Aids Related Nmr Research

Environmental Health Sciences

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Abstract

This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. During the past year, we synthesized U-[15N] and U-[13C,15N] RNase H domain of HIV-1 reverse transcriptase (RT). The isolated 15 kD domain has previously been studied by X-ray crystallography, however no solution structure and no inhibitor complexes have been reported. We have now assigned the resonances and determined the solution structure which is in general close to the previously reported crystal structure. However, the C-terminal helix which contains one of the active site aspartyl residues is disordered in solution. This result is somewhat surprising, since there are no interactions in the full RT enzyme which would be expected to lead to stabilization of this region of the protein. Thus, any conformational stabilization which occurs must result from substrate binding. We have found that Mg2+ ions and nucleosides such as AMP significantly affect the NMR spectrum of the enzyme. Titration with Mg2+ ions indicate the presence of two binding sites, with dissociation constants of ~ 3 mM and 35 mM. In order to better understand the nuclease inhibition process, we have recently worked on a model system - the NuiA inhibitor of Nuclease A from Anabaena sp. We have determined the structure of NuiA and are currently studying the NuiA-NucA complex. We have continued to study the P51 subunit of RT using [methyl-13C]methionine. Problems with this system led us to evaluate [methyl-13C]methionine polymerase beta as a model system. Due to difficulties encountered with this system, we have recently worked on beta polymerase as a model for the reverse transcriptase. The protein was similarly labeled with [methyl-13C]methionine, and we are currently making methionine -> valine mutants in order to assign these resonances.