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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES050150-01
Application #
2452861
Study Section
Special Emphasis Panel (LMB)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1996
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Dhungana, Suraj; Williams, Jason G; Fessler, Michael B et al. (2009) Epitope mapping by proteolysis of antigen-antibody complexes. Methods Mol Biol 524:87-101
Robinette, David; Neamati, Nouri; Tomer, Kenneth B et al. (2006) Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics. Expert Rev Proteomics 3:399-408
Williams, Jason G; Tomer, Kenneth B; Hioe, Catarina E et al. (2006) The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry. J Am Soc Mass Spectrom 17:1560-9
Hager-Braun, Christine; Katinger, Hermann; Tomer, Kenneth B (2006) The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen. J Immunol 176:7471-81
Hager-Braun, Christine; Tomer, Kenneth B (2005) Determination of protein-derived epitopes by mass spectrometry. Expert Rev Proteomics 2:745-56
Hager-Braun, Christine; Tomer, Kenneth B (2004) Determination of epitopes by mass spectrometry. Methods Mol Med 94:109-20
Cutalo, Jenny M; Deterding, Leesa J; Tomer, Kenneth B (2004) Characterization of glycopeptides from HIV-I(SF2) gp120 by liquid chromatography mass spectrometry. J Am Soc Mass Spectrom 15:1545-55
Hager-Braun, Christine; Tomer, Kenneth B (2002) Characterization of the tertiary structure of soluble CD4 bound to glycosylated full-length HIVgp120 by chemical modification of arginine residues and mass spectrometric analysis. Biochemistry 41:1759-66
Parker, C E; Tomer, K B (2000) Epitope mapping by a combination of epitope excision and MALDI-MS. Methods Mol Biol 146:185-201
Hochleitner, E O; Borchers, C; Parker, C et al. (2000) Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis. Protein Sci 9:487-96

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