Final Report Summary - EPCABO (Engineered Protein Capsids as Artificial Bacterial Organelles)
Many proteins self-assemble into regular, shell-like, polyhedral structures. The most prominent examples are the sturdy protein capsules that viruses utilize to protect and deliver genetic information. In some bacteria, similar protein shells encapsulate multiple enzymes and act as chemical nanoreactors. In the laboratory, such capsids are useful as molecular containers for diverse cargo molecules, including proteins, nucleic acids, metal nanoparticles, quantum dots, and low molecular weight drugs. They can serve as delivery vehicles, bioimaging agents, reaction vessels, and templates for the controlled synthesis of novel materials. In this project, we have applied our experience with protein design and laboratory evolution to extend the properties of protein containers to create practical, non-viral encapsulation systems for applications in the test tube and in living cells.
Specifically, we have produced versatile protein cages derived from Aquifex aeolicus lumazine synthase (AaLS), called AaLS-neg and AaLS-13, which possess negatively supercharged interiors and spontaneously encapsulate positively charged cargo molecules. In contrast to the closed-shell structure of native AaLS, we have discovered that these cages adopt unprecedented expanded architectures with large keyhole-shaped pores in their shells, thus facilitating rapid and quantitative loading of a wide variety of guests. For example, GFP(+36), a positively supercharged variant of green fluorescent protein, is spontaneously loaded into empty AaLS-13 cages in quantitative yields at diffusion-limited rates simply by mixing guest and cage in aqueous solution. Exploiting such simple and efficient host-guest interactions, active enzymes can be loaded into AaLS-neg and AaLS-13 assemblies by genetic fusion to GFP(+36), which acts as a localization tag. The utility of the resulting nanoreactors has been illustrated by the templated synthesis of polymers and mimicry of the carboxysome, a natural bacterial microcompartment. Although we did not observe significant kinetic advantages for sequentially active enzymes loaded in the AaLS-13 cage, proteases encapsulated within AaLS-13 cleave positively charged peptides two orders of magnitude faster than negatively charged analogues, suggesting an alternative function for AaLS-13 cages as substrate sorters based on electrostatic interactions. This type of substrate sorting system could be a powerful strategy to regulate enzyme activity in multicomponent systems including intracellular environments, analogous to the action of natural housekeeping proteases like the proteasome.
In parallel with our work on negatively supercharged protein cages, we have created positively supercharged AaLS variants that bind negatively charged cargo, functionalized the surface of the protein shells to facilitate uptake by mammalian cells, and created circularly permuted constructs that make inside-outside genetic modification possible. In perhaps the most exciting application of such systems, we have engineered and subsequently evolved circularly permuted AaLS cages to encapsulate their own full-length RNA genome in vivo and protect the cargo molecules from nucleases. The bottom-up construction of virus-like nucleocapsids from nonviral materials has great potential to shed light on the assembly and evolution of natural virions while providing safe alternatives to natural viruses for diverse nano- and biotechnological applications. Indeed, we have already successfully applied the principles learned in these studies to create computationally designed protein cages that efficiently deliver siRNA and hydrophobic small molecules to cells.
Specifically, we have produced versatile protein cages derived from Aquifex aeolicus lumazine synthase (AaLS), called AaLS-neg and AaLS-13, which possess negatively supercharged interiors and spontaneously encapsulate positively charged cargo molecules. In contrast to the closed-shell structure of native AaLS, we have discovered that these cages adopt unprecedented expanded architectures with large keyhole-shaped pores in their shells, thus facilitating rapid and quantitative loading of a wide variety of guests. For example, GFP(+36), a positively supercharged variant of green fluorescent protein, is spontaneously loaded into empty AaLS-13 cages in quantitative yields at diffusion-limited rates simply by mixing guest and cage in aqueous solution. Exploiting such simple and efficient host-guest interactions, active enzymes can be loaded into AaLS-neg and AaLS-13 assemblies by genetic fusion to GFP(+36), which acts as a localization tag. The utility of the resulting nanoreactors has been illustrated by the templated synthesis of polymers and mimicry of the carboxysome, a natural bacterial microcompartment. Although we did not observe significant kinetic advantages for sequentially active enzymes loaded in the AaLS-13 cage, proteases encapsulated within AaLS-13 cleave positively charged peptides two orders of magnitude faster than negatively charged analogues, suggesting an alternative function for AaLS-13 cages as substrate sorters based on electrostatic interactions. This type of substrate sorting system could be a powerful strategy to regulate enzyme activity in multicomponent systems including intracellular environments, analogous to the action of natural housekeeping proteases like the proteasome.
In parallel with our work on negatively supercharged protein cages, we have created positively supercharged AaLS variants that bind negatively charged cargo, functionalized the surface of the protein shells to facilitate uptake by mammalian cells, and created circularly permuted constructs that make inside-outside genetic modification possible. In perhaps the most exciting application of such systems, we have engineered and subsequently evolved circularly permuted AaLS cages to encapsulate their own full-length RNA genome in vivo and protect the cargo molecules from nucleases. The bottom-up construction of virus-like nucleocapsids from nonviral materials has great potential to shed light on the assembly and evolution of natural virions while providing safe alternatives to natural viruses for diverse nano- and biotechnological applications. Indeed, we have already successfully applied the principles learned in these studies to create computationally designed protein cages that efficiently deliver siRNA and hydrophobic small molecules to cells.