Characterising the interior and shell of recombinant encapsulin systems using structural biology and biochemistry

Abstract

Encapsulins are protein-based nanocompartments used by archaea and bacteria as a means of spatially controlling and organising the interior of the cell. Encapsulins can be classified based on their phylogenetic relationships and the functions of the cargo proteins that they encapsulate. Recent advances in cryo-electron microscopy and 3D single particle reconstructions have allowed for the visualisation of the dynamics and cargo loading of these nanocompartments. In this thesis I use cryo-EM to determine the structure of a family 1 encapsulin from the Gramnegative bacteria Rhodospirillum rubrum and show that the cargo loading is variable, with compartments possessing 0, 1, 3, or 5 encapsulated ferritin decamers. Work in this thesis demonstrates the impact of differential expression systems on cargo loading. Other features of the encapsulin shell are conserved, such as the flexibility of the five-fold pore on the encapsulin surface. The encapsulated ferritin enzyme found within the R. rubrum encapsulin which represents a distinct branch of the ferritin family. The mechanism and pathway for Fe2+ oxidation has recently become a point of contention, with different groups proposing different models. Using site directed mutagenesis of putative entry and exit sites I investigated the roles played by these sites using ferroxidase assays. The results presented in this thesis demonstrate that the conserved ferroxidase centre of the encapsulated ferritin is not the only site capable of iron oxidation. There is a surface site which is able to act in this capacity. Here I describe the structural characterisation of a newly discovered T=4 encapsulin from the Gram-positive methylotropic bacterium Bacillus methanolicus. This thesis focused on the recombinant production of the family 1 archaeal encapsulin systems for structural and biochemical characterisation. Greater understanding of these functional relationships between the encapsulin and their cargo proteins will aim to shed light on the apparent operon and structural paradoxes observed in these systems and their relevance to biotechnology and synthetic biology applications.