CONFORMATIONS OF LIGANDS AT ANTIBODY BINDING SITES

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This research will introduce tools offered by contemporary monoclonal antibody (Mab) technology into physical studies of ligand-macromolecular binding. The advantageous use of the large quantities of structurally homogeneous antibody binding sites available in the form of Fab fragments of monoclonal immunoglobulins forms the basis of the work. We plan a series of experiments which are aimed at elucidating, in submolecular detail, the binding of some biologically functional haptens to monoclonal antibodies raised against them and their structural analogs. The two haptenic systems chosen for initial study are the opiates and the chemotactic tripeptides. In preliminary work we have obtained 4 lines of monoclonal antibodies directed against pharmacologically important epitopes on morphine and have demonstrated their low dissociation constants from morphine and their differential cross-reactivities to opiate agonists and antagonists. One of our collaborators has done similar work in the chemotactic tripeptide system and this project has now been taken over into our laboratory. The physical methods of choice are high frequency nuclear magnetic resonance for solution studies and neutron diffraction for crystallized hapten-Fab complexes. In solution, using information from microscopic acidity constants, chemical shifts and proton-proton Nuclear Overhauser Effects for resonances from both haptens and immunoglobulin fragments we will form proximity maps connecting residues in immunoglobulin folds with atoms on the haptens. With the aid of specifically deuterated derivatives of the haptens we will determine their conformations at their own and cross-reacting binding sites using difference neutron diffraction performed on crystalline complexes. This work will be correlated with amino acid sequence analysis of the hypervariable regions of the light and heavy chains of the immunoglobulin fragments. Labeling, diffraction and sequencing work will each be done in collaboration with expert investigators. This work will differ significantly from previous work on macromolecular binding sites. We will be working with biologically functional ligands where the Mab tool allows us to examine the fine structures of the macromolecular binding sites in a detailed way. In addition, there is good evidence which is emerging in several systems, including the chemotactic peptide one, that antibody binding sites may be good models for the cellular receptor binding sites presented to haptens, such as we will be studying during their normal biological functions.