Doctor of Philosophy (PhD)
Chlamydia trachomatis (CT) is the most common, sexually transmitted bacterial disease (STD) in the world. In the developmental cycle of CT, specific chlamydia chaperone 4 (Scc4) is a unique protein with essential and multiple roles. Hence, Scc4 is significant as a virulence target for therapeutic approaches to treat chlamydial infections. A novel approach was discovered to purify tag free Scc4 by utilizing a 6X-histidine-tag on Scc1 in the co-expressed Scc4:Scc1 complex by capturing the complex on nickel-charged immobilized metal affinity chromatography resin, followed by dissociation of Scc4 with sarkosyl. Using triple resonance NMR experiments, backbone and sidechain resonances from 89% of the amino acids in Scc4 were assigned. With the assignments and the nuclear Overhauser effect (NOE) experiments, the dihedral angles, secondary structure, and the distance restraint data were determined and used to solve the 3D structure. X-ray crystallographic screening with the Scc4:Scc1 complex showed the optimum conditions to crystalize the protein complex with good diffraction to determine the structure of Scc1:Scc4 in the future. The experiments conducted with Scc4 with β flap showed that the interaction is weak. The NMR titration studies of Scc4 with the β flap tip helix (β-FTH) peptide showed a global structural change with 95% of the resonances shifting. To overcome the resonance overlap of the Scc4:Scc1 complex in NMR spectra, a novel technique was developed to produce the Scc4:Scc1 complex with chain selective labeling. The method was developed to observe NMR signals from one protein at a time by combining isotopically labeled and unlabeled proteins. This method is expected to lead to the 3D solution structure of the Scc4:Scc1 complex and the identification of the binding interface of the proteins. The comparison of free Scc4 and Scc4 bound to Scc1 showed that Scc4 undergoes a total conformational rearrangement to function as a T3SS chaperone protein. The molecular switching mechanism of Scc4 was investigated with metal ions and some small molecules, which showed some interactions between Scc4 and hemin chloride, nickel and ATP. Investigating these interactions with different methods produced conflicting results, such that conclusive evidence of these interactions requires further investigation.
Ukwaththage, Thilini Oshadhi Senarath, "Investigations of the Structure and Protein-Protein Interactions of Chlamydia trachomatis Scc4" (2021). LSU Doctoral Dissertations. 5492.
Macnaughtan, Megan A.
Available for download on Monday, March 11, 2024