Structural and Functional Studies of TraG From the F Plasmid and the Major Pilin From Coxiella Burnetii
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The Escherichia coli F-plasmid houses all genes necessary for the assembly of its conjugative machinery to facilitate successful DNA transfer. TraG is one of the largest proteins (102 kDa) encoded in the transfer (tra) region of the F-plasmid that facilitates conjugative transfer. The N-terminal region of TraG is involved in pilus synthesis and assembly, while the C-terminal periplasmic domain is required for plasmid-specific entry exclusion and mating pair stabilization. In order to provide structural insights into TraGs function, we have expressed, purified and initiated crystallization studies of the C-terminal region of TraG (designated TraG*, 52.7 kDa) and have obtained initial crystals of TraG*. Our experiments on functional and dynamic characterization of TraG* using liquid chromatography coupled mass spectrometry (LC-MS), size exclusion chromatography coupled multi-angle light scattering (SEC-MALS) and protein gel electrophoresis has given significant insight into TraG* crystallization strategies as well as potential TraG mechanism of action during conjugation.