Aids Related NMR Research

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This project utilizes NMR spectroscopy to study the molecular components of HIV. We have prepared [methyl-13C]methionine labeled P51 subunit of the viral reverse transcriptase, and studied this system by NMR spectroscopy. We were able to observe five of the six methionine residues in this subunit, and are currently performing studies to assign these resonances. Of particular interest is a comparison of the structure of the YMDD motif between the active, P66 subunit and the inactive P51 subunit. We also have begun studies on the RNase H domain of the reverse transcriptase. Previous NMR studies have demonstrated that the isolated domain exhibits extensive mobility throughout its structure. We are currently studying the effects of magnesium ion on the internal mobility of the isolated domain. In addition to the studies of HIV reverse transcriptase and its domains, we have been using NMR spectroscopy to study Type II Dihydrofolate Reductase. Patients with a compromised immune system are more susceptible to adventitious bacterial infections, leading to increased dependence on antibiotic treatments. One mechanism of bacterial resistance to anti-folate drug therapy involves the expression of a plasmid type II dihydrofolate reductase, which is structurally unrelated to chromosomal DHFR and is resistant to bacterial antifolates such as trimethoprim, which target the chromosomal enzyme. We have been studying the interaction of the type II DHFR with ligands in order to understand the catalytic mechanism and to develop new inhibitors. Inter-ligand Overhaser effects connecting the pyridine nucleotide and folate protons were observed and have been used to define the structure of the ternary DHFR-NADP-folate complex. We also have prepared [U-13C,15N]-DHFR and are studying the interaction of the labeled enzyme with substrate and cofactor.