Regarding the Stability and Permeability of Engineered Encapsulins

Abstract

Encapsulins are self-assembling protein nanocages that can be produced from a single gene. They have been studied and developed as a platform for engineering nanoreactors. This thesis explores the role of encapsulin pores in controlling chemical flux into encapsulins and how protein fusions affect encapsulin assembly. In Chapter 2, a molecular dynamics simulation was created to study the effects of encapsulin pore size and charge on ion flux through the pore. The Thermotoga maritima encapsulin and eight mutants were simulated and the ion pore flux was examined. It was found that Cl- ions flow in greater amounts through larger, more positively charged pores due to charge complementarity. In Chapter 3, new assays for measuring encapsulin pore flux were explored. Attempts were made to load fluorescent reporter proteins SNAP-tag and pFAST into encapsulins and assay them against their corresponding fluorophore reporter molecules. Both protein systems showed promise as reporters for encapsulin pore flux, and further development of the assays is required to determine their reliability. In Chapter 4, SUMO was fused to the Quasibacillus termotolerans encapsulin monomer and the resultant protein fusion was characterised. Its suitability for in vitro cargo loading was determined and was probed for dynamic behaviour. The encapsulin mutant was found to form assemblies that would not allow for cargo loading, and microscopy assays to determine dynamic behaviour were inconclusive. The computational and in vitro experiments in this thesis lay groundwork for our understanding of encapsulin pore flux and its dependence on the pore chemistry. The encapsulin mutants studied herein uncover the remarkable stability of encapsulin assemblies.