| Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which killed over 1.4 million people globally in 2010. The Mtb H37Rv genome contains approximately 4047 predicted genes, including two large families. The first is the toxin-antitoxin (TA) family, which is estimated to have 88 members, and the second is the PE/PPE family, which is estimated to have 168 members.;Within the TA family, the largest subfamily is the VapBC family, which is estimated to have 45 members. The first chapter details a 1.49 Å structure of the R.v0301-Rv0300 complex, a VapBC TA complex. The high resolution structure provides a wealth of data that explains probable mechanisms of action, including a coordinated Mg2+ ion at the putative ribonucleatic active site, and a possible RNA binding site. The structure also reveals a tryptophan of the antitoxin inserted into a cleft that may be a site for purines, and an arginine in the Mg2+-binding pocket.;The second chapter details an assay to test whether two proteins form a stable complex. The assay was developed from a split GFP system, and was successfully implemented to test a dozen PE/PPE pairs. Two of the dozen were conclusively determined to form complexes.;The third chapter details a spin-off of the split-GFP assay described in chapter 2. Based on an observation that a poorly expressing protein complex demonstrated enhanced soluble expression, I developed a system of vectors to enhance soluble expression of protein complexes, and I used the system to solve the structure of Rv1959c-Rv1960c, a ParDE TA complex. |
