Higher Order Assembly of Virus‐like Particles (VLPs) Mediated by Multi‐valent Protein Linkers

Abstract

Two- and three-dimensional assembly of nanoparticles has generated significant interest because these higher order structures could exhibit collective behaviors/properties beyond those of the individual nanoparticles. Highly specific interactions between molecules, which biology exploits to regulate molecular assemblies such as DNA hybridization, often provide inspiration for the construction of higher order materials using bottom-up approaches. In this study, higher order assembly of virus-like particles (VLPs) derived from the bacteriophage P22 is demonstrated by using a small adaptor protein, Dec, which binds to symmetry specific sites on the P22 capsid. Two types of connector proteins, which have different number of P22 binding sites and different geometries (ditopic linker with liner geometry and tetratopic linker with tetrahedral geometry) have been engineered through either a point mutation of Dec or genetic fusion with another protein, respectively. Bulk assembly and layer-by-layer deposition of P22 VLPs from solution was successfully achieved using both of the engineered multi-topic linker molecules, while Dec with only a single binding site does not mediate P22 assembly. Beyond the two types of linkers developed in this study, a wide range of different connector geometries could be envisioned using a similar engineering approach. This is a powerful strategy to construct higher order assemblies of VLP based nanomaterials.

Symmetry Controlled, Genetic Presentation of Bioactive Proteins on the P22 Virus-like Particle Using an External Decoration Protein

“…This insight can be used to design Dec constructs with alternative binding affinities to the capsid, allowing for control over processes such as Dec-mediated higher order assembly of capsids or release of the Dec-cargo from the capsid under controlled conditions. 42 Building on previous studies, demonstrating the broad utility of the P22 VLP as a modular encapsulation system through scaffold protein-fusion, Dec exterior functionalization allows for a similar utility on the exterior capsid surface. The unique use of accessory proteins (scaffold protein and Dec) as bioconjugation strategies while leaving the capsid protein unmodified allows for the preservation of the core capsid structure and the potential to add more complex cargos.…”

“…Dec has previously been shown to be useful in mediating the assembly of P22 into higher order structures. 42 Modulating the affinity of Dec linkers in these systems could provide a key control in the assembly process.…”

“…To remove the subpopulation from the PC sample we used a C-terminal cysteine mutant of Dec (DecS134C), which upon oxidation forms a linear head-to-head dimer of trimers that can cross-link and aggregate particles to which it binds 42 The percentage of this subpopulation in the P22-PC sample was found to vary from batch to batch and could be purified away through Dec-Dec aggregation. The remaining PC was shown to have no measurable affinity for Dec (Figure 8B).…”

Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology

“…This ditopic Dec–Dec linker, when mixed with the P22 VLP, lead to the assembly of an unstructured P22 assembly. However, a layer-by-layer approach, with alternating Dec–Dec and P22 layers could be readily formed using these materials (Uchida et al, 2015). The system could be extended to make a binary cage assembly through genetic fusion of Dec to the tetrahedral DPS protein leading to DPS VLPs with four exposed P22-binding Dec domains which could act as tetratopic linkers for the assembly of P22 VLPs.…”

Design and Applications of Protein‐Cage‐Based Nanomaterials

“…To construct two‐ and three‐dimensional architectures from protein‐cage building blocks, two general strategies have been developed. The first utilizes direct physical interaction, such as external surface electrostatics, between the protein cages, and the second employs either covalent or non‐covalent linker functionality …”