Structural Proteomics of Myobacterium Tuberculosis
EMSL Project ID
2445a
Abstract
Project Description: Due to the availability of the complete genome of M. tuberculosis and also in part to our development of a genetic system for studying gene function in M. tuberculosis, our understanding of M. tuberculosis is advancing rapidly and will allow us to accurately target proteins based on their importance in virulence, persistence, reactivation, and overall viability of the organism. Targeting of functionally important proteins:We are emphasizing structure determination of proteins known to be functionally important for pathenogenicity, although the precise function will in many cases not be known at the time structure determination is begun. It is our intent that the structures we determine will be useful not only as contributions to a database of folds and functions, but also as a basis for an in-depth understanding of biological processes. We view this targeting strategy as a key to maximizing the scientific returns from this structural genomics project. Accordingly, we are targeting based on known function, genetic screens, informatics and interest from the M. tuberculosis research community.We have developed methods for engineering proteins to make them highly soluble and expressed at high levels in E. coli and in vitro (Waldo et al., 1999). This technology will allow us to develop a standardized process to produce and purify most proteins in large quantity. We use extensive crystallization screening to maximize the yield of crystals and then use NMR to determine some of the structures of proteins that do not crystallize.NMR work:High through-put methods for engineering proteins with high solubility and expression allows for the ready determination of 3D structures by solution NMR spectroscopy. We are requesting 6 weeks of 600 MHz NMR time in order to determine the structures of proteins from M. tuberculosis that were not able to be crystallized. The first protein, coded by gene Rv3462c, is the likely homologue of E. coli translation initiation factor IF1. The structure of this protein will contribute to a better understanding of protein synthesis in mycobacterium. Two additional proteins, Rv2708c and Rv3321, are conserved hypothetical proteins. The former is predicted to be a novel fold. These proteins have many orthologues, but no sequence similarity to proteins with known 3D structure.Progress Summary: We are now taking advantage of high-throughput methods of protein expression and purification to determine good targets for NMR. We now have clones for three small (<10 kDa) M. tuberculosis protein targets which are well-expressed, highly soluble, and not able to be crystallized. This was also the method that suggested that DsrC from the hyperthermophilic archaebacterium Pyrobaculum aerophilum was a good candidate for NMR studies.Using EMSL NMR time granted for the prior period, we have completed acquisition of a complete set of spectra for Allochromatium vinosum DsrC. As expected, the data are of high quality and will certainly result in a high resolution structure. To date, the backbone resonances have been completely assigned. Assignment of side chain resonances is in progress. When the structure is complete, the structure of this mesophilic protein can be compared with our recently published structure of its thermophilic homolog from Pyrobaculum aerophilum (Cort et al., 2001).References:Waldo, G.S., Standish, B.M., Berendzen, J. & Terwilliger, T.C. (1999) Rapid protein-folding assay using green fluorescent protein. Nature Biotechnology 17, 691-695.Cort J.R., Mariappan S.V., Kim C.Y., Park M.S., Peat T.S., Waldo G.S., Terwilliger T.C., Kennedy M.A. (2001) Solution structure of Pyrobaculum aerophilum DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase. Eur. J. Biochem. 268(22), 5842-5850.
Project Details
Project type
Capability Research
Start Date
2004-09-22
End Date
2005-12-12
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members