Controlling the assembly and disassembly of nanoscale protein cages for the capture and internalization of protein or non-proteinaceous components is fundamentally important to a diverse range of bionanotechnological applications. Here, we study the reversible, pressure-induced dissociation of a natural protein nanocage, E. coli bacterioferritin (Bfr), using synchrotron radiation small-angle X-ray scattering (SAXS) and circular dichroism (CD). We demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely dissociate the Bfr 24-mer into protein dimers, and the reversibility and kinetics of the reassembly process can be controlled by selecting appropriate buffer conditions. We also demonstrate that the heme B prosthetic group present at the subunit dimer interface influences the stability and pressure lability of the cage, despite its location being discrete from the interdimer interface that is key to cage assembly. This indicates a major cage-stabilizing role for heme within this family of ferritins.

Here, we describe a facile route to the synthesis of enzymatically active highly fabricable plastics, where the enzyme is an intrinsic component of the material. This is facilitated by the formation of an electrostatically stabilized enzyme−polymer surfactant nanoconstruct, which, after lyophilization and melting, affords stable macromolecular dispersions in a wide range of organic solvents. A selection of plastics can then be co-dissolved in the dispersions, which provides a route to bespoke 3D enzyme plastic nanocomposite structures using a wide range of fabrication techniques, including melt electrowriting, casting, and pistondriven 3D printing. The resulting constructs comprising active phosphotriesterase (arPTE) readily detoxify organophosphates with persistent activity over repeated cycles and for long time periods. Moreover, we show that the protein guest molecules, such as arPTE or sfGFP, increase the compressive Young's modulus of the plastics and that the identity of the biomolecule influences the nanomorphology and mechanical properties of the resulting materials. Overall, we demonstrate that these biologically active nanocomposite plastics are compatible with state-of-the-art 3D fabrication techniques and that the methodology could be readily applied to produce robust and on-demand smart nanomaterial structures.

Catalytically active materials for the enhancement of personalized protective equipment (PPE) could be advantageous to help alleviate threats posed by neurotoxic organophosphorus compounds (OPs). Accordingly, a chimeric protein comprised of a supercharged green fluorescent protein (scGFP) and phosphotriesterase from Agrobacterium radiobacter (arPTE) was designed to drive the polymer surfactant (S − )-mediated selfassembly of microclusters to produce robust, enzymatically active materials. The chimera scGFP-arPTE was structurally characterized via circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering, and its biophysical properties were determined. Significantly, the chimera exhibited greater thermal stability than the native constituent proteins, as well as a higher catalytic turnover number (k cat ). Furthermore, scGFP-arPTE was electrostatically complexed with monomeric S − , driving selfassembly into [scGFP-arPTE][S − ] nanoclusters, which could be dehydrated and cross-linked to yield enzymatically active [scGFP-arPTE][S − ] porous films with a high-order structure. Moreover, these clusters could self-assemble within cotton fibers to generate active composite textiles without the need for the pretreatment of the fabrics. Significantly, the resulting materials maintained the biophysical activities of both constituent proteins and displayed recyclable and persistent activity against the nerve agent simulant paraoxon.

22Controlling the assembly and disassembly of nanoscale protein cages for the 23 capture and internalisation of protein or non-proteinaceous components is 24 fundamentally important to a diverse range of bionanotechnological 25 applications. Here, we study the reversible, pressure-induced dissociation of a 26 natural protein nanocage, E. coli bacterioferritin (Bfr), using synchrotron 27 radiation small angle X-ray scattering (SAXS) and circular dichroism (CD). We 28 demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely 29 dissociate the Bfr icositetramer into protein dimers, and the reversibility and 30 kinetics of the reassembly process can be controlled by selecting appropriate 31 buffer conditions. We also demonstrate that the heme B prosthetic group 32 present at the subunit dimer interface influences the stability and pressure 33 lability of the cage, despite its location being discrete from the inter-dimer 34 interface that is key to cage assembly. This indicates a major cage-stabilising 35 role for heme within this family of ferritins. 36 37 Nanoscale protein cages are attractive scaffolds for bionanotechnology and materials 38 science, where they can be exploited as platforms for constructing robust and 39 configurable therapeutic delivery vectors 1 , vaccines 2 , nanoreactors 3,4 and templates 40 for the synthesis of diverse nanomaterials 5-8 . These multifunctional containers, both 41 natural 9-13 and designed 14,15 , offer unparalleled control over size, shape, 42 microenvironment, surface functionalisation and stability when constructing novel 43 bionanomaterials. 44 45The ability to control the assembly of such nanocages is an invaluable tool in the 46 synthesis of complex materials, and can be instrumental in facilitating the natural [16][17][18] or engineered 19-21 cage metastability, the use of such nanocages could 49 ultimately compromise the robustness of the final assembled material. For nanocages 50 with higher relative stability, harsher environmental conditions 22,23 are required that 51 can adversely affect the protein cage, its functional modifications or the intended 52 payload for encapsulation. Therefore, new methods are required to circumvent the 53 necessity for harsh chemical conditions or specific interfacial engineering to promote 54 cage instability, and to realise the full potential of these cages in bionanotechnology. 55 56 Here, we report on how hydrostatic pressure can be employed to control the 57 disassembly and reassembly of the protein nanocage bacterioferritin from E. coli (Bfr). 58While hydrostatic pressure has been previously employed to dissociate the weakly 59 stable cage-like assembly HSP26 24 , the structure is not truly hollow 25 . There are 60 currently no reports of complete, reversible hydrostatic pressure-induced dissociation 61 in a highly robust nanocage such as ferritin 26,27 . While hydrostatic pressure has been 62 applied to human ferritin to facilitate the loading of doxorubicin and increase protein 63 recovery 27 , the assembly/...

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