Type of Document Dissertation Author Nguyen, Hau T.B. Author's Email Address HAU@MAGNET.FSU.EDU URN etd-04112008-112635 Title Structural Characterization of RV0008C – An Integral Membrane Protein from Mycobacterium Tuberculosis by Solution Nuclear Magnetic Resonance Spectroscopy Degree Doctor of Philosophy Department Molecular Biophysics, Institute of Advisory Committee
Advisor Name Title TIMOTHY A. CROSS Committee Chair P. BRYANT CHASE Committee Member PENNY GILMER Committee Member SAMUEL GRANT Committee Member TIMOTHY M. LOGAN Committee Member Keywords
- Membrane Protein
- Mycobacterium Tuberculosis
- Backbone Structure
- Functional Genomics
- Structural Genomics
- Cell Division
- Solution Nmr
Date of Defense 2008-04-09 Availability unrestricted AbstractTuberculosis, an infectious disease caused by Mycobacterium tuberculosis, has been a leading cause of death in the world infecting over one third of the world population (WHO 1999). A detailed structural understanding of the membrane proteins and cell envelope of M. tuberculosis will help unravel the mechanism through which bacteria infect and survive within human macrophages, and thus may help to facilitate the development of new treatments for tuberculosis. In fact, it is anticipated that more than 50% of all drug targets are membrane proteins. However, there is very little structural information on these proteins.
Our goal for this project is to characterize the three-dimensional backbone structure of Rv0008c, an integral membrane protein from M. tuberculosis by solution Nuclear Magnetic Resonance (NMR) spectroscopy. Rv0008c is a small, conserved membrane protein containing 145 residues and one transmembrane helix. It is in a gene cluster that we have shown to form a complex with Rv0011c - also known as a homologue of CrgA, an FtsZ inhibitor - a critical component of the cell division machinery. A detailed three-dimensional structure of this protein will help to understand its critical role in cell division process and therefore this will help to suggest a possible cell division control mechanism in M. tuberculosis. Controlling cell division is important since M. tuberculosis goes into a “latent” state where no cell division occurs and none of the current drugs are effective against this state. If M. tuberculosis can be prevented from entering the “latent” state, the drug regimen can be shortened dramatically.
Backbone assignments for Rv0008c were made using 2D and 3D- NMR experiments on uniformly 13C/15N–labeled Rv0008c protein in DPC (dodecyl phosphocholine) micelles. Dihedral angle restraints were derived from chemical shifts using TALOS (Torsion Angle Likelihood Obtained from Shifts and sequence similarity) program. Two sets of residual dipolar couplings were measured for proteins weakly aligned in compressed neutral and positive charged gels. Long-range paramagnetic relaxation enhancement (PRE) distances were obtained for protein samples at six spin label sites. NOE (nuclear Overhauser effect) distance restraints were also obtained to improve the quality of the structure. The three-dimensional backbone structure of Rv0008c was calculated and refined by the Xplor-NIH program using the dihedral angle, residual dipolar couplings, PRE and NOE distance restraints.
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