The reduced form of the antibody CH2 domain.

Protein Science : a Publication of the Protein Society
Zhaoyong XiRieko Ishima

Abstract

Among the immunoglobulin domains, the CH2 domain has the lowest thermal stability, which also depends on amino acid sequence and buffer conditions. To further identify factors that influence CH2 folding and stability, we characterized the domain in the reduced form using differential scanning fluorimetry and nuclear magnetic resonance. We show that the CH2 domain can fold, similarly to the disulfide-bridged form, without forming a disulfide-bridge, even though the protein contains two Cys residues. Although the reduced form exhibits thermal stability more than 15°C lower than the disulfide-bridged form, it does not undergo immediate full oxidization. To explain this phenomenon, we compared CH2 oxidization at different conditions and demonstrate a need for significant fluctuation of the folded conformation to enhance CH2 disulfide-bridge formation. We conclude that, since CH2 can be purified as a folded, semi-stable, reduced protein that can coexist with the oxidized form, verification of the level of oxidization at each step is critical in CH2 engineering studies.

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