Assembly of differently sized supercharged protein nanocages into superlattices for construction of binary nanoparticle-protein materials

This study focuses on the design and characterization of binary nanoparticle superlattices: Two differently sized, supercharged protein nanocages are used to create a matrix for nanoparticle arrangement. We have previously established the assembly of protein nanocages of the same size. Here, we present a novel approach for biohybrid material synthesis by successfully assembling two differently sized supercharged protein nanocages with different symmetries. Typically, the ordered assembly of objects with non-matching symmetry is challenging, but our electrostatic-based approach overcomes the symmetry mismatch by exploiting electrostatic interactions between oppositely charged cages. Moreover, our study showcases the use of nanoparticles as contrast enhancer in an elegant way to gain insights into the structural details of crystalline biohybrid materials. The assembled materials were characterized with various methods, including transmission electron microscopy (TEM) and single-crystal small-angle X-ray scattering (SAXS). We employed focused ion beam milling (FIB) under cryogenic temperatures to further characterize the nanoparticle sublattices via cryo-TEM. Notably, for the first time, we refined superlattice structure data obtained from single-crystal SAXS experiments, providing conclusive evidence of the final assembly type. Our findings highlight the versatility of protein nanocages for creating novel types of binary superlattices. Because the nanoparticles do not influence the type of assembly, protein cage matrices can combine various nanoparticle in the solid state. This study not only contributes to the expanding repertoire of nanoparticle assembly methods but also demonstrates the power of advanced characterization techniques in elucidating the structural intricacies of these biohybrid materials.