| Description |
Most proteins assemble into oligomeric complexes. These supramolecular associations may confer many advantages to the substituents. Polyhedral capsids represent a common, highly symmetric nanoscale architecture in which multiple subunits self-assemble to form a hollow three-dimensional surface which often serve as molecular containers or platforms for multivalent display of ligands. Capsids can be tailored to serve in applications such as drug delivery, biocatalysis, and materials synthesis. In this dissertation, I present a body of work undertaken on the Aquifex aeolicus lumazine synthase (AaLS) capsid to expand our knowledge of supramolecular protein associations and to generate capsids with novel functions. First, the construction and characterization of a novel nanoreactor is described. Using a previously established tagging system, an esterase was encapsulated in a laboratory-evolved variant of AaLS. Characterization of the purified complex shows an average loading of two esterases per capsid and an approximately 20-fold decrease in efficiency compared to the free esterase. This decrease is larger than most of the previously reported capsid-based nanoreactor systems which suggests that both the confinement molarity and the electrostatic environment of the capsid interior may significantly influence the kinetic parameters of guest enzymes. Second, I utilize charge complementarity to decorate the exterior of an AaLS capsid variant with green fluorescent protein (GFP). A new interface was engineered by negatively supercharging the five-fold symmetric capsid pores and appending a deca-arginine tag to the C-terminus of GFP. This interaction requires the engineered features of both binding partners and shows steep dependence on the buffer ionic strength, although it retains high affinity at physiological ionic strength. Thus, charge complementarity can provide a simple, powerful, and general method for designing protein associations de novo. Finally, I expand upon previous work in which a redox switch was developed to control capsid assembly. The original switch relies on the formation of a disulfide-bonded adduct between a pentameric variant of AaLS and thiophenol. I explore alterations to the prosthetic group structure which reveal that the three-fold symmetric interface of the assembled capsid is highly plastic and can tolerate a range of different adduct sizes and shapes. These studies also identified two new disassembly switches, providing greater control over the supramolecular chemistry of the AaLS capsid. |
