IDENTIFICATION OF RESIDUES IN MONOCLONAL-ANTIBODY NC10.8 THAT BIND TOTHE SWEETENER (P-CYANOPHENYL)-N'-(DIPHENYLMETHYL)GUANIDINOACETIC ACIDBY USING RADIOLIGAND BINDING, ABSORPTION AND FLUORESCENCE SPECTROSCOPY, COMPUTER-AIDED MOLECULAR MODELING, AND SITE-DIRECTED MUTAGENESIS

The interactive residues for mouse mAb NC10.8, which binds a superpote nt guanidinium sweetener (p-cyanophenyl)-N'-(diphenylmethyl)guanidinoa cetic acid with high affinity (K-d = 5 nM), were examined by using rad ioligand competitive binding, photoaffinity labeling, absorption and f luorescence spectroscopy, computer-aided molecular modeling, and site- directed mutagenesis. Competitive ligand analogue binding data reveale d important structural features and a pH sensitivity for ligand bindin g. Spectroscopy of the sweetener-mAb complex revealed ligand-induced f luorescence quenching and the presence of a charge-transfer band. Site -directed mutagenesis of L:96W abolished the ligand-induced fluorescen ce quenching and reduced Ab affinity. The apparent K-d increased from 5 nM to more than 200 nM after such modification. A theoretical model of the Fv region was generated with use of a knowledge-based algorithm , and this model was used to identify the locations of key residues in the complementarity determining regions. These experimental and theor etical studies support the prediction that the sweetener ligand coordi nates with the following residues: L:34H contacts the cyanophenyl ring , L:27(D)R forms a salt bridge with the acetic acid moiety, L:96W form s a pi-pi interaction with the cyanophenyl ring, and H:95E contacts th e positively charged aryl nitrogen. These studies are important to our understanding of Ab-ligand specificity and may also shed light on the important chemical motifs responsible for elevated levels of sweetnes s potency in organic compounds.