Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern stability and flux through their pores. In this work, we systematically designed 24 variants of the Thermotoga maritima encapsulin cage, featuring pores of different sizes and charges. Twelve pore variants were successfully assembled and purified, including eight designs with exceptional thermal stability. While negatively charged mutations were better tolerated, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures at 2.5- to 3.6-Å resolution. Molecular dynamics simulations and stopped-flow experiments revealed the importance of considering both pore size and charge, together with flexibility and rate-determining steps, when designing protein cages for controlling molecular flux.

Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. Pores lined with anionic residues resulted in more robust assemblies than their corresponding cationic variants. We then determined seven cryo-EM structures of pore variants at resolutions between 2.5-3.6 Å. Together with stopped-flow kinetics experiments for quantifying cation influx, we uncover the complex interplay between pore size, charge, and flexibility that controls molecular flux in protein cages, providing guidance for future nanoreactor designs.Abstract Figure

Self-assembling proteins can form porous compartments that adopt well-defined architectures at the nanoscale. In nature, protein compartments act as semipermeable barriers to enable spatial separation and organization of complex biochemical processes. The compartment pores play a key role in their overall function by selectively controlling the influx and efflux of important biomolecular species. By engineering the pores, the functionality of compartments can be tuned to facilitate non-native applications, such as artificial nanoreactors for catalysis. In this review, we analyze how protein structure determines the porosity and impacts the function of both native and engineered compartments, highlighting the wealth of structural data recently obtained by cryo-EM and X-ray crystallography. Through this analysis, we offer perspectives on how current structural insights can inform future studies into the design of artificial protein compartments as nanoreactors with tunable porosity and function.

The design of biomimetic systems in the laboratory is a long‐sought goal for systems chemists and synthetic biologists alike. Fundamental to this design is the generation of self‐assembled structures capable of mimicking compartmentalisation, which includes the encapsulation of molecular cargo as well as the display of molecules on the exterior. Protein nanocompartments are fast becoming popular scaffolds for these systems due to their robust self‐assembly, ability to encapsulate non‐native cargo, and amenability to surface modifications. In this Review, we discuss the primary methods for displaying a wide array of molecular motifs on compartment surfaces. We discuss benefits and drawbacks of each type of display and examine three recent case studies wherein molecular display was a critical design element in the construction of multi‐enzyme chemical systems. The analyses and case studies presented in this Review aim to provide a critical summary of the technologies currently used for molecular display to add another dimension to the design of chemical systems and nanoreactors.

This cover design highlights the two key properties of protein compartments relevant to this Review -the ability to encapsulate cargo within the interior and display molecules on the exterior. We hope that this cover inspires the readers of ChemSystemsChem to find out more about protein compartment display technologies and their applications in building novel catalytic systems. Who contributed to the idea behind the cover?ChemSystemsChem