Ferritin Crystals Research Articles

Where is the Macrocycle? Comment on “Simultaneous Organic and Inorganic Host‐Guest Chemistry within Pillararene‐Protein Cage Frameworks.”

Kostiainen and co-workers claim to have prepared crystals of ferritin by "electrostatically bridging" the protein with a cationic pillar[5]arene. Questions remain regarding the contribution of the pillar[5]arene to protein assembly and the putative role as a molecular glue.

Existence of twisting in dislocation-free protein single crystals

The growth of high-quality protein crystals is a prerequisite for the structure analysis of proteins by X-ray diffraction. However, dislocation-free perfect crystals such as silicon and diamond have been so far limited to only two kinds of protein crystals, such as glucose isomerase and ferritin crystals. It is expected that many other high-quality or dislocation-free protein crystals still exhibit some imperfection. The clarification of the cause of imperfection is essential for the improvement of crystallinity. Here, we explore twisting as a cause of the imperfection in high-quality protein crystals of hen egg-white lysozyme crystals with polymorphisms (different crystal forms) by digital X-ray topography with synchrotron radiation. The magnitude of the observed twisting is 10−6 to 10−5°/μm which is more than two orders smaller than 10−3 to 104°/μm in other twisted crystals owing to technique limitations with optical and electron microscopy. Twisting is clearly observed in small crystals or in the initial stage of crystal growth. It is uniformly relaxed with crystal growth and becomes smaller in larger crystals. Twisting is one of main residual defects in high-quality crystals and determines the crystal perfection. Furthermore, it is presumed that the handedness of twisting can be ascribed to the anisotropic interaction of chiral protein molecules associated with asymmetric units in the crystal forms. This mechanism of twisting may correspond to the geometric frustration proposed as a primary mechanism of twisting in molecular crystals. Our finding provides insights for the understanding of growth mechanism and the growth control of high-quality crystals.

Anisotropic Dynamics and Mechanics of Macromolecular Crystals Containing Lattice-Patterned Polymer Networks.

The mechanical and functional properties of many crystalline materials depend on cooperative changes in lattice arrangements in response to external perturbations. However, the flexibility and adaptiveness of crystalline materials are limited. Additionally, the bottom-up, molecular-level design of crystals with desired dynamic and mechanical properties at the macroscopic level remains a considerable challenge. To address these challenges, we had previously integrated mesoporous, cubic ferritin crystals with hydrogel networks, resulting in hybrid materials (polymer-integrated crystals or PIX) which could undergo dramatic structural changes while maintaining crystalline periodicity and display efficient self-healing. The dynamics and mechanics of these ferritin-PIX were devoid of directionality, which is an important attribute of many molecular and macroscopic materials/devices. In this study, we report that such directionality can be achieved through the use of ferritin crystals with anisotropic symmetries (rhombohedral or trigonal), which enable the templated formation of patterned hydrogel networks in crystallo. The resulting PIX expand and contract anisotropically without losing crystallinity, undergo prompt bending motions in response to stimuli, and self-heal efficiently, capturing some of the essential features of sophisticated biological devices like skeletal muscles.

Evaluation of crystal quality of thin protein crystals based on the dynamical theory of X-ray diffraction.

Knowledge of X-ray diffraction in macromolecular crystals is important for not only structural analysis of proteins but also diffraction physics. Dynamical diffraction provides evidence of perfect crystals. Until now, clear dynamical diffraction in protein crystals has only been observed in glucose isomerase crystals. We wondered whether there were other protein crystals with high quality that exhibit dynamical diffraction. Here we report the observation of dynamical diffraction in thin ferritin crystals by rocking-curve measurement and imaging techniques such as X-ray topography. It is generally known that in the case of thin crystals it is difficult to distinguish whether dynamical diffraction occurs from only rocking-curve profiles. Therefore, our results clarified that dynamical diffraction occurs in thin protein crystals because fringe contrasts similar to Pendellösung fringes were clearly observed in the X-ray topographic images. For macromolecular crystallography, it is hard to obtain large crystals because they are difficult to crystallize. For thin crystals, dynamical diffraction can be demonstrated by analysis of the equal-thickness fringes observed by X-ray topography.

Effect of the point mutation H54N on the ferroxidase process of Rana catesbeiana H′ ferritin

Human H and Rana catesbeiana H′ subunits in vertebrate ferritin protein cages catalyze the Fe(II) oxidation by molecular oxygen and promote the ferric oxide biomineral synthesis. By depositing iron biomineral, ferritins also prevent potentially toxic reactions products from Fe(II)-based Fenton chemistry. Recent work from our laboratory was aimed to describe the iron pathways within ferritin, from entrance into the cage to the ferroxidase site, and to understand the role played by amino-acid residues in iron trafficking and catalysis. Our approach exploits anomalous X-ray diffraction from ferritin crystals, exposed to a ferrous salt, to track transient iron binding sites along the path towards a well-defined di-iron site where they get oxidized by oxygen. Coupling structure determination with solution kinetic measurements on selected variants, allows validating the role played by key residues on the catalytic iron oxidation. Our previous studies on H′ ferritin indicated the regulatory role played by His54, and by its human counterpart Gln58, on guiding Fe(II) ions to the catalytic site. Here, we have investigated the effects induced by substituting the wild type His54 with Asn54, having different iron coordination properties. We have obtained a series of atomic-resolution crystal structures that provide time-dependent snapshots of iron bound at different locations in the H′ ferritin H54N variant. The comparison with H′ ferritin and H′ ferritin H54Q variant leads to identify a new iron binding site. Our kinetic and structural data support the role of H′ ferritin residue 54 in regulating the access of Fe(II) ions to the catalytic site.

A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements

A technique is described for generating large well diffracting crystals from conditions that yield microcrystals. Crystallization using this technique is both rapid (crystals appear in <1 h) and robust (48 out of 48 co-crystallized with a fragment library, compared with 26 out of 48 using conventional hanging drop). Agarose gel is used to exclude nucleation inducing elements from the remaining crystallization cocktail. The chemicals in the crystallization cocktail are partitioned into high concentration components (presumed to induce aggregation by reducing water activity) and low concentration nucleation agents (presumed to induce nucleation through direct interaction). The nucleation agents are then combined with 2% agarose gel and deposited on the crystallization shelf of a conventional vapor diffusion plate. The remaining components are mixed with the protein and placed in contact with the agarose drop. This technique yielded well diffracting crystals of lysozyme, cubic insulin, proteinase k, and ferritin (ferritin crystals diffracted to 1.43 Å). The crystals grew rapidly, reaching large size in less than one hour (maximum size was achieved in 1–12 h). This technique is not suitable for poorly expressing proteins because small protein volumes diffuse out of the agarose gel too quickly. However, it is a useful technique for situations where crystals must grow rapidly (such as educational applications and preparation of beamline test specimens) and in situations where crystals must grow robustly (such as co-crystallization with a fragment library).

Hyperexpandable, self-healing macromolecular crystals with integrated polymer networks.

The formation of condensed matter typically involves a trade-off between structural order and flexibility. As the extent and directionality of interactions between atomic or molecular components increase, materials generally become more ordered but less compliant, and vice versa. Nevertheless, high levels of structural order and flexibility are not necessarily mutually exclusive; there are many biological (such as microtubules1,2, flagella 3 , viruses4,5) and synthetic assemblies (for example, dynamic molecular crystals6-9 and frameworks10-13) that can undergo considerable structural transformations without losing their crystalline order and that have remarkable mechanical properties8,14,15 that are useful in diverse applications, such as selective sorption 16 , separation 17 , sensing 18 and mechanoactuation 19 . However, the extent of structural changes and the elasticity of such flexible crystals are constrained by the necessity to maintain a continuous network of bonding interactions between the constituents of the lattice. Consequently, even the most dynamic porous materials tend to be brittle and isolated as microcrystalline powders 14 , whereas flexible organic or inorganic molecular crystals cannot expand without fracturing. Owing to their rigidity, crystalline materials rarely display self-healing behaviour 20 . Here we report that macromolecular ferritin crystals with integrated hydrogel polymers can isotropically expand to 180 per cent of their original dimensions and more than 500 per cent of their original volume while retaining periodic order and faceted Wulff morphologies. Even after the separation of neighbouring ferritin molecules by 50 ångströms upon lattice expansion, specific molecular contacts between them can be reformed upon lattice contraction, resulting in the recovery of atomic-level periodicity and the highest-resolution ferritin structure reported so far. Dynamic bonding interactions between the hydrogel network and the ferritin molecules endow the crystals with the ability to resist fragmentation and self-heal efficiently, whereas the chemical tailorability of the ferritin molecules enables the creation of chemically and mechanically differentiated domains within single crystals.

Iron binding to human heavy-chain ferritin.

Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein from Rana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with its R. catesbeiana counterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species.

Time-lapse anomalous X-ray diffraction shows how Fe(2+) substrate ions move through ferritin protein nanocages to oxidoreductase sites.

Ferritin superfamily protein cages reversibly synthesize internal biominerals, Fe2O3·H2O. Fe(2+) and O2 (or H2O2) substrates bind at oxidoreductase sites in the cage, initiating biomineral synthesis to concentrate iron and prevent potentially toxic reactions products from Fe(2+)and O2 or H2O2 chemistry. By freezing ferritin crystals of Rana catesbeiana ferritin M (RcMf) at different time intervals after exposure to a ferrous salt, a series of high-resolution anomalous X-ray diffraction data sets were obtained that led to crystal structures that allowed the direct observation of ferrous ions entering, moving along and binding at enzyme sites in the protein cages. The ensemble of crystal structures from both aerobic and anaerobic conditions provides snapshots of the iron substrate bound at different cage locations that vary with time. The observed differential occupation of the two iron sites in the enzyme oxidoreductase centre (with Glu23 and Glu58, and with Glu58, His61 and Glu103 as ligands, respectively) and other iron-binding sites (with Glu53, His54, Glu57, Glu136 and Asp140 as ligands) reflects the approach of the Fe(2+) substrate and its progression before the enzymatic cycle 2Fe(2+) + O2 → Fe(3+)-O-O-Fe(3+) → Fe(3+)-O(H)-Fe(3+) and turnover. The crystal structures also revealed different Fe(2+) coordination compounds bound to the ion channels located at the threefold and fourfold symmetry axes of the cage.

Equilibrium forms of protein crystals

Models for equilibrium forms of protein crystals were suggested using classical methods, such as Stranski–Kaischew's mean-separation-work method and the Gibbs–Curie–Wulff law. Although absolute bonding energies in protein crystal lattices are unknown, their relative strength was estimated from the type of lattice contacts observed in the protein x-ray structures. Equilibrium forms of ferritin crystals deduced on this basis were compared with experimental observations of Yau and Vekilov.

Structural insights into the ferroxidase site of ferritins from higher eukaryotes.

The first step of iron biomineralization mediated by ferritin is the oxidation at the ferroxidase active site of two ferrous ions to a diferric oxo/hydroxo species. Metal-loaded ferritin crystals obtained by soaking crystals of frog ferritin in FeSO(4) and CuSO(4) solutions followed by flash freezing provided X-ray crystal structures of the tripositive iron and bipositive copper adducts at 2.7 and 2.8 Å resolution, respectively. At variance with the already available structures, the crystal form used in this study contains 24 independent subunits in the asymmetric unit permitting comparison between them. For the first time, the diferric species at the ferroxidase site is identified in ferritins from higher eukaryotes. Anomalous difference Fourier maps for crystals (iron crystal 1) obtained after long soaking times in FeSO(4) solution invariantly showed diferric species with a Fe-Fe average distance of 3.1 ± 0.1 Å, strongly indicative of the presence of a μ-oxo/hydroxo bridge between the irons; protein ligands for each iron ion (Fe1 and Fe2) were also unequivocally identified and found to be the same in all subunits. For copper bound ferritin, dicopper(II) centers are also observed. While copper at site 1 is essentially in the same position and has the same coordination environment as Fe1, copper at site 2 is displaced toward His54, now acting as a ligand; this results in an increased intermetal distance (4.3 ± 0.4 Å). His54 coordination and longer metal-metal distances might represent peculiar features of divalent cations at the ferroxidase site. This oxidation-dependent structural information may provide key features for the mechanistic pathway in ferritins from higher eukaryotes that drive uptake of bivalent cation and release of ferric products at the catalytic site. This mechanism is supported by the X-ray picture obtained after only 1 min of soaking in FeSO(4) solutions (iron crystal 2) which reasonably contain the metal at different oxidation states. Here two different di-iron species are trapped in the active site, with intermetal distances corresponding to those of the ferric dimer in crystal 1 and of the dicopper centers and corresponding rearrangement of the His54 side chain.

Irradiance regulates genotype-specific responses of Rhizobium-nodulated soybean to increasing iron and two manganese concentrations in solution culture

The growth of soybean plants were examined when subjected to three contrasting irradiance levels and to various combinations of nutrient solution Fe and Mn concentrations. Two Rhizobium-nodulated soybean genotypes (PI 227557 and Biloxi), which had been previously found to differ in their growth response to various Fe and Mn solutions, were studied. Both genotypes displayed the poorest growth, nodulation and the lowest chlorophyll and nodule ureide concentration at high irradiance (HI), regardless of the solution Fe and Mn concentrations. However, the genotypes differed under HI in their accumulation of Fe. For solution concentrations greater than 13 microM, PI 227557 accumulated up to 1200 microg Feg(-1) leaf dry wt mainly in the form of ferritin crystals within chloroplasts. In contrast, leaf Fe concentrations in Biloxi only reached 300 microg Feg(-1) dry wt and there were no ferritin crystals. Also, in PI 227557 HI induced more severe distortions in leaf cells and nodule ultrastructure than in Biloxi. Based on its poor growth under HI, PI 227557 could be categorized as an Fe-inefficient genotype prone to undergo photoinhibition at HI, in spite of the ferritin crystals in the chloroplasts. Enhanced growth, nodulation, chlorophyll and ureide concentrations in nodules as well as leaf ureide catabolism occurred in both genotypes grown at moderate irradiance (MI) in Fe solutions from 13 to 60 microM supplied with 20 microM Mn. At low irradiance (LI), plant growth and nodulation were lower than at MI values, but higher than those of plants at HI. Irradiance and solution Fe concentration did not alter leaf Cu and Zn concentration in either genotype, with the higher concentrations of these two elements detected in Biloxi. Solutions with Fe concentrations greater than 100 microM were deleterious for both genotypes at all irradiances. Low Fe and high Mn concentrations in leaves was bound to result in the best growth at HI.

Toward a Definition of X-ray Crystal Quality

Crystal X-ray quality is usually evaluated by looking at data quality parameters such as (relative) Wilson B-factor, resolution, R-factors, signal-to-noise ratio, and others. As these parameters are correlated, most studies focus on one or two of them. As part of a study of the effects of microgravity on X-ray quality, full data sets of 35 ferritin crystals (17 PromISS-4 “space” crystals and 18 from the ground control) were collected. Sixty-three parameters commonly used as indicative of X-ray data quality taken from the output of the processing, scaling, and merging software packages were analyzed. This highly dimensional “quality parameter dataset” was reduced using a principal component analysis. About 78% of the variability in the data set could be explained with the first four principal components. A score-plot in this four-dimensional space clearly showed two tendencies, one for the crystals grown in space and one for the ground crystals. The differences between the two groups are observed irrespective of the software package. They can be attributed to the first principal component and reflect the superior quality of the space crystals.

Nucleation of Protein Crystals under the Influence of Solution Shear Flow

Several recent theories and simulations have predicted that shear flow could enhance, or, conversely, suppress the nucleation of crystals from solution. Such modulations would offer a pathway for nucleation control and provide a novel explanation for numerous mysteries in nucleation research. For experimental tests of the effects of shear flow on protein crystal nucleation, we found that if a protein solution droplet of approximately 5 microL (2-3 mm diameter at base) is held on a hydrophobic substrate in an enclosed environment and in a quasi-uniform constant electric field of 2 to 6 kV cm(-1), a rotational flow with a maximum rate at the droplet top of approximately 10 microm s(-1) is induced. The shear rate varies from 10(-3) to 10(-1) s(-1). The likely mechanism of the rotational flow involves adsorption of the protein and amphiphylic buffer molecules on the air-water interface and their redistribution in the electric field, leading to nonuniform surface tension of the droplet and surface tension-driven flow. Observations of the number of nucleated crystals in 24- and 72-h experiments with the proteins ferritin, apoferritin, and lysozyme revealed that the crystals are typically nucleated at a certain radius of the droplet, that is, at a preferred shear rate. Variations of the rotational flow velocity resulted in suppression or enhancement of the total number of nucleated crystals of ferritin and apoferritin, while all solution flow rates were found to enhance lysozyme crystal nucleation. These observations show that shear flow may strongly affect nucleation, and that for some systems, an optimal flow velocity, leading to fastest nucleation, exists. Comparison with the predictions of theories and simulations suggest that the formation of ordered nuclei in a "normal" protein solution cannot be affected by such low shear rates. We conclude that the flow acts by helping or suppressing the formation of ordered nuclei within mesoscopic metastable dense liquid clusters. Such clusters were recently shown to exist in protein solutions and to constitute the first step in the nucleation mechanism of many protein and nonproteinsystems.

Adhesion of protein crystals: Measurement of the detachment force

The degree of adhesion of protein crystals, heterogeneously nucleated and grown on different supports (e.g. glass plates and plates coated with poly-L-lysine, hexamethyl-disilazane and silicon) is measured directly with a purposely-developed technique. The sticking force crystal/support is determined by means of a flexible glass fibre, which bending is calibrated by means of series of weights. In this way an elastic constant, specific for each glass fiber is determined individually. Appropriate glass fibres with relative bending less than 10% (Hook's law) are used. The force which is necessary to be exerted, by means of a micro-manipulator, in order to detach the crystal from the support is taken as a quantitative measure for the adhesion strength. Forces between 10 N cm-2 and 1 N cm-2 for differently oriented tetragonal hen-egg-white lysozyme and cubic ferritin crystals, and 0.1 N cm-2 for rhombohedral (porcine) insulin and orthorhombic trypsin crystals are measured. The tetragonal HEWL and rhombohedral insulin crystals show anisotropy of the adhesion strength. In contrast, the cubic ferritin crystals are isotropic also in this respect. For comparison purposes adhesion measurements are performed with NaCl and sugar crystals. An attempt is made to evaluate also the adhesion energy of the protein crystals. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Supramolecular Nanotube Hosts for Encapsulation of 10-nm-Scale Objects

AbstractSupramolecular nanotube hosts with precisely controlled inner or outer diameters have been synthesized by self-assembly of cardanol-based glycolipids, glucopyranosylamide lipids, or glucose-based unsymmetrical bolaamphiphiles. Time-resolved fluorescent measurement using 8-anilinonaphtahalene-1-sulfonate (ANS) as a probe revealed that the water confined in a cardanol-based glycolipid nanotube has relatively lower solvent polarity than bulk water, which corresponds to that of 1-propanol. Extensively developed hydrogen bond networks also characterize the confined water in comparison to the case in bulk water. Encapsulation ability of the glucopyranosylamide lipid nanotube, which depends on capillary action, has been examined by filling the lyophilized lipid nanotubes with aqueous dispersions of gold or silver nanocrystals, ferritin, or magnetic crystals. We have also succeeded in filling the unsymmetrical bolaamphiphile nanotube, which possesses positively charged inner surfaces, with negatively charged polymer beads or ferritin without depending on capillary action.

Radiation driven collapse of protein crystals

During coherent X-ray diffraction measurements on crystals of ferritin at room temperature using monochromatic undulator radiation from the Advanced Photon Source, a sudden lattice contraction was observed following a characteristic latent period and ultimately leading to the collapse of the crystal. The progression of this collapse is analysed using a two-state Hendricks-Teller model. It reveals that 55% of the layers collapse by 1.6% before the crystal completely stops diffracting.

Conceptions and first results on the electrocrystallization behaviour of ferritin

The role of electrochemical processes on Fe and CdSO(4) in the crystallization of horse spleen ferritin has been investigated using the cyclic voltammetry technique. It was found that although both species exhibit important redox properties in the presence of an external applied potential, CdSO(4) played a leading role not only in the nucleation process but also in the growth behaviour and morphology of ferritin crystals.

Hereditary hyperferritinaemia???cataract syndrome: a challenging diagnosis for the hepatogastroenterologist

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is a relatively rare disorder with an autosomal dominant trait. It can be caused by various mutations within the iron responsive element (IRE) of the L-ferritin gene. These mutations result in an increased translation of L-ferritin mRNA and consequently the accumulation of L-ferritin in different fluids and tissues. HHCS patients present with an isolated hyperferritinaemia in the absence of any sign of iron overload. Early onset bilateral cataract, probably due to accumulation of ferritin crystals in the lens, is the only presenting clinical manifestation. Internists, especially gastrohepatologists, should be aware of this syndrome and differentiate it from haemochromatosis which is much more frequent, in order to avoid unnecessary imaging procedures, liver biopsies and an eventual venesection therapy, which will only lead to microcytic anaemia. In the present paper we report the first cases with HHCS diagnosed in Belgium. At diagnosis, the seven known affected members of three different families had ferritin levels between 603 and 3432 microg/l (normal < 150 microg/l), and this in combination with normal iron and transferrin values. All of them were known with early-onset bilateral cataract and our postulated diagnosis of HHCS was confirmed after genetic sequencing of the L-ferritin gene, which showed a C39U point mutation in the first family, and an A40G point mutation in the IRE loop segment in the two other families. The other investigated members of the three families had normal ferritin values, no history of early-onset cataract and genetic analyses could not reveal a mutation in the IRE of their L-ferritin gene. In current clinical practice, gastroenterologists should remember that elevated ferritin levels in the absence of documented iron overload is not haemochromatosis.

The effects of temperature and radiation on holo and apo ferritin crystals

The effects of temperature and radiation on holo and apo ferritin crystals