Effects of secondary forces on the ligand binding and conformational state of antifluorescein monoclonal antibody 9-40.
Biochemical interactions that occur external to the antibody active site have been termed secondary forces. Secondary forces are supplemental to interactions within the antibody active site (i.e., primary interactions) and can affect ligand binding efficiency as well as variable domain conformation. The antifluorescein antibody system has been determined to be a superior method for delineating primary from secondary interactive components due to the active site-filling properties of the fluorescyl ligand. To date, all studies of secondary forces within the context of the antifluorescein system have been with the high-affinity monoclonal antibody 4-4-20 (mAb 4-4-20) (Mummert & Voss, 1995, 1996, 1997). In order to determine the generality of experimental observations and proposed models, we investigated the effects of secondary forces on the antifluorescein mAb 9-40. In addition to assessing the results of former studies, mAb 9-40 possesses properties unique from those of mAb 4-4-20, namely, a decreased affinity for fluorescein and increased conformational dynamics relative to mAb 4-4-20 (Carrero & Voss, 1996). Results of fluorescein and intrinsic mAb 9-40 tryptophan quenching as well as differential scanning calorimetric (DSC) studies indicated that secondary forces modulated the conformational (metatypic) state in accordance with previous investigations with mAb 4-4-20. Unlike mAb 4-4-20, mAb 9-40 did not exhibit altered ligand binding efficiency due to the inclusion of secondary interactive components. Models were developed that proposed that the increased malleability of mAb 9-40 variable domains could account for functional differences in properties between mAb 9-40 and mAb 4-4-20.
Mummert
M E
ME
Department of Microbiology, B103 Chemical and Life Sciences Laboratory, University of Illinois, 601 South Goodwin Avenue, Urbana, Illinois 61801-3704, USA.
Voss
E W
EW
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation; D014364:Tryptophan
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D014364:Tryptophan