Circular dichroism spectroscopy of monoclonal antibodies that bind a superpotent guanidinium sweetener ligand

Abstract

Three monoclonal antibodies (mAb) with nanomolar affinity to the superpotent trisubstituted guanidinium sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidineacetic acid were studied by circular dichroism (CD) spectroscopy. Two mAb, NC6.8 (IgG2b,kappa) and NC10.8 (IgG3, kappa) exhibited similar CD spectra, but mAb NC10.14 (IgG2b, lambda) had very different CD spectra in both far- and near-UV regions. Some of these differences may be due to effects of aromatic amino acid side chains, especially Trp and Tyr, located at the immunoglobulin intradomain surfaces. Heavy- and light-chain dissociation of reduced Fab fragments in 1 M acetic acid minimized these effects. Ligand binding changed the sign and amplitudes of the near-UV CD spectra of all three mAb. Calculation of the CD difference spectra (bound minus free) of stoichiometrically bound antibody-ligand complexes allowed us to visualize the net spectral changes. On the basis of the three-dimensional structures experimentally solved for NC6.8 and theoretical models of NC10.8 and NC10.14, we suggest that the p-cyanophenyl moiety on the sweetener ligand acts as a molecular pointer in the CD spectra and identifies contact aromatic residues in the different antibody binding pockets.