Biology shows many examples of spatially controlled assembly of cells and biomacromolecules into hierarchically organized structures, to which many of the complex biological functions are attributed. While such biological structures have inspired the design of synthetic materials, it is still a great challenge to control the spatial arrangement of individual building blocks when assembling multiple types of components into bulk materials. Here, we report self-assembly of multilayered, ordered protein arrays from mixed populations of virus-like particles (VLPs). We systematically tuned the magnitude of the surface charge of the VLPs via mutagenesis to prepare four different types of VLPs for mixing. A mixture of up to four types of VLPs selectively assembled into higher-order structures in the presence of oppositely charged dendrimers during a gradual lowering of the ionic strength of the solution. The assembly resulted in the formation of three-dimensional ordered VLP arrays with up to four distinct layers including a central core, with each layer comprising a single type of VLP. A coarse-grained computational model was developed and simulated using molecular dynamics to probe the formation of the multilayered, core-shell structure. Our findings establish a simple and versatile bottom-up strategy to synthesize multilayered, ordered materials by controlling the spatial arrangement of multiple types of nanoscale building blocks in a one-pot fabrication.
Encapsulation of enzymes inside protein cage structures, mimicking protein-based organelle structures found in nature, has great potential for the development of new catalytic materials with enhanced properties. In vitro and in vivo methodologies have been developed for the encapsulation of enzymes within protein cage structures of several types, particularly virus-like particles (VLPs), with the ability to retain the activity of the encapsulated enzymes. Here, we examine the in vivo encapsulation of enzymes within the bacteriophage P22 derived VLP and show that some enzymes may require a delay in encapsulation to allow proper folding and maturation before they can be encapsulated inside P22 as fully active enzymes. Using a sequential expression strategy, where enzyme cargoes are first expressed, allowed to fold, and later encapsulated by the expression of the P22 coat protein, altered enzymatic activities are obtained in comparison to enzymes encapsulated in P22 VLPs using a simultaneous coexpression strategy. The strategy and results discussed here highlight important considerations for researchers investigating the encapsulation of enzymes inside confined reaction environments via in vivo routes and provide a potential solution for those that have been unable to produce active enzymes upon encapsulation.
Hierarchical organization is one of the fundamental features observed in biological systems that allows for efficient and effective functioning. Virus-like particles (VLPs) are elegant examples of a hierarchically organized supramolecular structure, where many subunits are self-assembled to generate the functional cage-like architecture. Utilizing VLPs as building blocks to construct two-and threedimensional (3D) higher-order structures is an emerging research area in developing functional biomimetic materials. VLPs derived from P22 bacteriophages can be repurposed as nanoreactors by encapsulating enzymes and modular units to build higher-order catalytic materials via several techniques. In this study, we have used coiled-coil peptide interactions to mediate the P22 interparticle assembly into a highly stable, amorphous protein macromolecular framework (PMF) material, where the assembly does not depend on the VLP morphology, a limitation observed in previously reported P22 PMF assemblies. Many encapsulated enzymes lose their optimum functionalities under the harsh conditions that are required for the P22 VLP morphology transitions. Therefore, the coiled-coil-based PMF provides a fitting and versatile platform for constructing functional higher-order catalytic materials compatible with sensitive enzymes. We have characterized the material properties of the PMF and utilized the disordered PMF to construct a biocatalytic 3D material performing single-and multistep catalysis.
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