Assembly Requirements for the Construction of Large-Scale Binary Protein Structures

The precise assembly of multiple biomacromolecules into well-defined structures and materials is of great importance for various biomedical and nanobiotechnological applications. In this study, we investigate the assembly requirements for two-component materials using charged protein nanocages as building blocks. To achieve this, we designed several variants of ferritin nanocages to determine the surface characteristics necessary for the formation of large-scale binary three-dimensional (3D) assemblies. These nanocage variants were employed in protein crystallization experiments and macromolecular crystallography analyses, complemented by computational methods. Through the screening of nanocage variant combinations at various ionic strengths, we identified three essential features for successful assembly: (1) the presence of a favored crystal contact region, (2) the presence of a charged patch not involved in crystal contacts, and (3) sufficient distinctiveness between the nanocages. Surprisingly, the absence of noncrystal contact mediating patches had a detrimental effect on the assemblies, highlighting their unexpected importance. Intriguingly, we observed the formation of not only binary structures but also both negatively and positively charged unitary structures under previously exclusively binary conditions. Overall, our findings will inform future design strategies by providing some design rules, showcasing the utility of supercharging symmetric building blocks in facilitating the assembly of biomacromolecules into large-scale binary 3D assemblies.