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.
