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Tuberculosis and Rational Drug Design
Fatty Acid Bioysnthesis
We are studying the mechanism of the enoyl-reductase (ENR) enzymes from Mycobacterium tuberculosis (InhA) and Escherichia coli (FabI). The ENR >enzymes are targets for the anti-TB drug isoniazid and the antibacterial compound triclosan. Triclosan, previously thought to be a non-specific biocide, is an uncompetitive inhibitor of both FabI (Ki 3.8 pM) and InhA (Ki 0.2 µM). The X-ray structure of triclosan bound to InhA is being used as a starting point for inhibitor-design using computational methods. SAR studies are being performed using compounds based on the triclosan diphenyl ether skeleton. Recent studies have focused on the inhibition of InhA with the adduct formed from the reaction of NAD(H) and isoniazid. Significantly, the adduct binds with equal affinity to InhA mutants identified in clinically-resistant strains of M. tuberculosis and we are investigating whether InhA inhibition is modulated by the protein's oligomerization and/or protein-protein interactions with other FASII enzymes.
Menaquinone Biosynthesis
Menaquinone (vitamin K) is the sole quinone in M. tuberculosis and, thus, menaquinone biosynthesis is an attractive target for anti-TB drug discovery. We are cloning and expressing all the putative TB men enzymes and have recently solved the structure of MenB, the dihydroxynapthoyl-CoA synthetase. Gene knockouts are being used to probe the importance of the men genes in bacterial survival and pathogenesis
FtsZ and Cell Division
FtsZ is a bacterial tubulin homolog that polymerizes and depolymerizes during its normal biological function. Inhibitors of FtsZ (de)polymerization have antibacterial activity and we are using biophysical methods to study this process and to drive the development of novel FtsZ inhibitors.
Persistence of M. tuberculosis in Macrophages
We are probing the molecular basis for the ability of the TB bacillus to survive and replicate inside macrophages, cells normally tasked with destroying invading bacteria. Currently we are studying the role of the actin-binding protein coronin in phagosome maintenance using pull-down experiments to identify coronin-binding partners and RNA interference methods. .
Probing Electrostatic Forces in the Active Site
Raman, FTIR and NMR spectroscopy are being used in concert with site-directed mutagenesis, enzyme kinetics and X-ray crystallography to investigate the mechanism of the reactions catalyzed by three enzymes involved in fatty acid oxidation: enoyl-CoA hydratase, acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase. These methods probe alterations in the electronic structure of the substrate and substrate analogs that occur upon binding to the enzyme. Site-directed mutagenesis is then used to modify the protein and spectroscopic and kinetic characterization of the mutant enzymes enable structure-reactivity correlations to be generated. A specific aim of this project is to understand how enzymes cause and stabilize charge rearrangement.
Current studies are focusing on how ligand binding modulates the flavin redox potential and substrate pKa in acyl-CoA dehydrogenases. We are using Raman spectroscopy and direct detection 13C as well as two-dimensional HSQC NMR experiments to probe changes in the electronic structure of ligands caused by the enzyme's active site.
 Green and Red Fluorescent Protein
Green fluorescent proteins (GFPs) are intrinsically fluorescent and have a wide range of applications in molecular and cell biology. Importantly, it has been shown that light absorption causes structural changes in the GFP chromophore that result in alterations in the chromophore's fluorescence. In order that changes in fluorescence not be misinterpreted, it is important to determine the changes in chromophore structure resulting from irradiation and to analyze the spectroscopic properties of the different chromophore structures. Consequently, a complete understanding of the photophysical properties of the chromophore is fundamental to GFP applications. We are using a variety of spectroscopic methods coupled with site directed mutagenesis to determine how the protein environment controls the spectroscopic properties of the chromophore. In addition, we are constructing an expression system to introduce isotopic labels and unnatural amino acids into the GFP chromophore to study the mechanism of chromophore formation. These experiments will result in novel GFPs with faster rates of chromophore formation. |
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