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Abstract Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co‐assemble in different H and L ratios to form 24‐mer shell‐like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure–function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L‐rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H‐ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron‐induced protein thermal destabilization effect (ΔTmup to 20°C) is observed. To our knowledge, this is the first report of recombinant human homo‐ and hetero‐polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L‐rich ferritins may have great potential for biotechnological applications.
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Micromagnetic and morphological characterization of heteropolymer human ferritin cores
The physical properties of in vitro iron-reconstituted and genetically engineered human heteropolymer ferritins were investigated. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), electron energy-loss spectroscopy (EELS), and 57 Fe Mössbauer spectroscopy were employed to ascertain (1) the microstructural, electronic, and micromagnetic properties of the nanosized iron cores, and (2) the effect of the H and L ferritin subunit ratios on these properties. Mössbauer spectroscopic signatures indicate that all iron within the core is in the high spin ferric state. Variable temperature Mössbauer spectroscopy for H-rich (H 21 /L 3 ) and L-rich (H 2 /L 22 ) ferritins reconstituted at 1000 57 Fe/protein indicates superparamagnetic behavior with blocking temperatures of 19 K and 28 K, while HAADF-STEM measurements give average core diameters of (3.7 ± 0.6) nm and (5.9 ± 1.0) nm, respectively. Most significantly, H-rich proteins reveal elongated, dumbbell, and crescent-shaped cores, while L-rich proteins present spherical cores, pointing to a correlation between core shape and protein shell composition. Assuming an attempt time for spin reversal of τ 0 = 10 −11 s, the Néel–Brown formula for spin-relaxation time predicts effective magnetic anisotropy energy densities of 6.83 × 10 4 J m −3 and 2.75 × 10 4 J m −3 for H-rich and L-rich proteins, respectively, due to differences in surface and shape contributions to magnetic anisotropy in the two heteropolymers. The observed differences in shape, size, and effective magnetic anisotropies of the derived biomineral cores are discussed in terms of the iron nucleation sites within the interior surface of the heteropolymer shells for H-rich and L-rich proteins. Overall, our results imply that site-directed nucleation and core growth within the protein cavity play a determinant role in the resulting core morphology. Our findings have relevance to iron biomineralization processes in nature and the growth of designer's magnetic nanoparticles within recombinant apoferritin nano-templates for nanotechnology.
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- PAR ID:
- 10384224
- Date Published:
- Journal Name:
- Nanoscale Advances
- ISSN:
- 2516-0230
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d2na00544a
