The members of our library of anti-acetylcholine receptor monoclonal antibodies (mAbs) are unique in that their binding inhibits either agonists binding, antagonist binding, or channel opening. Thus the define a set of epitopes on the acetylcholine receptor (AcChR) molecule, each of which is important for the function of the AcChR. We propose to map their epitopes onto the primary structures of the AcChR subunits. Once the epitopes are precisely mapped, we will use the individual mAbs for probing, in a site-specific manner, the structure and function of the AcChR. To provide this primary structure analysis of the epitopes, we will determine, where possible, the particular protease-generated AcChR peptide that contains the epitope. The oligosaccharide contribution to the epitope will also be analyzed. However, the major thrust of this Project is to synthesize rationally designed oligopeptides from the primary sequence of the AcChR subunits. The peptides will be tested for binding to the mAbs in our present anti-AcChR library and will also be used as immunogens (in the Scientific Core) to produce new anti-peptide mAbs that crossreact with native AcChR. These peptides will be chosen to mimic both functionally important and structurally important sites on the AcChR. The mAbs raised against them will be analyzed for their effects on AcChR function in Projects 1-4. The epitopes they represent will also be localized in the three-dimensional model of AcChR by small angle X-ray diffraction and electron microscopic analysis of the mAbs bound to AcChR. The latter information will provide a mapping of epitopes in the primary structure onto sites in the three- dimensional model.
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