A STABLE CONFORMER OF IGG OBTAINED BY ACID TREATMENT - STUDY BY CALORIMETRY AND BINDING OF C1Q COMPONENT OF COMPLEMENT AND MONOSPECIFIC ANTI-IGG ANTIBODIES

Thermal stability and functional activity of rabbit IgG in its native conformation and after incubation at pH 2.0 were studied by differenti al scanning calorimetry and binding of conformational probes, i.e., Cl q component of complement and two monospecific monoclonal anti-IgG ant ibodies. The antibodies reacted selectively with the ''hinge'' region joining the Fab and Fc fragments of IgG or with the CH2 domain in the Fc fragment. At pH 2.0 complete unfolding of rabbit IgG does not occur and the protein demonstrates the presence of secondary and compact te rtiary structures, while it differs from the native conformation in de creased overall enthalpy and T-m of thermal unfolding transition and c hanged secondary structure parameters, as shown by CD spectroscopy and scanning calorimetry. Incubation at pH 2.0 followed by renaturation a t neutral pH leads to irreversible conformational changes in IgG. The most significant differences of the two IgG conformers were demonstrat ed by calorimetry at pH 3.5, which revealed that the acid-treated conf ormer differs from the native one in enhanced thermal stability of the CH2 domain. Using the combination of thermodynamic and functional stu dies, it was shown that the stabilization is due to increased interact ion between the CH2 domain of the Fc fragment and the CH1 domain in th e Fab fragment. This results in an increased functional link between t he antigen-binding domain and the Clq binding site in the CH2 domain o f the acid-induced IgG conformer. In parallel with the increase in sta bility of the CH2 domain, conformational changes in the ''hinge'' regi on were found, together with the absence of intrinsic conformational c hanges in the CH2 domain per se, judging from the Clq and monospecific anti-IgG binding assays. The results obtained demonstrate one of the possible mechanisms by which functionally significant rearrangements o f the IgG molecule can be achieved through changes in interactions of invariably folded domains, rather than intrinsic changes of domain con formation.