IDENTIFICATION OF THE EPITOPES OF CALCITONIN-GENE-RELATED PEPTIDE (CGRP) FOR 2 ANTI-CGRP MONOCLONAL-ANTIBODIES BY 2D NMR

The interactions between calcitonin gene-related peptide and FAB fragm ents prepared from two different high-affinity anti-CGRP monoclonal an tibodies (CB3 and CD1) have been studied at physiological pH using the ability of H-1 NMR to detect selectively regions of dynamic flexibili ty. The 37-residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more tha n half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However, the regions where substantia l broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the comp lex with CB3 the results indicate that the restricted residues lie exc lusively within the C-terminal half of the peptide, and include residu es 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CD1, the conformationally restricted residues appear to lie predominantly within the N-terminal half of the CGRP molecule, particularly residues 4-16, although several residues in the middle s ection of the sequence (22-31) have reduced conformational freedom. Th ese findings, consistent with the results from immunological assays, a dd considerably to our knowledge of the epitopes.