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

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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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

Indole and azaindole halogenation catalyzed by the RebH enzyme variant 3-LSR utilizing co-purified E. coli reductase

Biocatalytic C-H halogenation is becoming increasingly attractive due to excellent catalyst-controlled selectivity and environmentally benign reaction conditions. Significant efforts have been made on enzymatic halogenation of industrial arenes in a cost-effective manner. Here we report an unprecedented enzymatic halogenation of a panel of industrially important indole, azaindole and anthranilamide derivatives using a thermostable RebH variant without addition of any external flavin reductase enzyme. The reactions were catalyzed by the RebH variant 3-LSR enzyme with the help of a co-purified E. coli reductase identified as alkyl hydroperoxide reductase F (AhpF)

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 confirm their ability to enhance magnetic relaxation rate and suggests that the nanocomposites have potential application as MRI contrast agent.Published versio

A manganese–ferritin nanocomposite as an ultrasensitive T2 contrast agent

T 2 contrast is gaining importance in high field strength MRI. We report a strategy for developing a T2 contrast agent from paramagnetic metal ions synthesized within an engineered protein cage. The manganese–ferritin nanocomposite showed high T2 relaxivity indicating its potential as an ultrasensitive T2 contrast agent