How Pore Structure Affects the Stability and Function of Protein Cages

Abstract

Protein cages are ubiquitous organelles in biology that are involved in a wide range of biochemical processes such as free radical detoxification, anaerobic ammonium oxidation, and carbon fixation. Protein cages have garnered interest from biologists who wish to understand the functional role of cages in native organisms, and from engineers who wish to engineer cages to perform novel functions. Encapsulins are a class of protein cage organelles that are found throughout bacteria and archaea. The encapsulin system consists of a cage that is self-assembled from multiple copies of a single protein, and one or more types of cargo proteins that are tagged for encapsulation through a sequence-specific interaction between the tag and the cage protein. This thesis focuses on the encapsulin from Thermotoga maritima, with the overall aim of understanding how altering the size and charge of the encapsulin cage pores affects biochemical reactions occurring inside the cage. The effects of the pore structure on molecular flux through the cage and how the resulting changes affect the kinetics of encapsulated enzyme reaction are studied. The work presented in this thesis contributes to our understanding of how protein cages function, and will inform future efforts to engineer new functions into protein cages. The encapsulin pore mutant library presented in this work represents the most thorough examination of the mutability of an encapsulin cage to date. The structures obtained of the pore mutants provide insight into the structural basis of the size and charge of the encapsulin pore. Through a series of computational and experimental studies, it is revealed that the permeability of the encapsulin pore to ions and small molecules is dependent on both the size and charge of the pore. The work described in this thesis forms part of a growing body of research dedicated to understanding how protein cages are used in biology to improve the function of enzymes.