Hydrogen-deuterium exchange, adiabatic scanning microcalorimetry and difference sedimentation were used to detect ligand-induced conformational changes in a homogeneous rabbit antibody. Site-filling monovalent and bivalent ologosaccharide ligands, derived from the type III pneumococcal polysaccharide, were used. Only those antigen-antibody complexes which were monomeric with respect to antibody were included into this study. Hydrogen-deuterium exchange reflects the probability of solvent exposure of peptide hydrogens. In this study, the comparative analysis of the probability distributions for various antibody-ligand complexes demonstrates that most if not all of the observable exchange rates were affected by the bincling of hapten. Scanning microcalorimetry detects changes in the overall conformational stability of the antibody molecule. Biphasic heat absorption curves were obtained, reflecting biphasic heat-induced conformational transitions. The significant hapten-induced shift in the temperature of the first transition was interpreted as a consequence of tightening in the domain-domain interactions, while the slight shift in the second transition can be explained by a stabilization of the domains themselves. No difference could be detected in the hydrodynamic properties of the liganded and the unliganded antibodies by difference sedimentation. The effect of monovalent or bivalent hapten bincling upon antibody conformation manifests itself in restricted conformational motility and enhanced conformational stability, without any detectable change in the shape or the volume of the molecule.
ASJC Scopus subject areas
- Molecular Biology