Impact of Noncanonical Disulfide Bond on Thermal Resistance and Binding Affinity of Shark-Derived Single-Domain Antibodies

Single-domain antibodies (sdAbs) often exhibit superior thermal stability compared to traditional antibodies. Efforts are currently focused on enhancing their structural robustness and thermal refolding ability through protein engineering to achieve greater thermal properties and functionality in practical applications. Thermal aggregation is a key factor hindering the reversible thermal denaturation of sdAbs. While studies have explored the role of noncanonical disulfide bonds in camelid-derived VHH aggregation, research on thermal aggregation in shark-derived sdAbs (also known as VNARs) remains scarce, limiting their potential for further optimization. In this study, the role of noncanonical disulfide bonds in VNAR structural robustness, aggregation, and affinity has been simultaneously investigated. Enzyme-linked immunosorbent assay (ELISA), circular dichroism, and intrinsic fluorescence were carried out to compare thermal antigen-binding stability, refolding abilities, and melting temperatures of four wild VNARs B7, 1N9, 2E6, and 2E11 specific for different antigens. Meanwhile, nano differential scanning fluorimetry (nanoDSF) was applied, for the first time, to monitor the thermal aggregation of VNARs. Notably, 2E11, which lacked the noncanonical disulfide bond, demonstrated impressive performance in many aspects. When alanine mutation was engineered to remove the CDR1-CDR3 disulfide bond in 2E6, its refolding rate was increased, and thermal aggregation was prevented significantly. Furthermore, 2E6 exhibited enhanced thermal antigen-binding stability despite reduced structural robustness and affinity. This study provides deeper insights and theoretical support for improving VNAR biophysical properties, with potential applications in enhancing immunoassay performance.