Protein Nanocage as a pH-Switchable Pickering Emulsifier

Encapsulation of active compounds in Pickering emulsions using bioderived protein-based stabilizers holds potential for the development of novel formulations in the fields of foods and cosmetics. We employ a dodecahedron hollow protein nanocage as a pH-switchable Pickering emulsifier. E2 protein nanocages are derived from pyruvate dehydrogenase multienzyme complex from <i>Geobacillus stearothermophilus</i> which adsorb at the oil/water interface at neutral and basic pH’s and stabilize the Pickering emulsions, while in the acidic range, at pH ∼4, the emulsion separates into emulsion and serum phases due to flocculation. The observed process is reversible for at least five cycles. Optimal formulation of a Pickering emulsion composed of rosemary oil, an essential oil, and water has been achieved by ultrasonication and results in droplets of approximately 300 nm in diameter with an oil/water ratio of 0.11 (v/v) and 0.30–0.35% (wt %). Ionic stabilization is observed for concentrations up to 250 mM NaCl and pH values from 7 to 11. The emulsions are stable for at least 10 days when stored at different temperatures up to 50 °C. The resulting Pickering emulsions of different compositions also form a gel-like structure and show shear thinning behavior under shear stress at a higher oil/water ratio

Isolating a Trimer Intermediate in the Self-Assembly of E2 Protein Cage

Understanding the self-assembly mechanism of caged proteins provides clues to develop their potential applications in nanotechnology, such as a nanoscale drug delivery system. The E2 protein from Bacillus stearothermophilus, with a virus-like caged structure, has drawn much attention for its potential application as a nanocapsule. To investigate its self-assembly process from subunits to a spherical protein cage, we truncate the C-terminus of the E2 subunit. The redesigned protein subunit shows dynamic transition between monomer and trimer, but not the integrate 60-mer. The results indicate the role of the trimer as the intermediate and building block during the self-assembly of the E2 protein cage. In combination with the molecular dynamics simulations results, we conclude that the C-terminus modulates the self-assembly of the E2 protein cage from trimer to 60-mer. This investigation elucidates the role of the intersubunit interactions in engineering other functionalities in other caged structure proteins

The E2 porous protein cage.

<p>(A) and (B) E2 protein cage three-dimensional structure (adapted from PDB ID: 1b5s).[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162848#pone.0162848.ref042" target="_blank">42</a>] The inserts show typical electron microscopy images of the 5-fold axis (A) and 2-fold axis (B) orientations of the protein cage E2. The diameter <i>D</i> of the E2 protein cage is 25 nm. The diameter <i>d</i> of each pore is 6 nm. (C) E2 protein cage inner surface presenting RDGE loop sequences in blue. The diameter δ of the cage core is 13 nm.</p

Electron microscopy of wild-type and engineered E2 protein cages.

<p>Images of the E2 protein cages: (A, E) E2-WT; (B, F) E2-LH2; (C, G) E2-LH5; (D, H) E2-LH6. (A–D) scale bars are 100 nm. (E–H) scale bars are 50 nm.</p

Dynamic light scattering analysis of wild-type and engineered E2 protein cages with different oligohistidine sequences at the RDGE loop.

<p>Dynamic light scattering analysis of wild-type and engineered E2 protein cages with different oligohistidine sequences at the RDGE loop.</p

Oligonucleotides for plasmid construction.

<p>Oligonucleotides for plasmid construction.</p

Method for specific internalisation of peptide coated gold nanoparticles into engineered E2 protein cages.

<p>(a) Site-directed mutagenesis of E2 protein cage’s core with oligohistidine sequences (blue) at the RDGE loop (green) on E2 protein subunits; (b) Surface coating of 3.9 nm gold nanoparticles with a self-assembled monolayer made of peptidols and thiolated alkane ethylene glycol (EG) ligands, functionalised with Ni²⁺ nitrilotriacetic moieties (NTA, Ni²⁺); (c) Specific internalisation by affinity binding of Ni-NTA-functionalised peptide coated gold nanoparticles into E2 protein cages presenting oligohistidine sequences.</p

Ni-NTA-functionalised peptide coated gold nanoparticles.

<p>(A) Size distribution of 212 gold nanoparticles with an average diameter of 3.9 ± 0.8 nm. The insert shows a typical electron microscopy image used for size measurement. Immobilisation of Ni-NTA-functionalised peptide coated gold nanoparticles on a hexa-histidine loaded resin. (B) NTA-functionalised SAM coated gold nanoparticles not loaded with Ni²⁺ present no non-specific binding to hexa-histidine resin. (C) 10% (mol:mol) Ni-NTA-functionalised peptide coated gold nanoparticles fully bind to hexa-histidine resin as no free gold nanoparticles are found in the clear supernatant.</p

Transmission electron microscopy of samples of Ni-NTA-functionalised gold nanoparticles and E2 protein cages.

<p>Ni-NTA-functionalised gold nanoparticles mixed with (A) E2-WT, (B) E2-LH2, (C) E2-LH5, (D) E2-LH6. White circles indicate Ni-NTA-functionalised gold nanoparticles internalised into oligohistidine modified E2 protein cages. The samples were stained with 1% (w/v) phosphotungstic acid. Scale bars are 100 nm.</p

Temperature-Dependent Coherent Tunneling across Graphene–Ferritin Biomolecular Junctions

Understanding the mechanisms of charge transport (CT) across biomolecules in solid-state devices is imperative to realize biomolecular electronic devices in a predictive manner. Although it is well-accepted that biomolecule–electrode interactions play an essential role, it is often overlooked. This paper reveals the prominent role of graphene interfaces with Fe-storing proteins in the net CT across their tunnel junctions. Here, ferritin (AfFtn-AA) is adsorbed on the graphene by noncovalent amine–graphene interactions confirmed with Raman spectroscopy. In contrast to junctions with metal electrodes, graphene has a vanishing density of states toward its intrinsic Fermi level (“Dirac point”), which increases away from the Fermi level. Therefore, the amount of charge carriers is highly sensitive to temperature and electrostatic charging (induced doping), as deduced from a detailed analysis of CT as a function of temperature and iron loading. Remarkably, the temperature dependence can be fully explained within the coherent tunneling regime due to excitation of hot carriers. Graphene is not only demonstrated as an alternative platform to study CT across biomolecular tunnel junctions, but it also opens rich possibilities in employing interface electrostatics in tuning CT behavior