially attract calcium and phosphate ions and thus serve as possible nucleation sites for mineralization.
Assuntos
Cálcio/análise , Carbono/análise , Colágeno Tipo I/química , Oxigênio/análise , Fósforo/análise , Tilápia , Animais , Microscopia Eletrônica de Transmissão por Filtração de Energia , Tilápia/anatomia & histologiaRESUMO
Cavities formed by proteins have been utilized as the reaction chamber for the fabrication of a range of inorganic nanoparticles, providing control of the size of particles by limiting growth and preventing agglomeration. In crystal form, proteins construct molecular arrays that can provide regularly arranged sites for nanoparticles. Here we report the fabrication of nanometric iron and indium particles using ferritin, an iron-storage protein. The indium nanoparticles thus formed have uniform spherical shape with diameter of 6.6 +/- 0.5 nm, while the iron nanoparticles are somewhat irregular in shape (5.8 +/- 1.0 nm). Regular two-dimensional arrays of these nanoparticles are successfully produced by crystallizing ferritin molecules on a water-air interface using the denatured protein film method. The lattice constant of these nanoparticle arrays is 13 nm with hexagonal packing, and arrays of more than 1 microm in area can be obtained by transfer onto silicon wafer.
Assuntos
Cristalização/métodos , Ferritinas/química , Ferritinas/ultraestrutura , Índio/química , Ferro/química , Nanotecnologia/métodos , Nanotubos/química , Nanotubos/ultraestrutura , Adsorção , Ferritinas/análise , Índio/análise , Compostos Inorgânicos/análise , Compostos Inorgânicos/química , Ferro/análise , Nanotubos/análise , Tamanho da Partícula , Ligação ProteicaRESUMO
The iron storage protein, apoferritin, has a cavity in which iron is oxidized and stored as a hydrated oxide core. The size of the core is about 7 nm in diameter and is regulated by the cavity size. The cavity can be utilized as a nanoreactor to grow inorganic crystals. We incubated apoferritin in nickel or chromium salt solutions to fabricate hydroxide nanoparticles in the cavity. By using a solution containing dissolved carbon dioxide and by precisely controlling the pH, we succeeded in fabricating nickel and chromium cores. During the hydroxylation process of nickel ions a large portion of the apoferritin precipitated through bulk precipitation of nickel hydroxide. Bulk precipitation was suppressed by adding ammonium ions. However, even in the presence of ammonium ions the core did not form using a degassed solution. We concluded that carbonate ions were indispensable for core formation and that the ammonium ions prevented precipitation in the bulk solution. The optimized condition for nickel core formation was 0.3 mg/mL horse spleen apoferritin and 5 mM ammonium nickel sulfate in water containing dissolved carbon dioxide. The pH was maintained at 8.65 using two buffer solutions: 150 mM HEPES (pH 7.5) and 195 mM CAPSO (pH 9.5) with 20 mM ammonium at 23 degrees C. The pH had not changed after 48 h. After 24 h of incubation, all apoferritins remained in the supernatant and all of them had cores. Recombinant L-ferritin showed less precipitation even above a pH of 8.65. A chromium core was formed under the following conditions: 0.1 mg/mL apoferritin, 1 mM ammonium chromium sulfate, 100 mM HEPES (pH 7.5) with a solution containing dissolved carbon dioxide. About 80% of the supernatant apoferritin (0.07 mg/mL) formed a core. In nickel and chromium core formation, carbonate ions would play an important role in accelerating the hydroxylation in the apoferritin cavity compared to the bulk solution outside.