[A stable conformer of IgG, prepared by an acidic influence: study by calorimetry, binding of the C1q complement component, and monospecific anti-IgG].
Thermal stability and functional activity of rabbit IgG in its native conformation and after incubation at pH 2.0 were studied using differential scanning calorimetry and binding of conformational probes, i.e., the C1q component of the complement and two monospecific anti-IgG antibodies. The latter 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 did not occur: the protein demonstrated the presence of secondary and compact tertiary structures but differed from the native conformation by decreased overall enthalpy and TM of thermal denaturation as well as by changed secondary structure parameters as could be evidenced from CD spectroscopy and scanning calorimetry data. Incubation at pH 2.0 followed by renaturation at neutral pH led to irreversible conformational changes in IgG. The most significant differences between the two IgG conformers were demonstrated by calorimetry at pH 3.5 which revealed that the acid-treated conformer differs from the native one by enhanced thermal stability of the CH2 domain. Using the combination of thermodynamic and functional studies, it was shown that the origin of stabilization was the increase in the extent of interaction between the CH2 domain in the Fc fragment and the CHI domain in the Fab fragment. This resulted in the increase of the functional link between the antigen-binding domain and the C1q binding site in the CH2 domain of the acid-induced IgG conformer. In parallel with the increase in stability of the CH2 domain, conformational changes in the "hinge" region were found, together with the absence of intrinsic conformational changes in the CH2 domain proper as could be judged from C1q and monospecific anti-IgG binding assays. The results obtained demonstrate one of possible mechanisms whereby functionally significant rearrangements in the IgG molecule can be induced by changes in the interactions between invariably folded domains rather than by intrinsic changes in the domain conformation.
Kravchuk
Z I
ZI
Vlasov
A P
AP
Liakhnovich
G V
GV
Martsev
S P
SP
D000818:Animals; D000917:Antibody Formation; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D015922:Complement C1q / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007133:Immunoglobulin Joining Region / Q000276:immunology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007133:Immunoglobulin Joining Region; D015922:Complement C1q
Stabil'nyĭ konformer IgG, poluchennyĭ kislotnym vozdeĭstviem: issledovanie s pomoshch'iu kalorimetrii, sviazyvaniia C1q komponenta komplenta i monspetsificheskikh anti-IgG.