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Abstract Versatile methods to organize proteins in space are required to enable complex biomaterials, engineered biomolecular scaffolds, cell-free biology, and hybrid nanoscale systems. Here, we demonstrate how the tailored encapsulation of proteins in DNA-based voxels can be combined with programmable assembly that directs these voxels into biologically functional protein arrays with prescribed and ordered two-dimensional (2D) and three-dimensional (3D) organizations. We apply the presented concept to ferritin, an iron storage protein, and its iron-free analog, apoferritin, in order to form single-layers, double-layers, as well as several types of 3D protein lattices. Our study demonstrates that internal voxel design and inter-voxel encoding can be effectively employed to create protein lattices with designed organization, as confirmed by in situ X-ray scattering and cryo-electron microscopy 3D imaging. The assembled protein arrays maintain structural stability and biological activity in environments relevant for protein functionality. The framework design of the arrays then allows small molecules to access the ferritins and their iron cores and convert them into apoferritin arrays through the release of iron ions. The presented study introduces a platform approach for creating bio-active protein-containing ordered nanomaterials with desired 2D and 3D organizations.
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Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization
Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein’s surface, resulting in a shift of the putative net charge from −1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn–hCAII(+21) host–guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn–hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.
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- Award ID(s):
- 1905203
- PAR ID:
- 10357696
- Date Published:
- Journal Name:
- Biochemistry
- Volume:
- 60
- Issue:
- 47
- ISSN:
- 1520-4995
- Page Range / eLocation ID:
- 3596–3609
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/doi/10.1021/acs.biochem.1c00515
