Barindra Sana

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.

Bioengineered Tunable Memristor Based on Protein Nanocage

Bioengineered protein-based nanodevices with tunable and reproducible memristive performance are fabricated by combining the unique high loading capacity of Archaeoglobus fulgidus ferritin with OWL-generated nanogaps. By tuning the iron amount inside ferritin, the ON/OFF ratio of conductance switching can be modulated accordingly. Higher molecular loading exhibits better memristive performance owing to the higher electrochemical activity of the ferric complex core.

The role of nonconserved residues of Archaeoglobus fulgidus ferritin on its unique structure and biophysical properties.

Background: Archaeoglobus fulgidus ferritin (AfFtn) assembles with unique tetrahedral symmetry and four large pores. Results: The AfFtn K150A/R151A double mutant forms a closed octahedral assembly with reduced iron release rates relative to the tetrahedral assembly. Conclusion: The K150A/R151A substitution alters the symmetry type of the ferritin cage. Significance: The AfFtn can be modulated for tuning molecular release from the cavity. 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 Fe2+ 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.

Designing Non‐Native Iron‐Binding Site on a Protein Cage for Biological Synthesis of Nanoparticles

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self-assembly of 60 subunits, has been functionalized with non-native iron-mineralization capability by incorporating two types of iron-binding peptides. The non-native peptides introduced at the interior surface do not affect the self-assembly of E2 protein subunits. In contrast to the wild-type, the engineered E2 protein cages can serve as size- and shape-constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host-guest encapsulation properties of protein cages.

Investigation of electron transfer from isolated spinach thylakoids to indium tin oxide

The electrons generated by photosynthetic water splitting have been studied for direct electron transfer under light irradiation. Isolated thylakoids are incorporated as biocatalysts with indium tin oxide (ITO) as the electrode in a two-chamber photosynthetic electrochemical cell (PEC). The generated photocurrent is compared between deposited and dispersed thylakoids. The physical properties of deposited thylakoids are observed using field emission scanning electron microscopy (FESEM) and absorbance spectroscopy techniques. The results show the presence of thylakoids with bound photosystems including light harvesting antennas and other protein complexes. Further investigations reveal that the direction of electron transfer can be tuned by varying the applied potentials to promote bi-directional photocurrent. The maximum cathodic photocurrent is obtained at 50 mV vs. standard calomel electrode (SCE), while the maximum anodic photocurrent is enhanced with increasing positive potential applied. Our observation indicates the possibility of either reduction of O2 or H2O2 as the prominent cathodic photocurrent source, while electron transfer from thylakoids to ITO yields the anodic photocurrent.

Ecological Roles and Biotechnological Applications of Marine and Intertidal Microbial Biofilms

This review is a retrospective of ecological effects of bioactivities produced by biofilms of surface-dwelling marine/intertidal microbes as well as of the industrial and environmental biotechnologies developed exploiting the knowledge of biofilm formation. Some examples of significant interest pertaining to the ecological aspects of biofilm-forming species belonging to the Roseobacter clade include autochthonous bacteria from turbot larvae-rearing units with potential application as a probiotic as well as production of tropodithietic acid and indigoidine. Species of the Pseudoalteromonas genus are important examples of successful surface colonizers through elaboration of the AlpP protein and antimicrobial agents possessing broad-spectrum antagonistic activity against medical and environmental isolates. Further examples of significance comprise antiprotozoan activity of Pseudoalteromonas tunicata elicited by violacein, inhibition of fungal colonization, antifouling activities, inhibition of algal spore germination, and 2-n-pentyl-4-quinolinol production. Nitrous oxide, an important greenhouse gas, emanates from surface-attached microbial activity of marine animals. Marine and intertidal biofilms have been applied in the biotechnological production of violacein, phenylnannolones, and exopolysaccharides from marine and tropical intertidal environments. More examples of importance encompass production of protease, cellulase, and xylanase, melanin, and riboflavin. Antifouling activity of Bacillus sp. and application of anammox bacterial biofilms in bioremediation are described. Marine biofilms have been used as anodes and cathodes in microbial fuel cells. Some of the reaction vessels for biofilm cultivation reviewed are roller bottle, rotating disc bioreactor, polymethylmethacrylate conico-cylindrical flask, fixed bed reactor, artificial microbial mats, packed-bed bioreactors, and the Tanaka photobioreactor.

Protein Cages as Theranostic Agent Carriers

Protein cages can be engineered to tailor its function as carriers for therapeutic and diagnostic agents. They are formed by self-assembly of multiple subunits forming hollow spherical cage structures of nanometer size. Due to their proteinaceous nature, the protein cages allow facile modifications on its internal and external surfaces, as well as the subunit interfaces. Modifications on the internal surface allow conjugation of small molecule drugs or contrast agent while modifications on the external surface allow conjugation of various ligands including targeting ligands. The subunit interaction is of special interest in engineering controlled release property onto the protein cage. Two different protein cages, E2 protein and ferritin, are described.

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. GENERAL SIGNIFICANCE The iron-mediated self-assembly/disassembly property of AfFtn offers a new set of molecular trigger for formation and dissociation of the protein cage, which can potentially regulate uptake and release of molecular cargo from protein cages.

Bioresources for Control of Environmental Pollution

Environmental pollution is one of the biggest threats to human beings. For practical reasons it is not possible to stop most of the activities responsible for environmental pollution; rather we need to eliminate the pollutants. In addition to other existing means, biological processes can be utilized to get rid of toxic pollutants. Degradation, removal, or deactivation of pollutants by biological means is known as bioremediation. Nature itself has several weapons to deal with natural wastage and some of them are equally active for eliminating nonnatural pollutants. Several plants, microorganisms, and some lower eukaryotes utilize environmental pollutants as nutrients and some of them are very efficient for decontaminating specific types of pollutants. If exploited properly, these natural resources have enough potential to deal with most elements of environmental pollution. In addition, several artificial microbial consortia and genetically modified organisms with high bioremediation potential were developed by application of advanced scientific tools. On the other hand, natural equilibria of ecosystems are being affected by human intervention. Rapid population growth, urbanization, and industrialization are destroying ecological balances and the natural remediation ability of the Earth is being compromised. Several potential bioremediation tools are also being destroyed by biodiversity destruction of unexplored ecosystems. Pollution management by bioremediation is highly dependent on abundance, exploration, and exploitation of bioresources, and biodiversity is the key to success. Better pollution management needs the combined actions of biodiversity conservation, systematic exploration of natural resources, and their exploitation with sophisticated modern technologies.

Protein cage assisted metal-protein nanocomposite synthesis: Optimization of loading conditions

Ferritin is an iron-storage protein in most living systems with a cage-like structure. It has inherent property to form metallic nanocore within its cavity. The metallic core formed within the Archaeoglobus fulgidus ferritin cavity is stabilized by modulating the protein structure by site directed mutagenesis. Encapsulation protocol of various metals within the engineered ferritin cage (AfFtn-AA) is optimized. Dense metallic cores are visualized using electron microscopy and the bound metal was quantified by ICP-spectrometry. The AfFtn-AA is loaded with up to about 350 cobalt, 2000 chromium, and as high as 7000 iron atoms, separately. The metal-protein nanocomposites formed by encapsulation of cobalt, chromium, and iron are studied. Magnetic resonance imaging of the agarose embedded nanocomposites shows brightening of T1-weighted images and signal loss of T2-weighted images with increasing concentration of the nanocomposites. Shortening of magnetic relaxation times in the presence of the nanocomposites co...