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 site or pocket (i.e. secondary forces) that directly effect ligand binding efficiency, and the microenvironment-sensitive spectral properties of bound homologous ligand, residing within the active site of high affinity monoclonal antifluorescyl antibody (mAb) 4-4-20, have been previously reported. This study describes the synthesis and characterization of a series of specially designed and chemically distinct mono-fluoresceinated peptides of equal size (13-mer) as well as the changes in the spectral properties and free energy in the binding of each fluorescein derivatized peptide, upon interaction with mAb 4-4-20. Significant differences in binding efficiency and fluorescence quenching of the ligand, as well as the intrinsic tryptophan fluorescence, were observed for each monofluoresceinated peptide relative to one another and fluorescein ligand. In addition to the effects on the fluorescence quenching of fluorescein and intrinsic tryptophan residues, and the free energy of binding, the conformation of the variable domains of mAb 4-4-20 upon interaction with the fluoresceinated peptides was probed with polyclonal antimetatype (conformational dependent anti-liganded state) antibodies. Studies comparing the results of a solid-phase inhibition assay, with the binding of antimetatype antibodies in solution, suggested 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 proposed as a model and framework to interpret further the results. It was concluded that secondary forces can dictate conformer selection from the various substates. thereby modulating the primary antibody ligand interaction.
Mummert
M E
ME
University of Illinois, Department of Microbiology, Urbana 60801-3704, USA.
Voss
E W
EW
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001666:Binding Sites, Antibody; D019793:Fluorescein; D005452:Fluoresceins; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D007135:Immunoglobulin Variable Region; D008024:Ligands; D010455:Peptides; D019793:Fluorescein