Protein nanocage: A versatile molecular carrier

Protein nanocages can be engineered to tailor their functions as carriers for health (e.g. therapeutic and diagnostic agents), molecular electronic, and consumer care (e.g. cosmetics and food) applications. They are formed by the self-assembly of multiple subunits forming hollow cage-like structures of nanometer size. Due to their proteinaceous nature, the protein nanocages allow facile modifications on its internal and external surfaces, as well as the subunit interfaces designed for the intended applications. Protein nanocages loaded with metal have been shown to be promising MRI contrast agent or when loaded with drug, they can serve as drug carrier. Modifying the interface of the subunits render the nanocages sensitive to environmental changes, such as pH. Engineering of the external surface allows for the display of targeting ligands for selective accumulation on cancer cells as well as epitopes for modulating the immune system. Leveraging on its natural or engineered metal-chelating activities, protein nanocages serve a dual function as a reaction container and as facilitator in the deposition of monodispersed platinum nanoparticles on graphene surfaces for electrocatalysis in fuel cells. Long-range electron tunneling across metal-loaded protein nanocages has also been shown to be promising in the development of memristive devices and future molecular electronics. In the most recent works, we show that the protein nanocages are surface active with an ability to stabilize Pickering emulsion with pH-responsive behavior. Titrating the protein ratio allows for formation of gel-like structures. In summary, protein nanocages are versatile protein-based materials whose properties are tunable for various applications. Please click Additional Files below to see the full abstract

Protein nanocages as novel biosurfactant in the formulation of Pickering emulsion and gel

Protein nanocages have been shown to be versatile for multitude of applications in biomedicine [1]. Our group has recently reported that the self-assembling protein nanocages localize at oil-water interface and stabilize 200-400 nm nanoemulsions [2]. The protein nanocages are produced using microbial fermentation and purified using conventional chromatography technique. The protein nanocage-stabilized Pickering emulsion are produced by facile sonication technique. The emulsion has been shown to be pH responsive when the pH is switched between 4 and 8. The switch is reversible up to 6 times. The emulsion is stable for more than 2 years. Varying the mass fraction of the protein nanocages/oil results in a shift in rheology from emulsion to gel. The unique properties of the protein nanocages emulsions have attracted industrial interests and we are currently working with our industry collaborators to encapsulate their cosmetic ingredients. We have shown the potential of protein nanocages as a novel biosurfactant that are of interests to the cosmetic industry. Please click Additional Files below to see the full abstract

The unique self-assembly/disassembly property of Archaeoglobus fulgidus ferritin and its implications on molecular release from the protein cage

Background: In conventional in vitro encapsulation of molecular cargo, the multi-subunit ferritin protein cages are disassembled in extremely acidic pH and re-assembled in the presence of highly concentrated cargo materials, which results in poor yields due to the low-pH treatment. In contrast, Archaeoglobus fulgidus open-pore ferritin (AfFtn) and its closed-pore mutant (AfFtn-AA) are present as dimeric species in neutral buffers that self-assemble into cage-like structure upon addition of metal ions. Methods: To understand the iron-mediated self-assembly and ascorbate-mediated disassembly properties, we studied the iron binding and release profile of the AfFtn and AfFtn-AA, and the corresponding oligomerization of their subunits. Results: Fe^(2+) binding and conversion to Fe^(3+) triggered the self-assembly of cage-like structures from dimeric species of AfFtn and AfFtn-AA subunits, while disassembly was induced by dissolving the iron core with reducing agents. The closed-pore AfFtn-AA has identical iron binding kinetics but lower iron release rates when compared to AfFtn. While the iron binding rate is proportional to Fe^(2+) concentration, the iron release rate can be controlled by varying ascorbate concentrations. Conclusion: The AfFtn and AfFtn-AA cages formed by iron mineralization could be disassembled by dissolving the iron core. The open-pores of AfFtn contribute to enhanced reductive iron release while the small channels located at the 3-fold symmetry axis (3-fold channels) are used for iron uptake

Protein nanocages for cutaneous delivery

The skin protects the body from UV-induced DNA damage by the sun exposure through the pigment, melanin produced by the melanocytes. This pigment is sometimes over-expressed leading to pigmentation disorders such as melasma. Current treatment involves using tyrosinase inhibitors and lasers, leads to complications such as depigmentation, irritation, and dermatitis, with only 50% patient response. This is mainly due the inability of the delivery system to penetrate the stratum corneum layer of the skin and its non-specificity to the melanocytes. This project is aimed at engineering E2 protein nanocage for enhanced penetration into the stratum corneum layer of the epidermis and targeting/penetrating the melanocytes for the delivery of therapeutics. Genetic fusion of SPACE (Skin Penetrating And Cell Entering) peptide to the E2 nanocage helps its transduction through the stratum corneum layer, in vivo and to the interior of the melanocytes in vitro. Further modification of the E2 protein cage with targeting ligands can facilitate its uptake in melanocytes through the corresponding cell membrane receptors. Multiple modifications could also be imparted to the E2 protein cages without affecting its self-assembly, thereby aiding both penetration and targeting functions for drug delivery. Successful delivery of the engineered protein cages can aid the formulation of novel protein-based drug releasing molecules to be applied to the skin, which can be biocompatible with efficient pharmacokinetics

Structural integrity of protein nanocage at liquid-liquid Interface

Globular proteins adsorb at the interface of two immiscible liquids by maintaining thermodynamically favorable state which often results in a denatured structure and compromised functionalities. However, the behavior of highly structural proteins at the interface of two immiscible liquids is still unexplored. In this study, we focused on the structural behavior of supramolecular protein at the interface. Our previous studies show that highly structural protein adsorbs at the interface and act as a Pickering emulsifier. Theoretical analyses by Molecular Dynamic Simulation proved that the supramolecular protein E2, a highly structured protein nanocage, has retained structural integrity at the liquid-liquid interface. Further, experimental analyses by Small angle X-ray scattering (SAXS) and quartz crystal microbalance and dissipation (QCM-D) confirm the adsorption of E2 on the liquid-liquid interface with zero penetration depth. Moreover, molecular structural analyses using Circular Dichroism (CD) and tryptophan fluorescence for secondary and tertiary structures respectively, also suggest the structural integrity of the cage structure of E2 at the oil-water interface. This study brings new insights into the behavior of highly symmetrical supramolecular protein assembly at the liquid-liquid interface

Iron-based ferritin nanocore as a contrast agent

Self-assembling protein cages have been exploited as templates for nanoparticle synthesis. The ferritin molecule, a protein cage present in most living systems, stores excess soluble ferrous iron in the form of an insoluble ferric complex within its cavity. Magnetic nanocores formed by loading excess iron within an engineered ferritin from Archaeoglobus fulgidus (AfFtn-AA) were studied as a potential magnetic resonance (MR) imaging contrast agent. The self-assembly characteristics of the AfFtn-AA were investigated using dynamic light scattering technique and size exclusion chromatography. Homogeneous size distribution of the assembled nanoparticles was observed using transmission electron microscopy. The magnetic properties of iron-loaded AfFtn-AA were studied using vibrating sample magnetometry. Images obtained from a 3.0 T whole-body MRI scanner showed significant brightening of T1 images and signal loss of T2 images with increased concentrations of iron-loaded AfFtn-AA. The analysis of the MR image intensities showed extremely high R2 values (5300 mM^(−1) s^(−1)) for the iron-loaded AfFtn-AA confirming its potential as a T2 contrast agent

Next-generation plastic degrading enzymes

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Next-generation plastic degrading enzymes

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Electron Tunneling in Ferritin and Associated Biosystems

Ferritin is a 12 nanometer (nm) diameter iron storage protein complex that is found in most plants and animals. A substantial body of evidence has established that electrons can tunnel through and between ferritin protein nanoparticles and that it exhibits Coulomb blockade behavior, which is also seen in quantum dots and nanoparticles. This evidence can be used to understand the behavior of these particles for use in nanoelectronic devices, for biomedical applications and for investigation of quantum biological phenomena. Ferritin also has magnetic properties that make it useful for applications such as memristors and as a contrast agent for magnetic resonance imaging. This article provides a short overview of this evidence, as well as evidence of ferritin structures in vivo and of tunneling in those structures, with an emphasis on ferritin structures in substantia nigra pars compacta (SNc) neurons. Potential biomedical applications that could utilize these ferritin protein nanoparticles are also discussed.</p

The Role of Nonconserved Residues of Archaeoglobus fulgidus Ferritin on Its Unique Structure and Biophysical Properties

Archaeoglobus fulgidus ferritin (AfFtn) is the only tetracosameric ferritin known to form a tetrahedral cage, a structure that remains unique in structural biology. As a result of the tetrahedral (2-3) symmetry, four openings (∼45 Å in diameter) are formed in the cage. This open tetrahedral assembly contradicts the paradigm of a typical ferritin cage: a closed assembly having octahedral (4-3-2) symmetry. To investigate the molecular mechanism affecting this atypical assembly, amino acid residues Lys-150 and Arg-151 were replaced by alanine. The data presented here shed light on the role that these residues play in shaping the unique structural features and biophysical properties of the AfFtn. The x-ray crystal structure of the K150A/R151A mutant, solved at 2.1 Å resolution, indicates that replacement of these key residues flips a “symmetry switch.” The engineered molecule no longer assembles with tetrahedral symmetry but forms a typical closed octahedral ferritin cage. Small angle x-ray scattering reveals that the overall shape and size of AfFtn and AfFtn-AA in solution are consistent with those observed in their respective crystal structures. Iron binding and release kinetics of the AfFtn and AfFtn-AA were investigated to assess the contribution of cage openings to the kinetics of iron oxidation, mineralization, or reductive iron release. Identical iron binding kinetics for AfFtn and AfFtn-AA suggest that Fe^2+ ions do not utilize the triangular pores for access to the catalytic site. In contrast, relatively slow reductive iron release was observed for the closed AfFtn-AA, demonstrating involvement of the large pores in the pathway for iron release