In part 1 of this thesis, we discuss the characterization of a novel class of proteins belonging to the domain of unknown function (DUF) 3365 family. Bioinformatic analyses revealed that a subset of this family that contains heme c as a cofactor could be classified into two clusters based on properties predicted from genomic analysis. Proteins belonging to cluster 1 were predicted to have non-signal transduction or redox functions, whereas those belonging to cluster 2 were predicted to perform signal transduction. To better understand the properties and behaviors that may distinguish the two clusters, we selected one protein from each cluster (P. putida protein from cluster 1, and P. azotoformans receptor from cluster 2) and extensively characterized them in terms of their structures, reactivities, and responses to diatomic gas molecules. We discovered that the two proteins show striking differences in their response to O₂ and NO, and concluded that the P. putida protein perhaps behaves as a redox sensor within the cell. The P. azotoformans receptor showed similar spectroscopic and kinetic properties to NO sensors, which is unusual for a periplasmic protein and may have implications in terms of mediating a faster response to the environment. We further investigated this receptor as it has a unique architecture containing an isolated cytoplasmic HAMP domain, but no additional signaling domain normally associated with HAMP domains. We discovered that the receptor interacts with nanomolar affinity to its cognate Ser/Thr kinase through biochemical methods and cryo electron microscopy. We have also shown that NO binding to the receptor influences kinase activity. This work forms the basis for subsequent in vitro and bacterial studies on the role that this subset of DUF 3365 proteins plays in terms of NO binding and signal transmission.

In part 2, we present the development of an ELISA-based high-throughput screen to identify inhibitors of lysinoalanine (Lal) crosslinking in the hook protein FlgE of spirochetes. Spirochetes are defined by their unique morphology and rotational motility that is mediated by axial periplasmic flagella (PFs) that allow them to invade and colonize host tissues efficiently. Motility is essential for the virulence of several spirochete pathogens. Recently, we have shown that FlgE from the oral pathogen Treponema denticola catalyzes a unique inter-subunit post-translational protein cross-linkage in the form of lysinoalanine (Lal). Lal crosslinking within spirochete hooks is essential for spirochete motility and therefore pathogenesis. Interestingly, prevention of Lal crosslinking through mutation or inhibition with small molecules impairs motility in spirochetes. Lal formation thus represents a potential target for novel drug discovery to treat spirochaete-related diseases.

We have screened a small library ~800 compounds using the ELISA. The ELISA reconfirmed that hexachlorophene, a compound that was identified previously using a luciferase-based screen in our lab, is a potent inhibitor of Lal crosslinking. We have also identified two new compounds, honokiol and zafirlukast as potential inhibitors. The assay also detected several activator molecules, which surprisingly belong to some well-known commonly used families of antibiotics. Finally, our work demonstrates the utility of an ELISA-based sandwich assay for high-throughput monitoring of protein-protein interactions.