Improved understanding of the structure of HIV proteins can be useful in unraveling the function of the proteins and in understanding the mechanisms of the function of these proteins. This structural knowledge can also be useful in designing novel anti-HIV agents. X-Ray crystallography is the most accurate technique for determination of protein structures. Major drawbacks of the technique, however, are that it can be very time consuming and is dependent on the availability of protein crystals. Molecular modeling programs, on the other hand, can provide insights into the protein structure based on, amongst other things, homology with proteins whose structures are known. Although this approach is attractive, the results have often been less reliable than desired because insufficient information about the protein is available or the degree of homology with proteins of known structure is less than needed for the development of an accurate model. If information such as which amino acid residues are on the surface of the protein is available, however, the quality of the computer generated model can be significantly increased. This project is designed to probe the tertiary structure of HIV proteins using a combination of chemical modifications, enzymatic degradations and mass spectrometric identification. Recombinant HIV p24 was chosen as the first protein to examine. We decided to concentrate our initial efforts in this area on commercially available truncated p24, once the structure of this material was determined. (The molecular weight of the material did not match the catalog sequence. It was found that 11 of 12 amino acids in a leader sequence had been lost.) The first chemical reaction we used to probe the surface of the protein was glycinamidation of aspartic and glutamic acid residues. This reaction was carried out under non-denaturing conditions for one hour at the picomole level. The short reaction time was used to ensure that only partial reaction (most exposed residues) would occur. After the reaction was stopped, the resulting modified protein was digested with chymotrypsin. MALDI/MS analysis showed acidic residues at positions 188, 94, 172, 111, 165, and 176 were modified. A preliminary molecular structure was modeled using inverse folding algorithms so identify structure possessing folds compatible with p24. The identified folds, those belonging to the structure of the cytokine proteins interleukin 4 and interleukin 5, were used as a basis and point of departure for developing a model of the HIV p24 gag protein which also incorporates the surface residues identified in this project and the epitope determined in our epitope mapping project.
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