Determination of the role of aromatic residues in the binding pocket of an antibody that binds a sweetener lioand

The role of aromatics in the binding pocket of monoclonal antibody NC6.8 was studied by the means of radioimmunoassay, fluorescence spectroscopy, and circular dichroism. This mAb binds a tri-substituted guanidinium sweetener: N-(pcyanophenyl)-N'-diphenylmethyl)guanidine acetic acid), which is 200,000 times sweeter than sucrose. The Kj of the IgG and Fab was found to be 2 nM. In the VL and VH regions, there are 15 Tyr and 6 Trp. X- ray crystallograpic studies revealed that two key aromatics (L:96 Tyr and H:33 Trp) are in contact with the ligand. There was 24% quenching of intrinsic fluorescence of the IgG and 57% quenching of the Fab fluorescence due to ligand binding. To verify these findings, a single chain antibody (scFv) was created and analyzed. The protein was overexpressed in E. coli using pET I1A vector in a DE3 cell line with IPTG induction. A 6-His-tail was placed at the C-terminal for Ni2+ affinity column purification. To study the roles of these specific residues, PCR site directed mutagenesis was performed. These constructed proteins included: L96Tyr->L96Trp, H33Trp->H33Tyr, and a combination of both mutations. The mutants were purified and analyzed by CD spectra and fluorescence quenching with the ligand to the wild type antibody. Supported by NIH/GM46535.