Amyloid formation is typically associated with human neurodegenerative diseases by caus- ing aggregation-induced cytotoxicity in brain cells. However, non-pathological functional amyloid fibres are also produced in a wide range of organisms to serve for their advantages, including the formation of extracellular matrix for biofilm construction.
Curli fimbriae of Escherichia coli was the first bacterial functional amyloid identified. This secretion system is closely coordinated under the regulation of two divergently transcribed operons, which enables curli amyloid to be secreted extracellularly with minimal cytotoxicity. More recently, Functional Amyloid protein system in the Pseudomonas genus (Fap) was identified, in which Fap proteins are expressed from a single operon, fapABCDEF. Unlike the well-studied curli system, very limited information is known about the Fap system.
The work in this thesis seeks to characterise a range of proteins within the Fap operon, with particular focus on the periplasmic C39 peptidase FapD, its complex with the outer membrane transporter FapF (FapD-FapF), and the natively secreted Fap amyloid. FapD was predicted to be proteolytic and act as a chaperone within the Fap system. Different mutants of FapD were tested using secretion assay to assess their effects on FapC secretion. FapD C-terminal helix was also studied using coarse-grained molecular dynamics, NMR, and CD, in which a weak interaction with the membrane was revealed. Previous reports also demonstrated the co-evolution of FapF and FapD, and confirmed the existence of this complex using various methods. In continuation of this work, the complex was purified and optimised here for structural analysis using cryo-EM. Furthermore, natively secreted Fap fibres were extracted, purified and optimised for structural analysis by cryo-EM. Although the low-resolution reconstructed maps were insufficient for model fitting, they provided new insight into how native Fap fibres are formed. Understanding how these Fap proteins coordinate in the cell and how Fap fibres are assembled extracellularly will be key to aiding future research in the wider scope of functional amyloid secretion systems, and ultimately enabling us to control amyloid formation.