An ability to organize and encapsulate multiple active proteins into defined objects and spaces at the nanoscale has potential applications in biotechnology, nanotechnology, and synthetic biology. Previously, we have described the design, assembly, and characterization of peptide-based self-assembled cages (SAGEs). These ≈100 nm particles comprise thousands of copies of de novo designed peptide-based hubs that array into a hexagonal network and close to give caged structures. Here, we show that, when fused to the designed peptides, various natural proteins can be co-assembled into SAGE particles. We call these constructs pSAGE for protein-SAGE. These particles tolerate the incorporation of multiple copies of folded proteins fused to either the N or the C termini of the hubs, which modeling indicates form the external and internal surfaces of the particles, respectively. Up to 15% of the hubs can be functionalized without compromising the integrity of the pSAGEs. This corresponds to hundreds of copies giving mM local concentrations of protein in the particles. Moreover, and illustrating the modularity of the SAGE system, we show that multiple different proteins can be assembled simultaneously into the same particle. As the peptide-protein fusions are made via recombinant expression of synthetic genes, we envisage that pSAGE systems could be developed modularly to actively encapsulate or to present a wide variety of functional proteins, allowing them to be developed as nanoreactors through the immobilization of enzyme cascades or as vehicles for presenting whole antigenic proteins as synthetic vaccine platforms.

The design and assembly of peptide‐based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here a de novo peptide system is presented that forms nanoparticles or sheets depending on the strategic placement of a “disulfide pin” between two elements of secondary structure that drive self‐assembly. Specifically, homodimerizing and homotrimerizing de novo coiled‐coil α‐helices are joined with a flexible linker to generate a series of linear peptides. The helices are pinned back‐to‐back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling indicates, and advanced microscopy shows, that the proximally pinned hairpins self‐assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.

Better understanding of the dynamics of protein-based supramolecular capsids can be applied to synthetic biology and biotechnology.

The design and assembly of peptide based materials has advanced considerably, leading to a variety of fibrous, sheet and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here we present a straightforward de novo peptide system that forms nanoparticles or sheets depending on the strategic placement of a ‘disulfide pin’ between two elements of secondary structure that drive self-assembly. Specifically, we joined a homodimerizing and a homotrimerizing de novo coiled-coil α-helices with a flexible linker to generate a series of linear peptides. These helices were then pinned back-to-back and so constrained into a hairpin shape by a disulfide bond placed either proximal or distal to the linker. Computational modeling and extensive observations by advanced microscopy show that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.