|Keywords:||Chemistry; Biochemistry; Biology; chemistry; mass spectrometry; biochemistry; protein dynamics; type IV pilins; protein nanotubes; bacterial conjugation; time-resolved electrospray ionization; hydrogen-deuterium exchange|
|Full text PDF:||http://hdl.handle.net/10315/30744|
Persistent infections by Pseudomonas aeruginosa are initiated by interaction of a type IV pilus (T4P) with receptors on the mucosal cells of susceptible hosts. Here, we examine the structural changes occurring between the monomeric and dimeric states of ΔK122 using time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry (TRESI-HDX-MS). Based on levels of deuterium uptake, the N-terminal α-helix and the loop connecting the second and third strands of the anti-parallel β-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel β-sheet and αβ loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state. Additionally, Escherichia coli are able to adapt to changing environmental conditions and develop antibiotic resistance through a process called F-plasmid conjugation, carried out through a type IV secretion system (T4SS). The F-T4SS protein TraF is of particular interest due to its involvement in pilus assembly to mediate the transfer of DNA. Dynamic analysis of a GST-TraF construct through TRESI-HDX-MS was performed to gain further insights on its structure. These studies have revealed that the C-terminal region predicted to contain the thioredoxin-like domain is quite structured compared to the more solvent accessible N-terminal region predicted to form a protein-protein interaction with companion T4SS protein TraH. Structural analysis of a GST-TraF construct is on-going to further characterize the regions responsible for protein-protein interaction and the elucidation of its three-dimensional structure. Advisors/Committee Members: Audette, Gerald F..