Simple assay for adsorption of proteins to the air-water interface.

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air-water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.

Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development.

The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 Šresolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164-181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows.

Designed and biologically active protein lattices.

Versatile methods to organize proteins in space are required to enable complex biomaterials, engineered biomolecular scaffolds, cell-free biology, and hybrid nanoscale systems. Here, we demonstrate how the tailored encapsulation of proteins in DNA-based voxels can be combined with programmable assembly that directs these voxels into biologically functional protein arrays with prescribed and ordered two-dimensional (2D) and three-dimensional (3D) organizations. We apply the presented concept to ferritin, an iron storage protein, and its iron-free analog, apoferritin, in order to form single-layers, double-layers, as well as several types of 3D protein lattices. Our study demonstrates that internal voxel design and inter-voxel encoding can be effectively employed to create protein lattices with designed organization, as confirmed by in situ X-ray scattering and cryo-electron microscopy 3D imaging. The assembled protein arrays maintain structural stability and biological activity in environments relevant for protein functionality. The framework design of the arrays then allows small molecules to access the ferritins and their iron cores and convert them into apoferritin arrays through the release of iron ions. The presented study introduces a platform approach for creating bio-active protein-containing ordered nanomaterials with desired 2D and 3D organizations.

Design of metal-mediated protein assemblies via hydroxamic acid functionalities.

The self-assembly of proteins into sophisticated multicomponent assemblies is a hallmark of all living systems and has spawned extensive efforts in the construction of novel synthetic protein architectures with emergent functional properties. Protein assemblies in nature are formed via selective association of multiple protein surfaces through intricate noncovalent protein-protein interactions, a challenging task to accurately replicate in the de novo design of multiprotein systems. In this protocol, we describe the application of metal-coordinating hydroxamate (HA) motifs to direct the metal-mediated assembly of polyhedral protein architectures and 3D crystalline protein-metal-organic frameworks (protein-MOFs). This strategy has been implemented using an asymmetric cytochrome cb562 monomer through selective, concurrent association of Fe3+ and Zn2+ ions to form polyhedral cages. Furthermore, the use of ditopic HA linkers as bridging ligands with metal-binding protein nodes has allowed the construction of crystalline 3D protein-MOF lattices. The protocol is divided into two major sections: (1) the development of a Cys-reactive HA molecule for protein derivatization and self-assembly of protein-HA conjugates into polyhedral cages and (2) the synthesis of ditopic HA bridging ligands for the construction of ferritin-based protein-MOFs using symmetric metal-binding protein nodes. Protein cages are analyzed using analytical ultracentrifugation, transmission electron microscopy and single-crystal X-ray diffraction techniques. HA-mediated protein-MOFs are formed in sitting-drop vapor diffusion crystallization trays and are probed via single-crystal X-ray diffraction and multi-crystal small-angle X-ray scattering measurements. Ligand synthesis, construction of HA-mediated assemblies, and post-assembly analysis as described in this protocol can be performed by a graduate-level researcher within 6 weeks.

Iron stored in ferritin is chemically reduced in the presence of aggregating Aß(1-42).

Atypical low-oxidation-state iron phases in Alzheimer's disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide ß-amyloid (Aß) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aß and ferritin resulted in the conversion of ferritin's inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aß in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.

A mechanism of ferritin crystallization revealed by cryo-STEM tomography.

Protein crystallization is important in structural biology, disease research and pharmaceuticals. It has recently been recognized that nonclassical crystallization-involving initial formation of an amorphous precursor phase-occurs often in protein, organic and inorganic crystallization processes1-5. A two-step nucleation theory has thus been proposed, in which initial low-density, solvated amorphous aggregates subsequently densify, leading to nucleation4,6,7. This view differs from classical nucleation theory, which implies that crystalline nuclei forming in solution have the same density and structure as does the final crystalline state1. A protein crystallization mechanism involving this classical pathway has recently been observed directly8. However, a molecular mechanism of nonclassical protein crystallization9-15 has not been established9,11,14. To determine the nature of the amorphous precursors and whether crystallization takes place within them (and if so, how order develops at the molecular level), three-dimensional (3D) molecular-level imaging of a crystallization process is required. Here we report cryogenic scanning transmission microscopy tomography of ferritin aggregates at various stages of crystallization, followed by 3D reconstruction using simultaneous iterative reconstruction techniques to provide a 3D picture of crystallization with molecular resolution. As crystalline order gradually increased in the studied aggregates, they exhibited an increase in both order and density from their surface towards their interior. We observed no highly ordered small structures typical of a classical nucleation process, and occasionally we observed several ordered domains emerging within one amorphous aggregate, a phenomenon not predicted by either classical or two-step nucleation theories. Our molecular-level analysis hints at desolvation as the driver of the continuous order-evolution mechanism, a view that goes beyond current nucleation models, yet is consistent with a broad spectrum of protein crystallization mechanisms.

Crystallographic characterization of ferritin from Sinonovacula constricta.

Ferritins are ubiquitous iron-binding proteins that are mainly related to iron storage, detoxification and innate immunity. Here, we present the crystal structure of a marine invertebrate ferritin from Sinonovacula constricta at a resolution of 1.98 Å. The S. constricta ferritin (ScFer) possessed some structural similarities with vertebrate ferritins, and they shared a well-conserved architecture composed of five α-helical bundles that assembled into a cage-like structure with 24-subunits. The structure of ScFer also showed iron binding sites in the 3-fold channel, ferroxidase center, and putative nucleation sites. Further, electrostatic potential calculations suggested that the electrostatic gradient of the 3-fold channel could provide a guidance mechanism for iron entering the ferritin cavity.

Sub-cellular In-situ Characterization of Ferritin(iron) in a Rodent Model of Spinal Cord Injury.

Iron (Fe) is an essential metal involved in a wide spectrum of physiological functions. Sub-cellular characterization of the size, composition, and distribution of ferritin(iron) can provide valuable information on iron storage and transport in health and disease. In this study we employ magnetic force microscopy (MFM), transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS) to characterize differences in ferritin(iron) distribution and composition across injured and non-injured tissues by employing a rodent model of spinal cord injury (SCI). Our biophysical and ultrastructural analyses provide novel insights into iron distribution which are not obtained by routine biochemical stains. In particular, ferritin(iron) rich lysosomes revealed increased heterogeneity in MFM signal from tissues of SCI animals. Ultrastructural analysis using TEM elucidated that both cytosolic and lysosomal ferritin(iron) density was increased in the injured (spinal cord) and non-injured (spleen) tissues of SCI as compared to naïve animals. In-situ EELs analysis revealed that ferritin(iron) was primarily in Fe3+ oxidation state in both naïve and SCI animal tissues. The insights provided by this study and the approaches utilized here can be applied broadly to other systemic problems involving iron regulation or to understand the fate of exogenously delivered iron-oxide nanoparticles.

Programmed Self-Assembly of a Biochemical and Magnetic Scaffold to Trigger and Manipulate Microtubule Structures.

Artificial bio-based scaffolds offer broad applications in bioinspired chemistry, nanomedicine, and material science. One current challenge is to understand how the programmed self-assembly of biomolecules at the nanometre level can dictate the emergence of new functional properties at the mesoscopic scale. Here we report a general approach to design genetically encoded protein-based scaffolds with modular biochemical and magnetic functions. By combining chemically induced dimerization strategies and biomineralisation, we engineered ferritin nanocages to nucleate and manipulate microtubule structures upon magnetic actuation. Triggering the self-assembly of engineered ferritins into micrometric scaffolds mimics the function of centrosomes, the microtubule organizing centres of cells, and provides unique magnetic and self-organizing properties. We anticipate that our approach could be transposed to control various biological processes and extend to broader applications in biotechnology or material chemistry.

Simvastatin Promotes Hematoma Absorption and Reduces Hydrocephalus Following Intraventricular Hemorrhage in Part by Upregulating CD36.

We previously found that hematoma worsens hydrocephalus after intraventricular hemorrhage (IVH) via increasing iron deposition and aggravating ependymal cilia injury; therefore, promoting hematoma absorption may be a promising strategy for IVH. Recently, some investigations imply that simvastatin has the ability of accelerating hematoma absorption. Thus, this study was designed to examine the efficacy of simvastatin for IVH in rats. Intracerebral hemorrhage with ventricular extension was induced in adult male Sprague-Dawley rats after autologous blood injection. Simvastatin or vehicle was administered orally at 1 day after IVH and then daily for 1 week. MRI studies were performed to measure the volumes of intracranial hematoma and lateral ventricle at days 1, 3, 7, 14, and 28 after IVH. Motor and neurocognitive functions were assessed at days 1 to 7 and 23 to 28, respectively. Iron deposition, iron-related protein expression, ependymal damage, and histology were detected at day 28. Expression of CD36 scavenger receptor (facilitating phagocytosis) was examined at day 3 after IVH using western blotting and immunofluorescence. Simvastatin significantly increased hematoma absorption ratio, reduced ventricular volume, and attenuated neurological dysfunction post-IVH. In addition, less iron accumulation and more cilia survival was observed in the simvastatin group when compared with the control. What's more, higher expression of CD36 was detected around the hematoma after simvastatin administration. Simvastatin significantly enhanced brain hematoma absorption, alleviated hydrocephalus, and improved neurological recovery after experimental IVH, which may in part by upregulating CD36 expression. Our data suggest that early simvastatin use may be a novel therapy for IVH patients.

Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.

Towards instantaneous cellular level bio diagnosis: laser extraction and imaging of biological entities with conserved integrity and activity.

The prospect for spatial imaging with mass spectroscopy at the level of the cell requires new means of cell extraction to conserve molecular structure. To this aim, we demonstrate a new laser extraction process capable of extracting intact biological entities with conserved biological function. The method is based on the recently developed picosecond infrared laser (PIRL), designed specifically to provide matrix-free extraction by selectively exciting the water vibrational modes under the condition of ultrafast desorption by impulsive vibrational excitation (DIVE). The basic concept is to extract the constituent protein structures on the fastest impulsive limit for ablation to avoid excessive thermal heating of the proteins and to use strongly resonant 1-photon conditions to avoid multiphoton ionization and degradation of the sample integrity. With various microscope imaging and biochemical analysis methods, nanoscale single protein molecules, viruses, and cells in the ablation plume are found to be morphologically and functionally identical with their corresponding controls. This method provides a new means to resolve chemical activity within cells and is amenable to subcellular imaging with near-field approaches. The most important finding is the conserved nature of the extracted biological material within the laser ablation plume, which is fully consistent with in vivo structures and characteristics.

Distinguishing ferritin from apoferritin using magnetic force microscopy.

Estimating the amount of iron-replete ferritin versus iron-deficient apoferritin proteins is important in biomedical and nanotechnology applications. This work introduces a simple and novel approach to quantify ferritin by using magnetic force microscopy (MFM). We demonstrate how high magnetic moment probes enhance the magnitude of MFM signal, thus enabling accurate quantitative estimation of ferritin content in ferritin/apoferritin mixtures in vitro. We envisage MFM could be adapted to accurately determine ferritin content in protein mixtures or in small aliquots of clinical samples.

Iron-rich ferritin in the hypoxia-tolerant rodent Spalax ehrenbergi: a naturally-occurring biomarker confirms the internalization and pathways of intracellular macromolecules.

The discovery of pits/caveolae in the plasmalemma advanced the study of macromolecule internalization. "Transcytosis" describes the transport of macromolecular cargo from one front of a polarized cell to the other within membrane-bounded carrier(s), via endocytosis, intracellular trafficking and exocytosis. Clathrin-mediated transcytosis is used extensively by epithelial cells, while caveolae-mediated transcytosis mostly occurs in endothelial cells. The internalization pathways were monitored by various markers, including radioisotopes, nanoparticles, enzymes, immunostains, and fluorophores. We describe an internalization pathway identified using a naturally-occurring biomarker, in vivo assembled ferritin, containing electron-dense iron cores. Iron, an essential trace metal for most living species and iron homeostasis, is crucial for cellular life. Ferritin is a ubiquitous and highly conserved archeoprotein whose main function is to store a reserve iron supply inside the cytoplasm in a non-toxic form. Ferritin is present in all organisms which have a metabolic requirement for iron and in even in organisms whose taxonomic rank is very low. The newborns of the blind mole, Spalax ehrenbergi, are born and live in a hypoxic environment and have significant iron overload in their liver and heart, but their iron metabolism has not been previously studied. These newborns, which are evolutionarily adapted to fluctuations in the environmental oxygen, have a unique ability to sequester transplacental iron and store it in ferritin without any signs of iron toxicity. Using the ferrihydrite cores of ferritin, we were able to monitor the ferritin internalization from portals of its entry into the cytosol of hepatocytes and cardiomyocytes and into the lysosomes.

Electrostatic assembly of binary nanoparticle superlattices using protein cages.

Binary nanoparticle superlattices are periodic nanostructures with lattice constants much shorter than the wavelength of light and could be used to prepare multifunctional metamaterials. Such superlattices are typically made from synthetic nanoparticles, and although biohybrid structures have been developed, incorporating biological building blocks into binary nanoparticle superlattices remains challenging. Protein-based nanocages provide a complex yet monodisperse and geometrically well-defined hollow cage that can be used to encapsulate different materials. Such protein cages have been used to program the self-assembly of encapsulated materials to form free-standing crystals and superlattices at interfaces or in solution. Here, we show that electrostatically patchy protein cages--cowpea chlorotic mottle virus and ferritin cages--can be used to direct the self-assembly of three-dimensional binary superlattices. The negatively charged cages can encapsulate RNA or superparamagnetic iron oxide nanoparticles, and the superlattices are formed through tunable electrostatic interactions with positively charged gold nanoparticles. Gold nanoparticles and viruses form an AB(8)(fcc) crystal structure that is not isostructural with any known atomic or molecular crystal structure and has previously been observed only with large colloidal polymer particles. Gold nanoparticles and empty or nanoparticle-loaded ferritin cages form an interpenetrating simple cubic AB structure (isostructural with CsCl). We also show that these magnetic assemblies provide contrast enhancement in magnetic resonance imaging.

Visualizing macromolecular complexes with in situ liquid scanning transmission electron microscopy.

A central focus of biological research is understanding the structure/function relationship of macromolecular protein complexes. Yet conventional transmission electron microscopy techniques are limited to static observations. Here we present the first direct images of purified macromolecular protein complexes using in situ liquid scanning transmission electron microscopy. Our results establish the capability of this technique for visualizing the interface between biology and nanotechnology with high fidelity while also probing the interactions of biomolecules within solution. This method represents an important advancement towards allowing future high-resolution observations of biological processes and conformational dynamics in real-time.

Mass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata.

Ferritins with electrophoretic homogeneity were prepared from the visceral mass of Saccostrea cucullata in batch. The native PAGE approach showed similar electrophoretic mobility among pig pancreatic ferritin, liver ferritin of Dasyatis akajei, and visceral mass ferritin of Saccostrea cucullata. SDS-PAGE indicated that the Saccostrea cucullata visceral ferritin (SCVF) consisted of a single subunit type and had a molecular weight (MW) of approximately 20 kDa, suggesting that the protein shell in SCVF was composed of a single subunit. In addition, peptide mass fingerprinting and transmission electron microscopy were used to identify SCVF further, and to observe its molecular structure. We found that the molecular structure in SCVF was similar to that of most mammalian ferritins, which are composed of a protein shell and an iron core. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry under the assistance of an acidic matrix, sinapic acid, also showed that SCVF was composed of a single subunit type and its subunit MW was calculated to be 19871.042 Da in the absence of heme. Kinetics analysis revealed that the complete process of iron release fitted the law of a first-order reaction, which is similar to that of most ferritins in mammals. Similar to bacterial ferritin, studies indicated that the shell consisted of a single subunit type and showed similar kinetics of iron release, suggesting that this subunit plays two important roles in iron release and storage, and that it shows different stability and intensity of interaction in carrying out its physiological functions in SCVF.

Damage-free top-down processes for fabricating two-dimensional arrays of 7 nm GaAs nanodiscs using bio-templates and neutral beam etching.

The first damage-free top-down fabrication processes for a two-dimensional array of 7 nm GaAs nanodiscs was developed by using ferritin (a protein which includes a 7 nm diameter iron core) bio-templates and neutral beam etching. The photoluminescence of GaAs etched with a neutral beam clearly revealed that the processes could accomplish defect-free etching for GaAs. In the bio-template process, to remove the ferritin protein shell without thermal damage to the GaAs, we firstly developed an oxygen-radical treatment method with a low temperature of 280 °C. Then, the neutral beam etched the defect-free nanodisc structure of the GaAs using the iron core as an etching mask. As a result, a two-dimensional array of GaAs quantum dots with a diameter of ∼ 7 nm, a height of ∼ 10 nm, a high taper angle of 88° and a quantum dot density of more than 7 × 10(11) cm(-2) was successfully fabricated without causing any damage to the GaAs.

Stabilization of a protein nanocage through the plugging of a protein-protein interfacial water pocket.

The unique structural properties of the ferritin protein cages have provided impetus to focus on the methodical study of these self-assembling nanosystems. Among these proteins, Escherichia coli bacterioferritin (EcBfr), although architecturally very similar to other members of the family, shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states. Through computational analysis and comparison to its homologues, we have found that this protein has a smaller than average dimeric interface on its 2-fold symmetry axis mainly because of the existence of an interfacial water pocket centered around two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, we have used a semiempirical computational method to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to that of wild-type EcBfr. This computational study also converged on the water-bridged asparagines. Replacing these two asparagines with hydrophobic amino acids resulted in proteins that folded into α-helical monomers and assembled into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirmed that, in all cases, these proteins, in agreement with the calculations, possessed increased stability. One of the three mutations shifts the population in favor of the higher-order oligomerization state in solution as evidenced by both size exclusion chromatography and native gel electrophoresis. These results taken together suggest that our low-level design was successful and that it may be possible to apply the strategy of targeting water pockets at protein--protein interfaces to other protein cage and self-assembling systems. More generally, this study further demonstrates the power of jointly employing in silico and in vitro techniques to understand and enhance biostructural energetics.

A ferritin-based label for cellular electron cryotomography.

Electron cryotomography provides nanometer resolution structural detail of thin biological specimens in a near-native state. Currently, its application is limited by the lack of a specific label for the identification of molecules. Our aim was to develop such a label, analogous to GFP used in fluorescence microscopy. Here, we demonstrate the use of Escherichia coli ferritin FtnA protein as a clonable label for electron cryotomography. Overproduced ferritin is visible in E. coli cells using cryotomography and fusing this label to a short membrane targeting sequence correctly directs the ferritin fusion to the membrane. Using two proteins with known subcellular localization patterns with this ferritin tag, also including GFP, we obtained essentially the same labeling patterns using electron cryotomography as compared with fluorescence light microscopy. Hence, the ferritin label localizes efficiently and faithfully and it will be a valuable tool for the unambiguous identification of molecules in cellular electron cryotomograms.