Conformational and Colloidal Stabilities of Human Immunoglobulin G Fc and Its Cyclized Variant: Independent and Compensatory Participation of Domains in Aggregation of Multidomain Proteins

Monoclonal immunoglobulin G (IgG) is a multidomain protein. It has been reported that the conformational and colloidal stabilities of each domain are different, and it is predicted that limited domains participate in IgG aggregation. In contrast, the influence of interdomain interactions on IgG aggregation remains unclear. The fragment crystallizable (Fc) region is also a multidomain protein consisting of two sets of C(H)2 and C(H)3 domains. Here, we have analyzed the conformational change and aggregate size of an aglycosylated Fc region induced by both acid and salt stresses and have elucidated the influence of interdomain interactions between C(H)2 and C(H)3 domains on the conformational and colloidal stabilities of the aglycosylated Fc region. Singular value decomposition analyses demonstrated that the C(H)2 and C(H)3 domains unfolded almost independently from each other in the aglycosylated Fc region. Meanwhile, the colloidal stabilities of the C(H)2 and C(H)3 domains affect the aggregation process of the unfolded aglycosylated Fc region in a compensatory way. Moreover, the influence of an additional interdomain disulfide bond, introduced at the C-terminal end of the C(H)3 domains to produce the Fc variant, cyclized Fc, was evaluated. This interdomain disulfide bond increased the conformational stability of the C(H)3 domain. The stabilization of the C(H)3 domain in the cyclized Fc successfully improved aggregation tolerance following acid stress, although the sizes of aggregates produced were comparable to those of the aglycosylated Fc region.