Ferritin nanocages are promising platforms for drug encapsulation. However, extreme conditions (pH ≤ 2) required for dissociation limit their application. Here, we engineered protein interfaces to yield ferritin nanocages which disassemble at pH 4.0 and reassemble at pH 7.5. During this process, bioactive molecules can be encapsulated within the protein cavity.

Owing to diverse functionalities and metal binding abilities, proteins have been proven to be promising ligands in the synthesis of gold nanoclusters (Au NCs). In this work, we explored β-lactoglobulin (β-Lg), a protein byproduct generated during cheese processing, as a biotemplate for fabrication of Au NCs by a facile and green method for the first time. The as-prepared Au NCs are water soluble and highly fluorescent and exhibit high sensitivity and selectivity for Hg2+ detection in aqueous solution. Interestingly, we found that the fluorescence of these Au NCs is stable either in a variety of complex matrixes or over a broad pH range (5.0–13.0) and therefore can be explored as a cell and animal imaging agent. More importantly, we demonstrated that the β-lactoglobulin-stabilized Au NCs (β-Lg–Au NCs) could serve as a sensor for the detection and quantification of Hg2+ in beverages, urine, and serum with high sensitivity.

•The interaction of gallic acid and its derivatives with ferritin is structure-dependent.•These phenolic acids with three HO groups are prerequisite for the interaction.•Gallic acid, methyl gallate and propylgallate can bind to ferritin.•Such binding can protect ferritin from degradation by pepsin.Gallic acid and its derivatives co-exist with protein components in foodstuffs, but there is few report on their interaction with proteins. On the other hand, plant ferritin represents not only a novel class of iron supplement, but also a new nanocarrier for encapsulation of bioactive nutrients. However, plant ferritin is easy to be degraded by pepsin in the stomach, thereby limiting its application. Herein, we investigated the interaction of gallic acid and its derivatives with recombinant soybean seed H-2 ferritin (rH-2). We found that these phenolic acids interacted with rH-2 in a structure-dependent manner; namely, gallic acid (GA), methyl gallate (MEGA) and propyl gallate (PG) having three HO groups can bind to rH-2, while their analogues with two HO groups cannot. Consequently, such binding largely inhibited ferritin degradation by pepsin. These findings advance our understanding of the relationship between the structure and function of phenolic acids.

Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy—key subunit interface redesign (KSIR)—that alters a natural subunit–subunit interface by selective deletion of a small number of “silent” amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.Keywords: ferritin reassembly; geometry regulation; nanocage; subunit interface redesign; “silent” amino acid residues

The widespread occurrence of protein channels offers a good opportunity to fabricate protein architectures. Herein, we have developed a novel strategy for linear self-assembly of ferritin cages induced by poly(α, L-lysine) through channel-directed electrostatic interactions at pH 7.0. The length of the formed filaments can be controlled.

Reconstructed ferritin nanocages with expanded 4-fold channels can self-assemble into 2D square arrays through channel-directed electrostatic interactions with poly(α, L-lysine) at pH 7.0. Structurally, protein cages are aligned along their common 4-fold symmetry axis, imposing a fixed disposition of neighboring ferritins.

NH2-(α,L-lysine)5-COOH and SDS can self-assemble into nanodiscs or nanoribbons. We show that pH can regulate not only the diameter of nanodiscs but also the conversion between nanodiscs and nanoribbons. This system can be used as two different templates for fabricating platinum nanowires and nanodiscs.

•β-Carotene can be encapsulated within ferritin shell.•The β-carotene-containing ferritin nanocomposites become water-soluble.•Such encapsulation improves the thermal stability of β-carotene.Carotenoids may play a number of potential health benefits for human. However, their use in food industry is limited mostly because of their poor water-solubility and low thermal stability. Ferritins are widely distributed in nature with a shell-like structure which offers a great opportunity to improve the water-solubility and thermal stability of the carotenoids by encapsulation. In this work, recombinant human H-chain ferritin (rHuHF) was prepared and used to encapsulate β-carotene, a typical compound among carotenoids, by taking advantage of the reversible dissociation and reassembly characteristic of apoferritin in different pH environments. Results from high-performance liquid chromatography (HPLC), UV/Vis spectroscopy and transmission electron microscope (TEM) indicated that β-carotene molecules were successfully encapsulated within protein cages with a β-carotene/protein molar ratio of 12.4–1. Upon such encapsulation, these β-carotene-containing apoferritin nanocomposites were water-soluble. Interestingly, the thermal stability of the β-carotene encapsulated within apoferritin nanocages was markedly improved as compared to free β-carotene. These new properties might be favourable to the utilisation of β-carotene in food industry.

•Phenolic acids can bind to iron(III) in the presence of excess citrate.•The apparent thermodynamic and kinetic parameters are measured directly.•The condition-independent binding constants are calculated by displacement model.•Compared to GA and PCA, the iron(III) binding constant and rate constant of MEGA is the largest.Under physiological conditions, exogenous chelators such as polyphenols might interact with non-protein bound ferric complexes, such as Fe(III)–citrate. Additionally, Fe(III) and citrate are widely distributed in various fruits and vegetables which are also rich in phenolic acids. In this study, we focus on the interaction between phenolic acids (gallic acid, methyl gallate and protocatechuic acid) and Fe(III) in the presence of excessive citrate by isothermal titration calorimetry (ITC) for thermodynamic studies, and stopped-flow absorption spectrometry for fast kinetic studies. Results reveal that all of these three phenolic acids can bind to the Fe(III) with the same stoichiometry (3:1). Moreover, the binding constants of these three compounds with Fe(III) are greatly dependent on ligand structure, and are much higher than that of Fe(III)–citrate. Based on their stoichiometry and superhigh binding constants, it is most likely that these three phenolic acids can displace the citrate to bind with one iron(III) ion to form a stable octahedral geometric structure, albeit at different rates. These findings shed light on the interaction between phenolic acids and Fe(III) in the presence of citrate under either physiological conditions or in a food system.

From an evolutionary point of view, plant and animal ferritins arose from a common ancestor, but plant ferritin exhibits different features as compared with the animal analogue. One major difference is that the 4-fold channels naturally occurring in plant ferritin are hydrophilic, whereas the 4-fold channels in animal ferritin are hydrophobic. Prior to this study, however, the function of the 4-fold channels in oxidative deposition of iron in phytoferritin remained unknown. To elucidate the role of the 4-fold channels in iron oxidative deposition in ferritin, three mutants of recombinant soybean seed H-2 ferritin (rH-2) were prepared by site-directed mutagenesis, which contained H193A/H197A, a 4-fold channel mutant, E165I/E167A/E171A, a 3-fold channel mutant, and E165I/E167A/E171A/H193A/H197A, where both 3- and 4-channels were mutated. Stopped-flow, electrode oximetry, and transmission electron microscopy (TEM) results showed that H193A/H197A and E165I/E167A/E171A exhibited a similar catalyzing activity of iron oxidation with each other, but a pronounced low activity compared to rH-2, demonstrating that both the 4-fold and 3-fold hydrophilic channels are necessary for iron diffusion in ferritin, followed by oxidation. Indeed, among all tested ferritin, the catalyzing activity of E165I/E167A/E171A/H193A/H197A was weakest because its 3- and 4- fold channels were blocked. These findings advance our understanding of the function of 4-fold channels of plant ferritin and the relationship of the structure and function of ferritin.

Phytoferritin is a promising resource of non-heme iron supplementation, but it is not stable against degradation by proteases in the gastrointestinal tract. Therefore, how to improve the stability of ferritin in the presence of proteases is a challenge. Since (−)-epigallocatechin-3-gallate (EGCG) is rich in phenolic-hydroxyl groups, it could interact with ferritin through hydrogen bonds, thereby preventing protein from degradation. To confirm this idea, we focus on the interaction between EGCG and phytoferritin, and the consequence of such interaction. Results demonstrated that EGCG did interact with ferritin, and such interaction induced the change in the tertiary/quaternary structure of protein but not in its secondary structure. Furthermore, stopped-flow and dynamic light scattering (DLS) results showed that EGCG could trigger ferritin association. Consequently, such protein association markedly inhibited protein digestion by pepsin at pH 4.0 and by trypsin at pH 7.5. These findings raise the possibility to improve the stability of phytoferritin in the presence of proteases.

Iron deficiency is a major public health problem in the world. Phytoferritin as an alternative iron supplement has received increasing attentions recently. Plant ferritins are usually heteropolymers comprising two different H-type subunits, H-1 and H-2, while homopolymeric plant ferritin is rare. In the present study, a newly homopolymeric ferritin chickpea seed ferritin (CSF) was isolated and purified to homogeneity from chickpea seed (Cicer arietinum L.) by two consecutive anion exchange and size exclusion chromatography. SDS-PAGE result indicates that CSF only consists of 28.0 kDa (H-2) subunits, which was identified by Western blot analysis as well. N-terminal sequence and MALDITOF-MS analyses indicate that the subunit of CSF and H-2 subunit of heteropolymeric pea seed ferritin (PSF) share high identity in amino acid sequence. Subsequently, we demonstrated that homopolymeric CSF exhibits a higher catalyzing activity than heteropolymeric PSF at both low and high iron loadings. More importantly, the stability of homopolymeric CSF is much higher than all known plant ferritin due to its highest H-2 subunit content, which is favorable for its application as iron supplement.

•NADH molecules can bind on the outer surface of PSF.•NADH binding has a great effect on PSF senior structure.•NADH binding can induce iron release from holo PSF.•Such binding has no effect on iron uptake by PSF.Plant ferritin from legume seeds co-exists with coenzyme NADH (a reduced form of nicotinamide-adenine dinucleotide) in many foodstuffs. In the present study, the interaction of NADH with apo pea seed ferritin (PSF) was investigated by fluorescence resonance energy transfer (FRET), fluorescence titration, transmission electron microscope (TEM), and isothermal titration calorimetry (ITC). We found that NADH molecules bound on the outer surface of PSF close to the 4-fold channels, which was 1.58 nm from tryptophan residue (Trp). Consequently, such binding facilitates iron release from holo PSF, which might have a negative effect on PSF as an iron supplement, while NADH was oxidised into NAD+. However, the binding of NADH to the protein does not affect the entry of toxic ferrous ions into the apo protein shell, where these ferrous ions were oxidised into less toxic ferric ions. Moreover, NADH binding markedly affects the tertiary structure around Trp residues of PSF. These findings advanced our understanding of the interactions between different naturally occurring components in a complex food system.

The storage of large quantities of hydrogen at ambient temperature is a key factor in establishing a hydrogen-based economy. One strategy for hydrogen storage is to exploit the interaction between H2 and a solid material by physisorption of hydrogen on porous materials. However, physisorption materials containing MOF, porous carbons, zeolites, clathrates, and synthesized organic polymers physisorb only about 1 wt% of H2 at ambient temperature. One approach to solving this problem is to prepare new classes of physisorption materials which exhibits a mechanism different from the reported materials in hydrogen storage. Here we report the synthesis of apo cross-linked ferritin supramolecules by disulfide bonds, and their holo form. Unlike non-protein adsorbents, the hydrogen storage capacity of these protein materials increases as a function of temperature over the range of 20–40 °C. The holo supramolecules enable the adsorption of hydrogen up to 3.51 wt% at 40 °C and 40 bar H2. In contrast, non-protein physisorption materials such as activated carbon and nano Fe2O3 marginally adsorb hydrogen, and, as reported, their ability to adsorb hydrogen decreases with increasing temperature under the same experimental condition. These results demonstrate that protein materials have a unique hydrogen storage mechanism which offers new opportunities in exploration of physisorption materials at ambient temperature.Highlights► Novel apo/holo cross-linked ferritin supramolecules by disulfide bonds were prepared. ► The holo supramolecules enable the adsorption of hydrogen up to 3.51 wt% at 40 °C. ► Protein materials have a unique hydrogen storage mechanism at ambient temperature. ► Temperature has different effects on protein and other materials in hydrogen storage.

Phytoferritin from legume seeds is naturally compartmentalized in amyloplasts, where iron is takem up and released by ferritin during seed formation and germination. However, the effect of these two processes on starch granules remains unknown. No starch damage was visualized by SEM during iron uptake by apo soybean seed ferritin (SSF). In contrast, great damage was observed with the starch granules during iron release from holoSSF induced by ascorbic acid. Such a difference stems from different strategies to control HO• chemistry during these two processes. HO• is hardly formed during iron uptake by apoSSF, whereas a significant amount of HO• is generated during iron release due to the Fenton reaction. As a result, starch granules are kept intact during iron uptake, which might beneficial to the storage of the starch granules during seed formation. In contrast, these starch granules are dramatically hydrolyzed during the iron release process, which might favor seed germination.

•We reported the first case of using edible plant protein cages to encapsulate Ca2 + as calcium supplements.•These novel calcium supplements can be absorbed by Caco-2 cells through a unique pathway.•Such encapsulation could protect against the effect of dietary calcium absorption inhibitors.The consumption of milk is declining in industrialized countries, leading to inadequate calcium intake. Therefore, it is important to explore a new class of Ca-enriched nutrient for the fortification of food. In this work, we prepared a novel class of soluble and edible Ca–protein complexes where approximately 140 calcium ions were encapsulated within a phytoferritin nanocage. As an alternative to other organic and/or inorganic carriers, protein nanocages were found to provide a unique vehicle of biological origin for the intracellular delivery of calcium ions for supplementation. Such encapsulation can protect calcium ions within protein cages against dietary factors such as tannic acid (TA), oxalic acid (OA), and other divalent metal ions in foodstuffs. We demonstrated that the calcium-containing ferritin composites can be absorbed by Caco-2 cells through a process where a TfR1 receptor is involved, whereas the uptake of free calcium ions has been known to be associated with another receptor, DMT1, indicating that the calcium ions encapsulated in supramolecular protein cages can be internalized by the Caco-2 cells through a different pathway from its free analogs for calcium supplementation.Scheme of design and synthesis of edible calcium encapsulated phytoferritin complexes and its absorption by Caco-2 cells mediated by a ferritin receptor.Download high-res image (232KB)Download full-size image

Ferritin nanocages are promising platforms for drug encapsulation. However, extreme conditions (pH ≤ 2) required for dissociation limit their application. Here, we engineered protein interfaces to yield ferritin nanocages which disassemble at pH 4.0 and reassemble at pH 7.5. During this process, bioactive molecules can be encapsulated within the protein cavity.

The widespread occurrence of protein channels offers a good opportunity to fabricate protein architectures. Herein, we have developed a novel strategy for linear self-assembly of ferritin cages induced by poly(α, L-lysine) through channel-directed electrostatic interactions at pH 7.0. The length of the formed filaments can be controlled.

NH2-(α,L-lysine)5-COOH and SDS can self-assemble into nanodiscs or nanoribbons. We show that pH can regulate not only the diameter of nanodiscs but also the conversion between nanodiscs and nanoribbons. This system can be used as two different templates for fabricating platinum nanowires and nanodiscs.

Reconstructed ferritin nanocages with expanded 4-fold channels can self-assemble into 2D square arrays through channel-directed electrostatic interactions with poly(α, L-lysine) at pH 7.0. Structurally, protein cages are aligned along their common 4-fold symmetry axis, imposing a fixed disposition of neighboring ferritins.