Challenges in Characterizing Membrane Proteins and Intrinsically Disordered Regions Involved in Mycobacterium Tuberculosis Cell Division

Structural biology has been successful in the characterization of soluble protein, which has given insight in the inner working of several thousands of proteins. However, structural characterization of proteins has been limited in two areas. One area corresponds to the rise on interest on intrinsically disorder proteins. This kind of proteins has changed the structure-function paradigm since they can perform important cellular function without a defined three dimensional structure. The second are is the characterization of membrane proteins, which contrary to soluble proteins, they are embedded in an anisotropic environment that makes them difficult to characterize. This dissertation work was focused in the characterization of two important membrane proteins involved in cell division of Mycobacterium tuberculosis, ChiZ and FtsX. The first part of this work is related to the characterization of ChiZ, a cell division protein possibly involved in peptidoglycan remodeling and control of cell division progression. Structural characterization of ChiZ was mainly focused on the N-terminal soluble region that is intrinsically disordered. This region was previously identified to have peptidoglycan hydrolysis activity. Thus, a combination of solution and solid state NMR to characterize the structure of ChiZ N-terminal region in solution and in the full length protein reconstituted in liposomes. In addition to solution NMR, ChiZ N-terminal intrinsically disordered region was characterized with other method including circular dichroism, dynamic light scattering, size exclusion chromatography and small angle X-ray scattering. Thus, the objective was to obtain a clear picture of the behavior of the intrinsically disordered region in solution. Furthermore, characterization of ChiZ peptidoglycan hydrolysis activity was attempted with the goal of correlating structural properties of the N-terminal region and the protein function. The second part of this study focuses on the challenges of working with membrane proteins. The subject of study was FtsX, a membrane protein is one of the components of the cell division machinery or divisome. This part of the work attempts to give a general guideline for optimizing membrane protein purification and reconstitution with large emphasis on detergent selection. Detergent selection can affect each step of FtsX work, from protein purification to membrane protein reconstitution and sample preparation for solid state NMR experiments.