EFFECTS OF SECONDARY FORCES ON THE LIGAND-BINDING PROPERTIES AND VARIABLE DOMAIN CONFORMATIONS OF A MONOCLONAL ANTI-FLUORESCYL ANTIBODY

Biochemical interactions occurring external to the antibody active sit e or pocket (i.e. secondary forces) that directly effect ligand bindin g efficiency, and the microenvironment-sensitive spectral properties o f bound homologous ligand, residing within the active site of high aff inity monoclonal antifluorescyl antibody (mAb) 4-4-20, have been previ ously reported. This study describes the synthesis and characterizatio n of a series of specially designed and chemically distinct mono-fluor esceinated peptides of equal size (13-mer) as well as the changes in t he spectral properties and free energy in the binding of each fluoresc ein derivatized peptide, upon interaction with mAb 4-4-20. Significant differences in binding efficiency and fluorescence quenching of the l igand, as well as the intrinsic tryptophan fluorescence, were observed for each monofluoresceinated peptide relative to one another and fluo rescein ligand. In addition to the effects on the fluorescence quenchi ng of fluorescein and intrinsic tryptophan residues, and the free ener gy of binding, the conformation of the variable domains of mAb 4-4-20 upon interaction with the fluoresceinated peptides was probed with pol yclonal antimetatype (conformational dependent anti-liganded state) an tibodies. Studies comparing the results of a solid-phase inhibition as say, with the binding of antimetatype antibodies in solution, suggeste d that variant metatypic states of mAb 4-4-20 resulted from binding of the various fluorescein derivatized peptides. Depiction of the mAb 4- 4-20 active site as a series of thermally averaged substates is propos ed as a model and framework to interpret further the results. It was c oncluded that secondary forces can dictate conformer selection from th e various substates, thereby modulating the primary antibody-ligand in teraction. Copyright (C) 1996 Elsevier Science Ltd.