[Non-native conformational states of immunoglobulins: thermodynamic and functional analysis of rabbit IgG].

By changing pH within the pH range 2-7, the processes of intramolecular rearrangement in the multidomain structure of rabbit IgG were induced which resulted in the formation of four IgG conformers - N, NA, I and NI, differing in the amount of structure in the CH2 domain and as well as in its interaction with the neighboring domains. The stability of the IgG conformers was studied by differential scanning calorimetry and expressed in terms of thermodynamic parameters, delta H, Tm, and delta G. The pH-dependent changes in thermal stability of IgG and the range of stability for the conformers were described by conformational (phase) diagrams. The native N-conformer exists at pH 7.0-5.5. At pH < 5.5 the formation of the NA conformer showing significant decooperation (lower extent of interaction) of the domains occurs. Decooperation permits the CH2 domain in the NA conformer to undergo conformational transitions independently of the rest of the molecule-the property which cannot be observed for the more cooperative N-conformer of IgG. The formation of the intermediate I-state occurs at pH < 3 as a consequence of unfolding of the tertiary structure of the CH2 domain, while its secondary structure and compact tertiary structure of the remainder of the IgG domains remain unchanged. Such partially structured and stable states of immunoglobulins have not yet been described. Reversibility of pH-induced transitions in IgG was studied using calorimetry and ligand-binding assays involving the C1q component of the complement, protein A, antigen and monospecific anti-CH2 domain antibodies as conformational probes reporting local conformational changes in the CH2 domain and its interdomain interfaces. Refolding of IgG from the I-state by increasing pH results in the formation of a functionally active N1 conformer differing from native IgG by a changed balance of interdomain interactions. In addition to previously reported data suggesting a greater extent of the CH2 and CH1 domain interactions in the NI conformer, this conformer was shown to possess a lower extent of interaction of the CH2 and CH3 domains. The changed stability and a balance of domain interactions in the NI conformer results in changes in some of the effector functions including enhanced two-site binding to protein A and antigen-dependent binding to C1q with a concomitant decrease in the affinity of protein A binding site and reduced antigen binding capacity of the high affinity IgG. Low affinity IgG does not change the antigen-binding parameters after refolding to the N1 conformer.