Two HIV related proteins, TGF-beta1 and the HIV-1 protease, were studied by NMR spectroscopy. Studies were carried out to (1) elucidate the solution structure of TGF-beta1 and (2) determine the feasibility of an NMR structural determination of the HIV-1 protease in solution. (1) TGF-beta1 structure. TGF-beta1 up- and down-regulates proliferation of HIV infected cells. In order to understand the activity of this multifunctional cytokine, we have undertaken to determine the structure of TGF-beta1 in solution. We have sequentially assigned essentially all proton signals of the protein, and have measured several hundred NOEs. Using the NOE data we have determined the secondary structure of the protein in solution. The structure determined in solution is in agreement with an independently determined X-ray structure of TGF-beta2, except in a domain that distinguishes the activities of the beta1 and beta2 isoforms. (2) HIV-1 protease. Several months ago we obtained 2-dimensional NMR spectra of the HIV-1 protease complexed with an inhibitor. These spectra showed that the complex was stable for several weeks in solution, and had the hydrodynamic properties of a non-aggregating homodimer. Recently, our colleagues at DuPont-Merck have worked out conditions for increasing the solubility of the protease to a level of 1mM. This development opens the way to obtain 3- and 4-dimensional NMR spectra, from which detailed structural information can be obtained. The significance of this project arises from the unique, detailed structural information that is being obtained about HIV related proteins in solution. This information will form the basis for a rational drug design based upon the understanding of the function of these proteins in terms of interactions at the molecular level.
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