Supplementary Evidence for Structural Requirements at Position 9 for Glucagon Activity Abstract: Our previous work has established that position-9 aspartic acid in glucagon is a critical residue for transduction of the hormone response. An uncoupling of the binding interaction from adenylate cyclase activation was demonstrated by the observation that amino acid replacements at position 9 resulted in peptides that had no measurable adenylate cyclase activity yet was still recognized by the glucagon receptor. It was also later shown that His¹ played a major role in activation, and it was suggested that an electrostatic interaction between the aspartic acid carboxylate and the histidine imidazole occurs as part of the activation mechanism. This does not preclude intermolecular interactions of this aspartic acid with other residues within the receptor binding site. The observation that a conservative substitution of glutamic acid for aspartic acid at position 9 was sufficient to result in the potent antagonist, des-His¹d[Glu⁹]glucagon amide, implied that even glutamic acid possessed the minimum properties necessary for inhibition, and that the precise position of the carboxyl group at position 9 in glucagon is an absolute requirement for full agonist activity. This investigation was conducted to find out ab initio calculations and molecular modeling can shed some light on the source of this phenomenon.