Assembly Requirement for Binary Protein Structure and the Design of Anisotropic Protein Nanocages

Abstract

Precise assembly of multiple biomacromolecules into well-defined structures and materials is of great importance for various biomedical and nanobiotechnological applications. Biomaterials will be crucial for a sustainable bio-economy with uses in industry and healthcare alike. In the first part of this work, the assembly requirements for twocomponent materials using supercharged ferritin nanocages as building blocks were investigated. Several variants of ferritin nanocages were designed to determine the surface characteristics necessary for the formation of large-scale binary three-dimensional (3D) assemblies. The newly generated nanocage variants were employed in protein crystallization experiments and macromolecular crystallography analyses. Computational methods were used to complement the experimental findings. By screening of nanocage variant combinations at various ionic strengths, three essential features for successful assembly could be identified: (1) the presence of a favored crystal contact region, (2) the presence of a charged patch that is not involved in crystal contacts, and (3) sufficiently distinct surface characteristics between the nanocages. Surprisingly, the absence of non-crystalcontact-mediating patches had a detrimental effect on the assemblies, highlighting their unexpected importance. The formation of unitary, single building block, structures containing either negatively or positively charged nanocages under previously exclusively binary conditions was achieved. These findings will guide future design strategies by offering design principles and demonstrating how supercharging symmetric building blocks can aid in the assembly of biomacromolecules into extensive binary 3D structures. The second part aimed to extend the palette of available building blocks towards defined cylindrical nanocages. Toward this goal, a design approach focused on hollow dihedral architectures was conceptualized. These would allow the use of anisotropic nanoparticulate systems such as quantum rods as cargo previously inaccessible for the formation of biohybrid materials. Recently developed design tools were used to generate de novo in silico assemblies. Several existing protein structures were tested for their suitability as building blocks. Finally, assemblies were validated by physics-based simulations and deep-learning-based protein structure prediction tools.