Currently there is great interest in using site-directed mutagenesis to study structure-function relationships in proteins. Indeed, the ability to manipulate protein structures by substitution of a specific amino acid(s) has spurred a revival in interest in protein-protein and protein-ligand interactions including studies on the molecular bases of enzyme activity, protein folding, receptor-ligand interaction and in antigen- antibody interaction. Quite recently, the crystal structure of several antigen-antibody (Ag-Ab) complexes have been solved at resolutions that should permit detailed analysis of specific amino acids (on Ag or Ab) in determination of specificity and affinity. Three of these crystal structures involve complexes of monoclonal antibody Fab fragments and the antigen- Hen Egg-white Lysozyme (HEL). The overall goal of this project is to combine the power of site- directed mutagenesis with immunochemical, chemical, physical and, in certain select cases, crystallographic analysis to provide a more complete knowledge of antigen-antibody interactions in particular and protein-protein interactions in general. Two different, yet complementary systems for which cloned expression systems are on hand, will be used: a) Hen Egg White Lysozyme -- a disulfide-bonded, relatively rigid molecule--for which the crystal structure of complexes with three different antibodies have been determined and, b) Staphylococcal nuclease -- a non-disulfide- bonded and presumably more flexible molecule -- for which a large panel of monoclonal antibodies are on hand. Specific questions to be considered include: 1) doe antigen- antibody interaction result in significant conformational changes in antigen and/or antibody?; 2) what structural constraints does an inflexible antigen impose on antigen-antibody union?; 3) what is the relative contribution of given amino acids in antigen to the overall binding energy. Few diseases are without an immunological component including cancer, autoimmune disorders, infectious diseases, etc. The ability to manipulate the immune system for the benefit of patients with such diseases may require the ability to manipulate the structure of the antigen, antibody, or lymphocyte receptor. Such manipulations may permit the shifting of the response in the direction of more effective immunity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020745-06
Application #
3130558
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1984-09-30
Project End
1993-06-30
Budget Start
1991-07-01
Budget End
1993-06-30
Support Year
6
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Benjamin, D C (1995) B-cell epitopes: fact and fiction. Adv Exp Med Biol 386:95-108
Huczko, E L; Bodnar, W M; Benjamin, D et al. (1993) Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling. J Immunol 151:2572-87
Sosnick, T R; Benjamin, D C; Novotny, J et al. (1992) Distances between the antigen-binding sites of three murine antibody subclasses measured using neutron and X-ray scattering. Biochemistry 31:1779-86
Benjamin, D C; Williams Jr, D C; Smith-Gill, S J et al. (1992) Long-range changes in a protein antigen due to antigen-antibody interaction. Biochemistry 31:9539-45
Benjamin, D C (1991) Molecular approaches to the study of B cell epitopes. Int Rev Immunol 7:149-64
Smith, A M; Woodward, M P; Hershey, C W et al. (1991) The antigenic surface of staphylococcal nuclease. I. Mapping epitopes by site-directed mutagenesis. J Immunol 146:1254-8
Smith, A M; Benjamin, D C (1991) The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis. J Immunol 146:1259-64