HIV protease derived from recombinant sources will be subjected to crystallization studies aimed at producing crystals suitable for a three-dimensional structure determination by X-ray crystallographic methods. A wide variety of crystallization protocols will be employed. Particular attention will be paid to the possible effects of pH, temperature, ionic strength and composition on the structure and activity of the protease. Quaternary structure will be monitored by analytical ultracentrifugation and fluorescence methods and correlated with enzyme activity which will be assayed. Secondary structure will be estimated using circular dichroism and structure prediction methods. A structural model of HIV protease will be constructed based upon secondary structure estimates and limited sequence homology with aspartic proteinases. The model will be used to make predictions for site-directed mutagenesis, inhibitor design and may also be useful for molecular replacement phasing. Protease inhibitors will be soaked into crystals of native protease for structure determination using difference Fourier methods. In addition, the protease will be co-crystallized with selected inhibitors in parallel with the protease crystallization experiments. The crystal structure determination of a protease- inhibitor complex will be facilitated by incorporating heavy atoms at specific sites in the inhibitor structures. The structures of native and inhibited HIV protease will be refined and used to design potent and specific inhibitors of HIV protease. These studies will be extended to other HIV protease sequence variants and mutants as well as to HIV-2 in order to understand the structural basis for any sequence dependent variations in inhibitor binding. Eventually the insights gained in these studies may lead to the design of antiviral agents against viruses other than HIV which require a virally encoded proteolytic function during their life cycle.
