of Work: 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 primary and tertiary structures of HIV proteins using a combination of chemical modifications, enzymatic degradations and mass spectrometric identification. In using commercially available sources of recombinant HIV proteins, we rapidly became aware of discrepancies between the catalog structures and the actual molecular weights of the proteins. We have developed a technique, direct analysis of affinity-bound analytes, and applied it to recombinant His-tagged proteins that are affinity bound to immobilized metal ion affinity columns to determine the actual sequence of two of these HIV proteins, rp24 and rvif. We are continuing to probe the tertiary structure of recombinant HIV. Although portions of the molecule have been crystallized and the structure solved, the manner in which the two domains are configured together is not known. We are proceeding with acetylation of lysine residues on the intact native protein. Relative reactivities of five lysines have been determined, (positions 70, 131, 140, 170 and 158) while the relative reactivities of six other lysines have not been determined as yet. At this point the relative reactivities do not correlate exactly with the solution structures of the constituent domains. This may be due to the microenvironment around the lysine residues or may be due to the manner in which the two domains are connected in the intact molecule. With the present MS/MS capabilities, the acetylation reactions will be repeated and the reactivities of all lysines will be elucidated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES050150-03
Application #
6106724
Study Section
Special Emphasis Panel (LSB)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
1998
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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