Controlling protein nanocage assembly with hydrostatic pressure

Abstract

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/...

Characterizing the Dynamic Disassembly/Reassembly Mechanisms of Encapsulin Protein Nanocages

“…For example, by identifying critical interactions between nanocage subunits that may affect the assembly process, future genetic engineering efforts of subunits may lead to enhanced control over conditions that trigger disassembly/ reassembly, size, structure, and cargo-loading efficiency of encapsulins. 50,51 In summary, the findings of this study advance our understanding of encapsulins by providing critical insight into their unique disassembly/reassembly dynamics. This knowledge provides a roadmap toward an encapsulin "tool kit" comprising nanocages with varying structural architectures and biochemical/biophysical properties, which can be readily selected and further customized for a specific nanobiotechnological application.…”

Ferritin: A Multifunctional Nanoplatform for Biological Detection, Imaging Diagnosis, and Drug Delivery

“…41 It was demonstrated that a hydrostatic pressure of 450 MPa is sufficient to completely dissociate the ferritin nanocage into protein dimers, and the reassembly process could be controlled by selecting appropriate buffer conditions. 42…”

Effect of high hydrostatic pressure processing on the structure, functionality, and nutritional properties of food proteins: A review

“…Structural alterations in proteins under HHP treatment HHP is known to induce changes in protein folding and oligomerization, and this change primarily occurs by influencing weak bonds, especially those in hydrophobic interactions and electrostatic interactions (Le Vay et al, 2020). Moreover, pressures below 1 GPa rarely break covalent bonds; therefore, the primary structures of proteins are unaffected by HHP (Silva et al, 2014).…”

“…Although protein unfolding may also expose hydrophobic AA residues and consequently increase the hydration volume, the net changes in V Φ upon unfolding are mostly negative (Chen & Makhatadze, 2017). In addition, pressurization favors the dissociation of oligomeric proteins, and this process can be ascribed to the disruption of interfacial protein-protein interactions due to protein unfolding (Le Vay et al, 2020).…”

“…According to Le Chatelier's principle, high pressure favors biophysical states that occupy a smaller overall volume. Consequently, under pressure treatments, it is likely that protein chains will unfold and oligomeric proteins will disassociate (Knorr et al, 2011;Le Vay et al, 2020). Upon decompression, the altered protein structure may not recover; thus, the protein properties are modified (Roche & Royer, 2018).…”